Dr Direk Limmathurotsakul

Research Area: Microbiology
Technology Exchange: Bioinformatics and Medical statistics
Scientific Themes: Tropical Medicine & Global Health and Clinical Trials & Epidemiology
Keywords: Infection, Immunology, Microbiology, Bioinformatics, Clinical Epidemiology and Infectious Diseases
Web Links:

The Head of Microbiology at Mahidol-Oxford Tropical Medicine Research Unit (MORU), Mahidol University (http://www.tropmedres.ac), since January 2012, Direk Limmathurotsakul also holds a Wellcome Trust Intermediate Fellowship in Public Health and Tropical Medicine. Besides facilitating other researchers’ work, Direk runs his own fellowship-funded research programme, “Reducing the global burden of melioidosis”. From January 2014 to December 2015, Direk was also the director of Southeast Asia Infectious Clinical Research Network (SEAICRN, http://www.seaicrn.org).

Melioidosis, an often-fatal infectious disease caused by bacteria Burkholderia pseudomallei, is one of Direk’s main research areas. He led a series of laboratory and clinical studies, and demonstrated for the first time that ingestion and inhalation are important infection routes for melioidosis in northeast Thailand. He also developed the first evidence-based guidelines for the prevention of melioidosis for people living in endemic areas. Direk is a chair of International Melioidosis Society [IMS] (http://www.melioidosis.info) with a mission to facilitate communication among member of IMS and people with an interest in melioidosis, and to create awareness about melioidosis among medical and veterinary professionals, scientists, health planners, policy makers and the general public in different parts of the world and to support the melioidosis research community.

Antimicrobial resistance is also one of Direk’s main research areas. By integrating routinely collected data from a range of databases he estimate that around an extra 19,000 deaths are caused by multi-drug resistant bacteria in Thailand each year. Thailand has a population of about 70 million, and so, per capita, this estimate is about 3 to 5 times larger than those for the United States and European Union (which have a populations of about 300 million and 500 million, respectively). He also shows that more of the bacteria collected from patients are resistant to multiple antimicrobial drugs and that the burden of antimicrobial resistance in Thailand is worsening over time. These findings suggest that more studies with a systematic approach need to be done in other low- and middle-income countries, especially in countries where microbiological laboratories are readily available and routinely used. Further work is also needed to identify where resources and attentions are most needed to effectively fight against antimicrobial resistance in low- and middle-income countries.

Direk is also an expert in Bayesian statistics and statistics for evaluating diagnostic tests. He developed a user-friendly open-access web-based application, http://mice.tropmedres.ac, that allows general researchers to apply imperfect gold standard Bayesian latent class models to their own data sets. 

Name Department Institution Country
Professor Sharon Peacock London School of Hygiene and Tropical Medicine United Kingdom
Gouliouris T, Blane B, Brodrick HJ, Raven KE, Ambridge KE, Kidney AD, Hadjirin NF, Török ME, Limmathurotsakul D, Peacock SJ. 2016. Comparison of two chromogenic media for the detection of vancomycin-resistant enterococcal carriage by nursing home residents. Diagn Microbiol Infect Dis, 85 (4), pp. 409-412. | Show Abstract | Read more

We compared ChromID VRE and Brilliance VRE media for the detection of vancomycin-resistant enterococci (VRE). Using a panel of 28 enterococcal isolates, 10 vanA Enterococcus faecium and three vanA Enterococcus faecalis isolates grew as per manufacturers' instructions whilst growth of two vanC and eight vancomycin-susceptible enterococci was inhibited on both media. Important differences were noted in the selectivity and chromogenic properties of the two media for vanA Enterococcus raffinosus and vanB E. faecium. The two media were further evaluated using 295 stool samples from nursing home residents, 34 of which grew VRE (11.5%). ChromID and Brilliance had comparable sensitivity, which was increased markedly by prolonging incubation to 48 hours (from 29% to 82%, and from 41% to 85%, respectively) and by a pre-enrichment step (to 97% and 100%, respectively). Brilliance VRE agar had higher selectivity at 48 hours, and after pre-enrichment.

Blacksell SD, Lim C, Tanganuchitcharnchai A, Jintaworn S, Kantipong P, Richards AL, Paris DH, Limmathurotsakul D, Day NP. 2016. Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay. J Clin Microbiol, 54 (6), pp. 1472-1478. | Show Abstract | Read more

The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.

Suttisunhakul V, Wuthiekanun V, Brett PJ, Khusmith S, Day NP, Burtnick MN, Limmathurotsakul D, Chantratita N. 2016. Development of Rapid Enzyme-Linked Immunosorbent Assays for Detection of Antibodies to Burkholderia pseudomallei. J Clin Microbiol, 54 (5), pp. 1259-1268. | Show Abstract | Read more

Burkholderia pseudomallei, the causative agent of melioidosis, is an environmental bacillus found in northeast Thailand. The mortality rate of melioidosis is ∼40%. An indirect hemagglutination assay (IHA) is used as a reference serodiagnostic test; however, it has low specificity in areas where the background seropositivity of healthy people is high. To improve assay specificity and reduce the time for diagnosis, four rapid enzyme-linked immunosorbent assays (ELISAs) were developed using two purified polysaccharide antigens (O-polysaccharide [OPS] and 6-deoxyheptan capsular polysaccharide [CPS]) and two crude antigens (whole-cell [WC] antigen and culture filtrate [CF] antigen) of B. pseudomallei The ELISAs were evaluated using serum samples from 141 culture-confirmed melioidosis patients from Thailand along with 188 healthy donors from Thailand and 90 healthy donors from the United States as controls. The areas under receiver operator characteristic curves (AUROCC) using Thai controls were high for the OPS-ELISA (0.91), CF-ELISA (0.91), and WC-ELISA (0.90), while those of CPS-ELISA (0.84) and IHA (0.72) were lower. AUROCC values using U.S. controls were comparable to those of the Thai controls for all ELISAs except IHA (0.93). Using a cutoff optical density (OD) of 0.87, the OPS-ELISA had a sensitivity of 71.6% and a specificity of 95.7% for Thai controls; for U.S. controls, specificity was 96.7%. An additional 120 serum samples from tuberculosis, scrub typhus, or leptospirosis patients were evaluated in all ELISAs and resulted in comparable or higher specificities than using Thai healthy donors. Our findings suggest that antigen-specific ELISAs, particularly the OPS-ELISA, may be useful for serodiagnosis of melioidosis in areas where it is endemic and nonendemic.

Limmathurotsakul D, Golding N, Dance DA, Messina JP, Pigott DM, Moyes CL, Rolim DB, Bertherat E, Day NP, Peacock SJ, Hay SI. 2016. Predicted global distribution of Burkholderia pseudomallei and burden of melioidosis. Nat Microbiol, 1 (1), pp. 15008-15008. | Show Abstract | Read more

Burkholderia pseudomallei, a highly pathogenic bacterium that causes melioidosis, is commonly found in soil in Southeast Asia and Northern Australia(1,2). Melioidosis can be difficult to diagnose due to its diverse clinical manifestations and the inadequacy of conventional bacterial identification methods(3). The bacterium is intrinsically resistant to a wide range of antimicrobials, and treatment with ineffective antimicrobials may result in case fatality rates (CFRs) exceeding 70%(4,5). The importation of infected animals has, in the past, spread melioidosis to non-endemic areas(6,7). The global distribution of B. pseudomallei and burden of melioidosis, however, remain poorly understood. Here, we map documented human and animal cases, and the presence of environmental B. pseudomallei, and combine this in a formal modelling framework(8-10) to estimate the global burden of melioidosis. We estimate there to be 165,000 (95% credible interval 68,000-412,000) human melioidosis cases per year worldwide, of which 89,000 (36,000-227,000) die. Our estimates suggest that melioidosis is severely underreported in the 45 countries in which it is known to be endemic and that melioidosis is likely endemic in a further 34 countries which have never reported the disease. The large numbers of estimated cases and fatalities emphasise that the disease warrants renewed attention from public health officials and policy makers.

Bicanic T, Bottomley C, Loyse A, Brouwer AE, Muzoora C, Taseera K, Jackson A, Phulusa J et al. 2015. Toxicity of Amphotericin B Deoxycholate-Based Induction Therapy in Patients with HIV-Associated Cryptococcal Meningitis. Antimicrob Agents Chemother, 59 (12), pp. 7224-7231. | Show Abstract | Read more

Amphotericin B deoxycholate (AmBd) is the recommended induction treatment for HIV-associated cryptococcal meningitis (CM). Its use is hampered by toxicities that include electrolyte abnormalities, nephrotoxicity, and anemia. Protocols to minimize toxicity are applied inconsistently. In a clinical trial cohort of AmBd-based CM induction treatment, a standardized protocol of preemptive hydration and electrolyte supplementation was applied. Changes in blood counts, electrolyte levels, and creatinine levels over 14 days were analyzed in relation to the AmBd dose, treatment duration (short course of 5 to 7 days or standard course of 14 days), addition of flucytosine (5FC), and outcome. In the 368 patients studied, the hemoglobin levels dropped by a mean of 1.5 g/dl (95% confidence interval [CI], 1.0 to 1.9 g/dl) following 7 days of AmBd and by a mean of 2.3 g/dl (95% CI, 1.1 to 3.6 g/dl) after 14 days. Serum creatinine levels increased by 37 μmol/liter (95% CI, 30 to 45 μmol/liter) by day 7 and by 49 μmol/liter (95% CI, 35 to 64μmol/liter) by day 14 of AmBd treatment. Overall, 33% of patients developed grade III/IV anemia, 5.6% developed grade III hypokalemia, 9.5% had creatinine levels that exceeded 220 μmol, and 6% discontinued AmBd prematurely. The addition of 5FC was associated with a slight increase in anemia but not neutropenia. Laboratory abnormalities stabilized or reversed during the second week in patients on short-course induction. Grade III/IV anemia (adjusted odds ratio [aOR], 2.2; 95% CI, 1.1 to 4.3; P = 0.028) and nephrotoxicity (aOR, 4.5; 95% CI, 1.8 to 11; P = 0.001) were risk factors for 10-week mortality. In summary, routine intravenous saline hydration and preemptive electrolyte replacement during AmBd-based induction regimens for HIV-associated CM minimized the incidence of hypokalemia and nephrotoxicity. Anemia remained a concerning adverse effect. The addition of flucytosine was not associated with increased neutropenia. Shorter AmBd courses were less toxic, with rapid reversibility.

Hantrakun V, Chierakul W, Chetchotisakd P, Anunnatsiri S, Currie BJ, Peacock SJ, Day NP, Cheah PY, Limmathurotsakul D, Lubell Y. 2015. Cost-effectiveness analysis of parenteral antimicrobials for acute melioidosis in Thailand. Trans R Soc Trop Med Hyg, 109 (12), pp. 803. | Read more

Blane B, Brodrick HJ, Gouliouris T, Ambridge KE, Kidney AD, Ludden CM, Limmathurotsakul D, Török ME, Peacock SJ. 2016. Comparison of 2 chromogenic media for the detection of extended-spectrum β-lactamase producing Enterobacteriaceae stool carriage in nursing home residents. Diagn Microbiol Infect Dis, 84 (3), pp. 181-183. | Show Abstract | Read more

ChromID ESBL agar and Brilliance ESBL agar were compared for the isolation of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae from 298 stools. These had comparable sensitivity and selectivity for the 116 positive samples. Pre-enrichment with cefpodoxime and extending incubation to 48 hours after direct plating both significantly increased sensitivity but reduced selectivity of both agars.

Jenjaroen K, Chumseng S, Sumonwiriya M, Ariyaprasert P, Chantratita N, Sunyakumthorn P, Hongsuwan M, Wuthiekanun V et al. 2015. T-Cell Responses Are Associated with Survival in Acute Melioidosis Patients. PLoS Negl Trop Dis, 9 (10), pp. e0004152. | Show Abstract | Read more

BACKGROUND: Melioidosis is an increasingly recognised cause of sepsis and death across South East Asia and Northern Australia, caused by the bacterium Burkholderia pseudomallei. Risk factors include diabetes, alcoholism and renal disease, and a vaccine targeting at-risk populations is urgently required. A better understanding of the protective immune response in naturally infected patients is essential for vaccine design. METHODS: We conducted a longitudinal clinical and immunological study of 200 patients with melioidosis on admission, 12 weeks (n = 113) and 52 weeks (n = 65) later. Responses to whole killed B. pseudomallei were measured in peripheral blood mononuclear cells (PBMC) by interferon-gamma (IFN-γ) ELIspot assay and flow cytometry and compared to those of control subjects in the region with diabetes (n = 45) and without diabetes (n = 43). RESULTS: We demonstrated strong CD4+ and CD8+ responses to B. pseudomallei during acute disease, 12 weeks and 52 weeks later. 28-day mortality was 26% for melioidosis patients, and B. pseudomallei-specific cellular responses in fatal cases (mean 98 IFN-γ cells per million PBMC) were significantly lower than those in the survivors (mean 142 IFN-γ cells per million PBMC) in a multivariable logistic regression model (P = 0.01). A J-shaped curve association between circulating neutrophil count and mortality was seen with an optimal count of 4000 to 8000 neutrophils/μl. Melioidosis patients with known diabetes had poor diabetic control (median glycated haemoglobin HbA1c 10.2%, interquartile range 9.2-13.1) and showed a stunted B. pseudomallei-specific cellular response during acute illness compared to those without diabetes. CONCLUSIONS: The results demonstrate the role of both CD4+ and CD8+ T-cells in protection against melioidosis, and an interaction between diabetes and cellular responses. This supports development of vaccine strategies that induce strong T-cell responses for the control of intracellular pathogens such as B. pseudomallei.

Lim C, Peacock SJ, Limmathurotsakul D. 2016. Association between activities related to routes of infection and clinical manifestations of melioidosis. Clin Microbiol Infect, 22 (1), pp. 79.e1-79.e3. | Show Abstract | Read more

We sought associations between route of infection by Burkholderia pseudomallei and clinical manifestations in 330 cases of melioidosis in northeast Thailand using bivariate multivariable logistic regression models. Activities related to skin inoculation were negatively associated with bacteraemia, activities related to ingestion were associated with bacteraemia, and activities related to inhalation were associated with pneumonia. Our study suggests that route of infection is one of the factors related to clinical manifestations of melioidosis.

Suttisunhakul V, Chantratita N, Wikraiphat C, Wuthiekanun V, Douglas Z, Day NP, Limmathurotsakul D, Brett PJ, Burtnick MN. 2015. Evaluation of Polysaccharide-Based Latex Agglutination Assays for the Rapid Detection of Antibodies to Burkholderia pseudomallei. Am J Trop Med Hyg, 93 (3), pp. 542-546. | Show Abstract | Read more

Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Diagnosis of melioidosis currently relies on the isolation of B. pseudomallei from clinical samples, which can take several days. An indirect hemagglutination assay (IHA) is widely used for serodiagnosis, but it has a short shelf life, is poorly standardized, and requires a viable bacteria culture performed in a biosafety level 3 (BSL-3) laboratory. To improve the diagnostic methods, we have developed two rapid latex agglutination tests based on purified B. pseudomallei O-polysaccharide (OPS) and capsular polysaccharide (CPS) antigens. The immunodiagnostic potential of these tests was evaluated using serum from culture-confirmed melioidosis patients (N = 143) and healthy donors from either endemic (N = 199) or non-endemic areas (N = 90). The sensitivity of the OPS-based latex agglutination assay (OPS-latex; 84.4%) was significantly higher than both the CPS-latex (69.5%) (P < 0.001) and IHA (69.5%) (P = 0.001). When evaluated with Thai donor serum, the OPS-latex had comparable specificity (56.9%) to the CPS-latex (63.8%) (P = 0.053), but was significantly lower than the IHA (67.6%) (P = 0.002). In contrast, all tests with U.S. donor serum were highly specific (≥ 97.8%). These results suggest that polysaccharide-based latex agglutination assays may be useful for serodiagnosis of melioidosis in non-endemic areas.

Wuthiekanun V, Amornchai P, Langla S, White NJ, Day NP, Limmathurotsakul D, Peacock SJ. 2015. Antimicrobial Disk Susceptibility Testing of Leptospira spp. Using Leptospira Vanaporn Wuthiekanun (LVW) Agar. Am J Trop Med Hyg, 93 (2), pp. 241-243. | Show Abstract | Read more

Leptospira Vanaporn Wuthiekanun (LVW) agar was used to develop a disk diffusion assay for Leptospira spp. Ten pathogenic Leptospira isolates were tested, all of which were susceptible to 17 antimicrobial agents (amoxicillin/clavulanic acid, amoxicillin, azithromycin, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, doripenem, doxycycline, gentamicin, linezolid, nitrofurantoin, penicillin, piperacillin/tazobactam, and tetracycline). All 10 isolates had no zone of growth inhibition for four antimicrobials (fosfomycin, nalidixic acid, rifampicin, and trimethoprim/sulfamethoxazole). Of the ten Leptospira, seven had a growth inhibition zone of ≤ 21 mm for aztreonam, the zone diameter susceptibility break point for Enterobacteriaceae. This assay could find utility as a simple screening method during the epidemiological surveillance of antimicrobial resistance in Leptospira spp.

Limmathurotsakul D, Funnell SG, Torres AG, Morici LA, Brett PJ, Dunachie S, Atkins T, Altmann DM, Bancroft G, Peacock SJ, Steering Group on Melioidosis Vaccine Development. 2015. Consensus on the development of vaccines against naturally acquired melioidosis. Emerg Infect Dis, 21 (6), pp. e1-e7. | Show Abstract | Read more

Several candidates for a vaccine against Burkholderia pseudomallei, the causal bacterium of melioidosis, have been developed, and a rational approach is now needed to select and advance candidates for testing in relevant nonhuman primate models and in human clinical trials. Development of such a vaccine was the topic of a meeting in the United Kingdom in March 2014 attended by international candidate vaccine developers, researchers, and government health officials. The focus of the meeting was advancement of vaccines for prevention of natural infection, rather than for protection from the organism's known potential for use as a biological weapon. A direct comparison of candidate vaccines in well-characterized mouse models was proposed. Knowledge gaps requiring further research were identified. Recommendations were made to accelerate the development of an effective vaccine against melioidosis.

Saunderson RB, Gouliouris T, Nickerson EK, Cartwright EJ, Kidney A, Aliyu SH, Brown NM, Limmathurotsakul D, Peacock SJ, Török ME. 2015. Impact of routine bedside infectious disease consultation on clinical management and outcome of Staphylococcus aureus bacteraemia in adults. Clin Microbiol Infect, 21 (8), pp. 779-785. | Show Abstract | Read more

Staphylococcus aureus bacteraemia (SAB) is a common, serious infection that is associated with high rates of morbidity and mortality. Evidence suggests that infectious disease consultation (IDC) improves clinical management in patients with SAB. We examined whether the introduction of a routine bedside IDC service for adults with SAB improved clinical management and outcomes compared to telephone consultation. We conducted an observational cohort study of 571 adults with SAB at a teaching hospital in the United Kingdom between July 2006 and December 2012. A telephone consultation was provided on the day of positive blood culture in all cases, but an additional bedside IDC was provided after November 2009 (routine IDC group). Compared to patients in the pre-IDC group, those in the routine IDC group were more likely to have a removable focus of infection identified, echocardiography performed and follow-up blood cultures performed. They also received longer courses of antimicrobial therapy, were more likely to receive combination antimicrobial therapy and were more likely to have SAB recorded in the hospital discharge summary. There was a trend towards lower mortality at 30 days in the routine IDC group compared to the pre-IDC group (12% vs. 22%, p 0.07). Our findings suggest that routine bedside IDC should become the standard of care for adults with SAB.

Hantrakun V, Chierakul W, Chetchotisakd P, Anunnatsiri S, Currie BJ, Peacock SJ, Day NP, Cheah PY, Limmathurotsakul D, Lubell Y. 2015. Cost-effectiveness analysis of parenteral antimicrobials for acute melioidosis in Thailand. Trans R Soc Trop Med Hyg, 109 (6), pp. 416-418. | Show Abstract | Read more

BACKGROUND: Melioidosis is a common community-acquired infectious disease in northeast Thailand associated with overall mortality of approximately 40% in hospitalized patients, and over 70% in severe cases. Ceftazidime is recommended for parenteral treatment in patients with suspected melioidosis. Meropenem is increasingly used but evidence to support this is lacking. METHODS: A decision tree was used to estimate the cost-effectiveness of treating non-severe and severe suspected acute melioidosis cases with either ceftazidime or meropenem. RESULTS: Empirical treatment with meropenem is likely to be cost-effective providing meropenem reduces mortality in severe cases by at least 9% and the proportion with subsequent culture-confirmed melioidosis is over 20%. CONCLUSIONS: In this context, treatment of severe cases with meropenem is likely to be cost-effective, while the evidence to support the use of meropenem in non-severe suspected melioidosis is not yet available.

Saiprom N, Amornchai P, Wuthiekanun V, Day NPJ, Limmathurotsakul D, Peacock SJ, Chantratita N. 2015. Trimethoprim/sulfamethoxazole resistance in clinical isolates of Burkholderia pseudomallei from Thailand International Journal of Antimicrobial Agents, 45 (5), pp. 557-559. | Read more

Cooper BS, Kotirum S, Kulpeng W, Praditsitthikorn N, Chittaganpitch M, Limmathurotsakul D, Day NP, Coker R, Teerawattananon Y, Meeyai A. 2015. Mortality attributable to seasonal influenza A and B infections in Thailand, 2005-2009: a longitudinal study. Am J Epidemiol, 181 (11), pp. 898-907. | Show Abstract | Read more

Influenza epidemiology differs substantially in tropical and temperate zones, but estimates of seasonal influenza mortality in developing countries in the tropics are lacking. We aimed to quantify mortality due to seasonal influenza in Thailand, a tropical middle-income country. Time series of polymerase chain reaction-confirmed influenza infections between 2005 and 2009 were constructed from a sentinel surveillance network. These were combined with influenza-like illness data to derive measures of influenza activity and relationships to mortality by using a Bayesian regression framework. We estimated 6.1 (95% credible interval: 0.5, 12.4) annual deaths per 100,000 population attributable to influenza A and B, predominantly in those aged ≥60 years, with the largest contribution from influenza A(H1N1) in 3 out of 4 years. For A(H3N2), the relationship between influenza activity and mortality varied over time. Influenza was associated with increases in deaths classified as resulting from respiratory disease (posterior probability of positive association, 99.8%), cancer (98.6%), renal disease (98.0%), and liver disease (99.2%). No association with circulatory disease mortality was found. Seasonal influenza infections are associated with substantial mortality in Thailand, but evidence for the strong relationship between influenza activity and circulatory disease mortality reported in temperate countries is lacking.

Saiprom N, Amornchai P, Wuthiekanun V, Day NP, Limmathurotsakul D, Peacock SJ, Chantratita N. 2015. Trimethoprim/sulfamethoxazole resistance in clinical isolates of Burkholderia pseudomallei from Thailand. Int J Antimicrob Agents, 45 (5), pp. 557-559. | Read more

Birnie E, Wiersinga WJ, Limmathurotsakul D, Grobusch MP. 2015. Melioidosis in Africa: should we be looking more closely? Future Microbiol, 10 (2), pp. 273-281. | Show Abstract | Read more

Melioidosis is a life-threatening infection caused by the Gram-negative bacterium Burkholderia pseudomallei, mainly found in Southeast Asia. Recently, African foci have been identified, although reports remain mostly anecdotal. In Africa, multiple febrile diseases have been erroneously attributed to malaria in the past, and many cases of fever remain mis- or undiagnosed. Vigilance for previously under-recognized pathogens may enhance our understanding of disease epidemiology and facilitate improvement of patient care. Melioidosis may be such a condition. We summarize data on melioidosis in Africa and discuss the future directions for epidemiological, clinical and bacteriological studies. We conclude that searching for old bugs in new places is no academic treasure hunt but a clinically relevant activity to pursue.

Thaipadungpanit J, Amornchai P, Nickerson EK, Wongsuvan G, Wuthiekanun V, Limmathurotsakul D, Peacock SJ. 2015. Clinical and molecular epidemiology of Staphylococcus argenteus infections in Thailand. J Clin Microbiol, 53 (3), pp. 1005-1008. | Show Abstract | Read more

Molecular typing of 246 Staphylococcus aureus isolates from unselected patients in Thailand showed that 10 (4.1%) were actually Staphylococcus argenteus. Contrary to the suggestion that S. argenteus is less virulent than S. aureus, we demonstrated comparable rates of morbidity, death, and health care-associated infection in patients infected with either of these two species.

Lim C, Blacksell SD, Laongnualpanich A, Kantipong P, Day NP, Paris DH, Limmathurotsakul D. 2015. Optimal Cutoff Titers for Indirect Immunofluorescence Assay for Diagnosis of Scrub Typhus. J Clin Microbiol, 53 (11), pp. 3663-3666. | Show Abstract | Read more

We determined the optimal cutoff titers in admission and convalescent-phase samples for scrub typhus indirect immunofluorescence assay using Bayesian latent class models. Cutoff titers of ≥1:3,200 in an admission sample or of a ≥4-fold rise to ≥1:3,200 in a convalescent-phase sample provided the highest accuracy (sensitivity, 81.6%; specificity, 100%).

Birnie E, Wiersinga WJ, Limmathurotsakul D, Grobusch MP. 2015. Melioidosis in Africa: should we be looking more closely? Future microbiology, 10 (2), pp. 273-281. | Show Abstract | Read more

Melioidosis is a life-threatening infection caused by the Gram-negative bacterium Burkholderia pseudomallei, mainly found in Southeast Asia. Recently, African foci have been identified, although reports remain mostly anecdotal. In Africa, multiple febrile diseases have been erroneously attributed to malaria in the past, and many cases of fever remain mis- or undiagnosed. Vigilance for previously under-recognized pathogens may enhance our understanding of disease epidemiology and facilitate improvement of patient care. Melioidosis may be such a condition. We summarize data on melioidosis in Africa and discuss the future directions for epidemiological, clinical and bacteriological studies. We conclude that searching for old bugs in new places is no academic treasure hunt but a clinically relevant activity to pursue.

Luangasanatip N, Hongsuwan M, Limmathurotsakul D, Lubell Y, Lee AS, Harbarth S, Day NP, Graves N, Cooper BS. 2015. Comparative efficacy of interventions to promote hand hygiene in hospital: systematic review and network meta-analysis. BMJ, 351 pp. h3728. | Show Abstract | Read more

OBJECTIVE: To evaluate the relative efficacy of the World Health Organization 2005 campaign (WHO-5) and other interventions to promote hand hygiene among healthcare workers in hospital settings and to summarize associated information on use of resources. DESIGN: Systematic review and network meta-analysis. DATA SOURCES: Medline, Embase, CINAHL, NHS Economic Evaluation Database, NHS Centre for Reviews and Dissemination, Cochrane Library, and the EPOC register (December 2009 to February 2014); studies selected by the same search terms in previous systematic reviews (1980-2009). REVIEW METHODS: Included studies were randomised controlled trials, non-randomised trials, controlled before-after trials, and interrupted time series studies implementing an intervention to improve compliance with hand hygiene among healthcare workers in hospital settings and measuring compliance or appropriate proxies that met predefined quality inclusion criteria. When studies had not used appropriate analytical methods, primary data were re-analysed. Random effects and network meta-analyses were performed on studies reporting directly observed compliance with hand hygiene when they were considered sufficiently homogeneous with regard to interventions and participants. Information on resources required for interventions was extracted and graded into three levels. RESULTS: Of 3639 studies retrieved, 41 met the inclusion criteria (six randomised controlled trials, 32 interrupted time series, one non-randomised trial, and two controlled before-after studies). Meta-analysis of two randomised controlled trials showed the addition of goal setting to WHO-5 was associated with improved compliance (pooled odds ratio 1.35, 95% confidence interval 1.04 to 1.76; I(2)=81%). Of 22 pairwise comparisons from interrupted time series, 18 showed stepwise increases in compliance with hand hygiene, and all but four showed a trend for increasing compliance after the intervention. Network meta-analysis indicated considerable uncertainty in the relative effectiveness of interventions, but nonetheless provided evidence that WHO-5 is effective and that compliance can be further improved by adding interventions including goal setting, reward incentives, and accountability. Nineteen studies reported clinical outcomes; data from these were consistent with clinically important reductions in rates of infection resulting from improved hand hygiene for some but not all important hospital pathogens. Reported costs of interventions ranged from $225 to $4669 (£146-£3035; €204-€4229) per 1000 bed days. CONCLUSION: Promotion of hand hygiene with WHO-5 is effective at increasing compliance in healthcare workers. Addition of goal setting, reward incentives, and accountability strategies can lead to further improvements. Reporting of resources required for such interventions remains inadequate.

Saunderson RB, Gouliouris T, Nickerson EK, Cartwright EJP, Kidney A, Aliyu SH, Brown NM, Limmathurotsakul D, Peacock SJ, Török ME. 2015. Impact of routine bedside infectious disease consultation on clinical management and outcome of Staphylococcus aureus bacteraemia in adults Clinical Microbiology and Infection, 21 (8), pp. 779-785. | Show Abstract | Read more

© 2015 The Authors.Staphylococcus aureus bacteraemia (SAB) is a common, serious infection that is associated with high rates of morbidity and mortality. Evidence suggests that infectious disease consultation (IDC) improves clinical management in patients with SAB. We examined whether the introduction of a routine bedside IDC service for adults with SAB improved clinical management and outcomes compared to telephone consultation. We conducted an observational cohort study of 571 adults with SAB at a teaching hospital in the United Kingdom between July 2006 and December 2012. A telephone consultation was provided on the day of positive blood culture in all cases, but an additional bedside IDC was provided after November 2009 (routine IDC group). Compared to patients in the pre-IDC group, those in the routine IDC group were more likely to have a removable focus of infection identified, echocardiography performed and follow-up blood cultures performed. They also received longer courses of antimicrobial therapy, were more likely to receive combination antimicrobial therapy and were more likely to have SAB recorded in the hospital discharge summary. There was a trend towards lower mortality at 30 days in the routine IDC group compared to the pre-IDC group (12% vs. 22%, p 0.07). Our findings suggest that routine bedside IDC should become the standard of care for adults with SAB.

Birnie E, Wiersinga WJ, Limmathurotsakul D, Grobusch MP. 2015. Corrigendum Future Microbiology, 10 (6), pp. 1101-1101. | Read more

Lim C, Paris DH, Blacksell SD, Laongnualpanich A, Kantipong P, Chierakul W, Wuthiekanun V, Day NP, Cooper BS, Limmathurotsakul D. 2015. How to Determine the Accuracy of an Alternative Diagnostic Test when It Is Actually Better than the Reference Tests: A Re-Evaluation of Diagnostic Tests for Scrub Typhus Using Bayesian LCMs. PLoS One, 10 (5), pp. e0114930. | Show Abstract | Read more

BACKGROUND: The indirect immunofluorescence assay (IFA) is considered a reference test for scrub typhus. Recently, the Scrub Typhus Infection Criteria (STIC; a combination of culture, PCR assays and IFA IgM) were proposed as a reference standard for evaluating alternative diagnostic tests. Here, we use Bayesian latent class models (LCMs) to estimate the true accuracy of each diagnostic test, and of STIC, for diagnosing scrub typhus. METHODS/PRINCIPAL FINDINGS: Data from 161 patients with undifferentiated fever were re-evaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar, and tested with blood culture for Orientia tsutsugamushi, three different PCR assays, IFA IgM, and the Panbio IgM immunochromatographic test (ICT). True sensitivity and specificity of culture (24.4% and 100%), 56kDa PCR assay (56.8% and 98.4%), 47kDa PCR assay (63.2% and 96.1%), groEL PCR assay (71.4% and 93.0%), IFA IgM (70.0% and 83.8%), PanBio IgM ICT (72.8% and 96.8%), presence of eschar (42.7% and 98.9%) and STIC (90.5% and 82.5%) estimated by Bayesian LCM were considerably different from those obtained when using STIC as a reference standard. The IgM ICT had comparable sensitivity and significantly higher specificity compared to IFA (p=0.34 and p<0.001, respectively). CONCLUSIONS: The low specificity of STIC was caused by the low specificity of IFA IgM. Neither STIC nor IFA IgM can be used as reference standards against which to evaluate alternative diagnostic tests. Further evaluation of new diagnostic tests should be done with a carefully selected set of diagnostic tests and appropriate statistical models.

Chansrichavala P, Wongsuwan N, Suddee S, Malasit M, Hongsuwan M, Wannapinij P, Kitphati R, Day NP, Michie S, Peacock SJ, Limmathurotsakul D. 2015. Public awareness of melioidosis in Thailand and potential use of video clips as educational tools. PLoS One, 10 (3), pp. e0121311. | Show Abstract | Read more

BACKGROUND: Melioidosis causes more than 1,000 deaths in Thailand each year. Infection occurs via inoculation, ingestion or inhalation of the causative organism (Burkholderia pseuodmallei) present in soil and water. Here, we evaluated public awareness of melioidosis using a combination of population-based questionnaire, a public engagement campaign to obtain video clips made by the public, and viewpoints on these video clips as potential educational tools about the disease and its prevention. METHODS: A questionnaire was developed to evaluate public awareness of melioidosis, and knowledge about its prevention. From 1 March to 31 April 2012, the questionnaire was delivered to five randomly selected adults in each of 928 districts in Thailand. A video clip contest entitled "Melioidosis, an infectious disease that Thais must know" was run between May and October 2012. The best 12 video clips judged by a contest committee were shown to 71 people at risk from melioidosis (diabetics). Focus group interviews were used to evaluate their perceptions of the video clips. RESULTS: Of 4,203 Thais who completed our study questionnaire, 74% had never heard of melioidosis, and 19% had heard of the disease but had no further knowledge. Most participants in all focus group sessions felt that video clips were beneficial and could positively influence them to increase adherence to recommended preventive behaviours, including drinking boiled water and wearing protective gear if in contact with soil or environmental water. Participants suggested that video clips should be presented in the local dialect with simple words rather than medical terms, in a serious manner, with a doctor as the one presenting the facts, and having detailed pictures of each recommended prevention method. CONCLUSIONS: In summary, public awareness of melioidosis in Thailand is very low, and video clips could serve as a useful medium to educate people and promote disease prevention. PRESENTED IN PART: World Melioidosis Congress 2013, Bangkok, Thailand, 18-20 September 2013 (abstract OS VII-04).

Hoffmaster AR, AuCoin D, Baccam P, Baggett HC, Baird R, Bhengsri S, Blaney DD, Brett PJ et al. 2015. Melioidosis diagnostic workshop, 2013. Emerg Infect Dis, 21 (2), pp. 1-9. | Show Abstract | Read more

Melioidosis is a severe disease that can be difficult to diagnose because of its diverse clinical manifestations and a lack of adequate diagnostic capabilities for suspected cases. There is broad interest in improving detection and diagnosis of this disease not only in melioidosis-endemic regions but also outside these regions because melioidosis may be underreported and poses a potential bioterrorism challenge for public health authorities. Therefore, a workshop of academic, government, and private sector personnel from around the world was convened to discuss the current state of melioidosis diagnostics, diagnostic needs, and future directions.

Lim YT, Jobichen C, Wong J, Limmathurotsakul D, Li S, Chen Y, Raida M, Srinivasan N, MacAry PA, Sivaraman J, Gan YH. 2015. Extended loop region of Hcp1 is critical for the assembly and function of type VI secretion system in Burkholderia pseudomallei. Sci Rep, 5 pp. 8235. | Show Abstract | Read more

The Type VI Secretion System cluster 1 (T6SS1) is essential for the pathogenesis of Burkholderia pseudomallei, the causative agent of melioidosis, a disease endemic in the tropics. Inside host cells, B. pseudomallei escapes into the cytosol and through T6SS1, induces multinucleated giant cell (MNGC) formation that is thought to be important for bacterial cell to cell spread. The hemolysin-coregulated protein (Hcp) is both a T6SS substrate, as well as postulated to form part of the T6SS secretion tube. Our structural study reveals that Hcp1 forms hexameric rings similar to the other Hcp homologs but has an extended loop (Asp40-Arg56) that deviates significantly in position compared to other Hcp structures and may act as a key contact point between adjacent hexameric rings. When two residues within the loop were mutated, the mutant proteins were unable to stack as dodecamers, suggesting defective tube assembly. Moreover, infection with a bacterial mutant containing in situ substitution of these hcp1 residues abolishes Hcp1 secretion inside infected cells and MNGC formation. We further show that Hcp has the ability to preferentially bind to the surface of antigen-presenting cells, which may contribute to its immunogenicity in inducing high titers of antibodies seen in melioidosis patients.

Wiersinga WJ, Birnie E, Weehuizen TA, Alabi AS, Huson MA, Huis in 't Veld RA, Mabala HK, Adzoda GK et al. 2015. Clinical, environmental, and serologic surveillance studies of melioidosis in Gabon, 2012-2013. Emerg Infect Dis, 21 (1), pp. 40-47. | Show Abstract | Read more

Burkholderia pseudomallei, an environmental gram-negative bacillus, is the causative agent of melioidosis and a bio-threat agent. Reports of B. pseudomallei isolation from soil and animals in East and West Africa suggest that melioidosis might be more widely distributed than previously thought. Because it has been found in equatorial areas with tropical climates, we hypothesized that B. pseudomallei could exist in Gabon. During 2012-2013, we conducted a seroprevalance study in which we set up microbiology facilities at a large clinical referral center and prospectively screened all febrile patients by conducting blood cultures and testing for B. pseudomallei and related species; we also determined whether B. pseudomallei could be isolated from soil. We discovered a novel B. pseudomallei sequence type that caused lethal septic shock and identified B. pseudomallei and B. thailandensis in the environment. Our data suggest that melioidosis is emerging in Central Africa but is unrecognized because of the lack of diagnostic microbiology facilities.

Tong SY, Holden MT, Nickerson EK, Cooper BS, Köser CU, Cori A, Jombart T, Cauchemez S et al. 2015. Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting. Genome Res, 25 (1), pp. 111-118. | Show Abstract | Read more

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of nosocomial infection. Whole-genome sequencing of MRSA has been used to define phylogeny and transmission in well-resourced healthcare settings, yet the greatest burden of nosocomial infection occurs in resource-restricted settings where barriers to transmission are lower. Here, we study the flux and genetic diversity of MRSA on ward and individual patient levels in a hospital where transmission was common. We repeatedly screened all patients on two intensive care units for MRSA carriage over a 3-mo period. All MRSA belonged to multilocus sequence type 239 (ST 239). We defined the population structure and charted the spread of MRSA by sequencing 79 isolates from 46 patients and five members of staff, including the first MRSA-positive screen isolates and up to two repeat isolates where available. Phylogenetic analysis identified a flux of distinct ST 239 clades over time in each intensive care unit. In total, five main clades were identified, which varied in the carriage of plasmids encoding antiseptic and antimicrobial resistance determinants. Sequence data confirmed intra- and interwards transmission events and identified individual patients who were colonized by more than one clade. One patient on each unit was the source of numerous transmission events, and deep sampling of one of these cases demonstrated colonization with a "cloud" of related MRSA variants. The application of whole-genome sequencing and analysis provides novel insights into the transmission of MRSA in under-resourced healthcare settings and has relevance to wider global health.

Limmathurotsakul D, Paeyao A, Wongratanacheewin S, Saiprom N, Takpho N, Thaipadungpanit J, Chantratita N, Wuthiekanun V, Day NPJ, Peacock SJ. 2014. Role of burkholderia pseudomallei biofilm formation and lipopolysaccharide in relapse of melioidosis Clinical Microbiology and Infection, 20 (11), pp. O854-O856. | Show Abstract | Read more

© 2014 The Authors.We examined whether quantitative biofilm formation and/or lipopolysaccharide type of Burkholderia pseudomallei was associated with relapsing melioidosis. We devised a 1: 4 nested case-control study in which both cases and controls were drawn from a cohort of patients with primary melioidosis. Paired isolates from 80 patients with relapse and single isolates from 184 patients without relapse were tested. Relapse was associated with biofilm formation of the primary infecting isolate (conditional OR 2.03; 95% CI 1.27-3.25; p 0.003), but not with lipopolysaccharide type (p 0.74). This finding highlights the importance of biofilm formation in relapsing melioidosis.

Thaipadungpanit J, Chierakul W, Pattanaporkrattana W, Phoodaeng A, Wongsuvan G, Huntrakun V, Amornchai P, Chatchen S et al. 2014. Burkholderia pseudomallei in water supplies, southern Thailand. Emerg Infect Dis, 20 (11), pp. 1947-1949. | Read more

Teerawattanasook N, Limmathurotsakul D, Day NP, Wuthiekanun V. 2014. Short report: Failure of Burkholderia pseudomallei to grow in an automated blood culture system. Am J Trop Med Hyg, 91 (6), pp. 1173-1175. | Show Abstract | Read more

We compared the organisms isolated from 30,210 pairs of blood culture bottles by using BacT/Alert system and the conventional system. Overall, 2,575 (8.5%) specimens were culture positive for pathogenic organisms. The sensitivity for detection of pathogenic organisms with the BACT/Alert system (85.6%, 2,203 of 2,575) was significantly higher than that with the conventional method (74.1%, 1,908 of 2,575; P < 0.0001). However, Burkholderia pseudomallei was isolated less often with the BacT/ALERT system (73.5%, 328 of 446) than with the conventional system (90.3%, 403 of 446; P < 0.0001). This finding suggests that use of the conventional culture method in conjunction with the BacT/Alert system may improve the isolation rate for B. pseudomallei in melioidosis-endemic areas.

Wuthiekanun V, Amornchai P, Langla S, Oyuchua M, Day NP, Limmathurotsakul D. 2014. Maintenance of leptospira species in leptospira Vanaporn Wuthiekanun agar. J Clin Microbiol, 52 (12), pp. 4350-4352. | Show Abstract | Read more

The maintenance of Leptospira species in liquid or semisolid medium is time-consuming and at risk of contamination due to the needs of routine subculture and dark field microscopy. Using Leptospira Vanaporn Wuthiekanun (LVW) agar, we maintained 100 pathogenic Leptospira isolates for 12 months without the need for subculture and confirmed the viability of all isolates by the naked eye.

West TE, Myers ND, Chantratita N, Chierakul W, Limmathurotsakul D, Wuthiekanun V, Miao EA, Hajjar AM, Peacock SJ, Liggitt HD, Skerrett SJ. 2014. NLRC4 and TLR5 each contribute to host defense in respiratory melioidosis. PLoS Negl Trop Dis, 8 (9), pp. e3178. | Show Abstract | Read more

Burkholderia pseudomallei causes the tropical infection melioidosis. Pneumonia is a common manifestation of melioidosis and is associated with high mortality. Understanding the key elements of host defense is essential to developing new therapeutics for melioidosis. As a flagellated bacterium encoding type III secretion systems, B. pseudomallei may trigger numerous host pathogen recognition receptors. TLR5 is a flagellin sensor located on the plasma membrane. NLRC4, along with NAIP proteins, assembles a canonical caspase-1-dependent inflammasome in the cytoplasm that responds to flagellin (in mice) and type III secretion system components (in mice and humans). In a murine model of respiratory melioidosis, Tlr5 and Nlrc4 each contributed to survival. Mice deficient in both Tlr5 and Nlrc4 were not more susceptible than single knockout animals. Deficiency of Casp1/Casp11 resulted in impaired bacterial control in the lung and spleen; in the lung much of this effect was attributable to Nlrc4, despite relative preservation of pulmonary IL-1β production in Nlrc4(-/-) mice. Histologically, deficiency of Casp1/Casp11 imparted more severe pulmonary inflammation than deficiency of Nlrc4. The human NLRC4 region polymorphism rs6757121 was associated with survival in melioidosis patients with pulmonary involvement. Co-inheritance of rs6757121 and a functional TLR5 polymorphism had an additive effect on survival. Our results show that NLRC4 and TLR5, key components of two flagellin sensing pathways, each contribute to host defense in respiratory melioidosis.

Schweizer HP, Limmathurotsakul D, Peacock SJ. 2014. New insights from the 7th World Melioidosis Congress 2013. Emerg Infect Dis, 20 (7), | Read more

Limmathurotsakul D, Holden MT, Coupland P, Price EP, Chantratita N, Wuthiekanun V, Amornchai P, Parkhill J, Peacock SJ. 2014. Microevolution of Burkholderia pseudomallei during an acute infection. J Clin Microbiol, 52 (9), pp. 3418-3421. | Show Abstract | Read more

We used whole-genome sequencing to evaluate 69 independent colonies of Burkholderia pseudomallei isolated from seven body sites of a patient with acute disseminated melioidosis. Fourteen closely related genotypes were found, providing evidence for the rapid in vivo diversification of B. pseudomallei after inoculation and systemic spread.

Tonpitak W, Sornklien C, Chawanit M, Pavasutthipaisit S, Wuthiekanun V, Hantrakun V, Amornchai P, Thaipadungpanit J et al. 2014. Fatal melioidosis in goats in Bangkok, Thailand. Am J Trop Med Hyg, 91 (2), pp. 287-290. | Show Abstract | Read more

Bangkok, Thailand, is a city considered to be at low risk for melioidosis. We describe 10 goats that died of melioidosis in Bangkok. Half of them were born and reared in the city. Multilocus sequence typing ruled out an outbreak. This finding challenges the assumption that melioidosis is rarely acquired in central Thailand.

Török ME, Harris SR, Cartwright EJ, Raven KE, Brown NM, Allison ME, Greaves D, Quail MA et al. 2014. Zero tolerance for healthcare-associated MRSA bacteraemia: is it realistic? J Antimicrob Chemother, 69 (8), pp. 2238-2245. | Show Abstract | Read more

BACKGROUND: The term 'zero tolerance' has recently been applied to healthcare-associated infections, implying that such events are always preventable. This may not be the case for healthcare-associated infections such as methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia. METHODS: We combined information from an epidemiological investigation and bacterial whole-genome sequencing to evaluate a cluster of five MRSA bacteraemia episodes in four patients in a specialist hepatology unit. RESULTS: The five MRSA bacteraemia isolates were highly related by multilocus sequence type (ST) (four isolates were ST22 and one isolate was a single-locus variant, ST2046). Whole-genome sequencing demonstrated unequivocally that the bacteraemia cases were unrelated. Placing the MRSA bacteraemia isolates within a local and global phylogenetic tree of MRSA ST22 genomes demonstrated that the five bacteraemia isolates were highly diverse. This was consistent with the acquisition and importation of MRSA from the wider referral network. Analysis of MRSA carriage and disease in patients within the hepatology service demonstrated a higher risk of both initial MRSA acquisition compared with the nephrology service and a higher risk of progression from MRSA carriage to bacteraemia, compared with patients in nephrology or geriatric services. A root cause analysis failed to reveal any mechanism by which three of five MRSA bacteraemia episodes could have been prevented. CONCLUSIONS: This study illustrates the complex nature of MRSA carriage and bacteraemia in patients in a specialized hepatology unit. Despite numerous ongoing interventions to prevent MRSA bacteraemia in healthcare settings, these are unlikely to result in a zero incidence in referral centres that treat highly complex patients.

Duval BD, Elrod MG, Gee JE, Chantratita N, Tandhavanant S, Limmathurotsakul D, Hoffmaster AR. 2014. Evaluation of a latex agglutination assay for the identification of Burkholderia pseudomallei and Burkholderia mallei. Am J Trop Med Hyg, 90 (6), pp. 1043-1046. | Show Abstract | Read more

Cases of melioidosis and glanders are rare in the United States, but the etiologic agents of each disease (Burkholderia pseudomallei and Burkholderia mallei, respectively) are classified as Tier 1 select agents because of concerns about their potential use as bioterrorism agents. A rapid, highly sensitive, and portable assay for clinical laboratories and field use is required. Our laboratory has further evaluated a latex agglutination assay for its ability to identify B. pseudomallei and B. mallei isolates. This assay uses a monoclonal antibody that specifically recognizes the capsular polysaccharide produced by B. pseudomallei and B. mallei, but is absent in closely related Burkholderia species. A total of 110 B. pseudomallei and B. mallei were tested, and 36 closely related Burkholderia species. The latex agglutination assay was positive for 109 of 110 (99.1% sensitivity) B. pseudomallei and B. mallei isolates tested.

Limmathurotsakul D, Paeyao A, Wongratanacheewin S, Saiprom N, Takpho N, Thaipadungpanit J, Chantratita N, Wuthiekanun V, Day NP, Peacock SJ. 2014. Role of Burkholderia pseudomallei biofilm formation and lipopolysaccharide in relapse of melioidosis. Clin Microbiol Infect, 20 (11), pp. O854-O856. | Show Abstract | Read more

We examined whether quantitative biofilm formation and/or lipopolysaccharide type of Burkholderia pseudomallei was associated with relapsing melioidosis. We devised a 1:4 nested case-control study in which both cases and controls were drawn from a cohort of patients with primary melioidosis. Paired isolates from 80 patients with relapse and single isolates from 184 patients without relapse were tested. Relapse was associated with biofilm formation of the primary infecting isolate (conditional OR 2.03; 95% CI 1.27-3.25; p 0.003), but not with lipopolysaccharide type (p 0.74). This finding highlights the importance of biofilm formation in relapsing melioidosis.

Houghton RL, Reed DE, Hubbard MA, Dillon MJ, Chen H, Currie BJ, Mayo M, Sarovich DS et al. 2014. Development of a prototype lateral flow immunoassay (LFI) for the rapid diagnosis of melioidosis. PLoS Negl Trop Dis, 8 (3), pp. e2727. | Show Abstract | Read more

Burkholderia pseudomallei is a soil-dwelling bacterium and the causative agent of melioidosis. Isolation of B. pseudomallei from clinical samples is the "gold standard" for the diagnosis of melioidosis; results can take 3-7 days to produce. Alternatively, antibody-based tests have low specificity due to a high percentage of seropositive individuals in endemic areas. There is a clear need to develop a rapid point-of-care antigen detection assay for the diagnosis of melioidosis. Previously, we employed In vivo Microbial Antigen Discovery (InMAD) to identify potential B. pseudomallei diagnostic biomarkers. The B. pseudomallei capsular polysaccharide (CPS) and numerous protein antigens were identified as potential candidates. Here, we describe the development of a diagnostic immunoassay based on the detection of CPS. Following production of a CPS-specific monoclonal antibody (mAb), an antigen-capture immunoassay was developed to determine the concentration of CPS within a panel of melioidosis patient serum and urine samples. The same mAb was used to produce a prototype Active Melioidosis Detect Lateral Flow Immunoassay (AMD LFI); the limit of detection of the LFI for CPS is comparable to the antigen-capture immunoassay (∼0.2 ng/ml). The analytical reactivity (inclusivity) of the AMD LFI was 98.7% (76/77) when tested against a large panel of B. pseudomallei isolates. Analytical specificity (cross-reactivity) testing determined that 97.2% of B. pseudomallei near neighbor species (35/36) were not reactive. The non-reactive B. pseudomallei strain and the reactive near neighbor strain can be explained through genetic sequence analysis. Importantly, we show the AMD LFI is capable of detecting CPS in a variety of patient samples. The LFI is currently being evaluated in Thailand and Australia; the focus is to optimize and validate testing procedures on melioidosis patient samples prior to initiation of a large, multisite pre-clinical evaluation.

Suntornsut P, Wongsuvan G, Wuthiekanun V, Kasemsupat K, Jutrakul Y, Day NP, Peacock SJ, Limmathurotsakul D. 2014. In response. Am J Trop Med Hyg, 90 (2), pp. 386. | Read more

Limmathurotsakul D, Wongsuvan G, Aanensen D, Ngamwilai S, Saiprom N, Rongkard P, Thaipadungpanit J, Kanoksil M, Chantratita N, Day NP, Peacock SJ. 2014. Melioidosis caused by Burkholderia pseudomallei in drinking water, Thailand, 2012. Emerg Infect Dis, 20 (2), pp. 265-268. | Show Abstract | Read more

We identified 10 patients in Thailand with culture-confirmed melioidosis who had Burkholderia pseudomallei isolated from their drinking water. The multilocus sequence type of B. pseudomallei from clinical specimens and water samples were identical for 2 patients. This finding suggests that drinking water is a preventable source of B. pseudomallei infection.

Hongsuwan M, Srisamang P, Kanoksil M, Luangasanatip N, Jatapai A, Day NP, Peacock SJ, Cooper BS, Limmathurotsakul D. 2014. Increasing incidence of hospital-acquired and healthcare-associated bacteremia in northeast Thailand: a multicenter surveillance study. PLoS One, 9 (10), pp. e109324. | Show Abstract | Read more

BACKGROUND: Little is known about the epidemiology of nosocomial bloodstream infections in public hospitals in developing countries. We evaluated trends in incidence of hospital-acquired bacteremia (HAB) and healthcare-associated bacteremia (HCAB) and associated mortality in a developing country using routinely available databases. METHODS: Information from the microbiology and hospital databases of 10 provincial hospitals in northeast Thailand was linked with the national death registry for 2004-2010. Bacteremia was considered hospital-acquired if detected after the first two days of hospital admission, and healthcare-associated if detected within two days of hospital admission with a prior inpatient episode in the preceding 30 days. RESULTS: A total of 3,424 patients out of 1,069,443 at risk developed HAB and 2,184 out of 119,286 at risk had HCAB. Of these 1,559 (45.5%) and 913 (41.8%) died within 30 days, respectively. Between 2004 and 2010, the incidence rate of HAB increased from 0.6 to 0.8 per 1,000 patient-days at risk (p<0.001), and the cumulative incidence of HCAB increased from 1.2 to 2.0 per 100 readmissions (p<0.001). The most common causes of HAB were Acinetobacter spp. (16.2%), Klebsiella pneumoniae (13.9%), and Staphylococcus aureus (13.9%), while those of HCAB were Escherichia coli (26.3%), S. aureus (14.0%), and K. pneumoniae (9.7%). There was an overall increase over time in the proportions of ESBL-producing E. coli causing HAB and HCAB. CONCLUSIONS: This study demonstrates a high and increasing incidence of HAB and HCAB in provincial hospitals in northeast Thailand, increasing proportions of ESBL-producing isolates, and very high associated mortality.

Saunderson RB, Gouliouris T, Cartwright EJ, Nickerson EJ, Aliyu SH, O'Donnell DR, Kelsall W, Limmathurotsakul D, Peacock SJ, Török ME. 2014. Impact of infectious diseases consultation on the management of Staphylococcus aureus bacteraemia in children. BMJ Open, 4 (7), pp. e004659. | Show Abstract | Read more

OBJECTIVES: Infectious diseases consultation (IDC) in adults with Staphylococcus aureus bacteraemia (SAB) has been shown to improve management and outcome. The aim of this study was to evaluate the impact of IDC on the management of SAB in children. STUDY DESIGN: Observational cohort study of children with SAB. SETTING: Cambridge University Hospitals National Health Service (NHS) Foundation Trust, a large acute NHS Trust in the UK. PARTICIPANTS: All children with SAB admitted to the Cambridge University Hospitals NHS Foundation Trust between 16 July 2006 and 31 December 2012. METHODS: Children with SAB between 2006 and 31 October 2009 were managed by routine clinical care (pre-IDC group) and data were collected retrospectively by case notes review. An IDC service for SAB was introduced in November 2009. All children with SAB were reviewed regularly and data were collected prospectively (IDC group) until 31 December 2012. Baseline characteristics, quality metrics and outcome were compared between the pre-IDC group and IDC group. RESULTS: There were 66 episodes of SAB in 63 children-28 patients (30 episodes) in the pre-IDC group, and 35 patients (36 episodes) in the IDC group. The median age was 3.4 years (IQR 0.2-10.7 years). Patients in the IDC group were more likely to have echocardiography performed, a removable focus of infection identified and to receive a longer course of intravenous antimicrobial therapy. There were no differences in total duration of antibiotic therapy, duration of hospital admission or outcome at 30 or 90 days following onset of SAB. CONCLUSIONS: IDC resulted in improvements in the investigation and management of SAB in children.

Chantratita N, Tandhavanant S, Myers ND, Chierakul W, Wuthiekanun V, Mahavanakul W, Limmathurotsakul D, Peacock SJ, West TE. 2014. Common TLR1 genetic variation is not associated with death from melioidosis, a common cause of sepsis in rural Thailand. PLoS One, 9 (1), pp. e83285. | Show Abstract | Read more

Melioidosis, infection caused by the Gram-negative bacterium Burkholderia pseudomallei, is a common cause of sepsis in northeast Thailand. In white North Americans, common functional genetic variation in TLR1 is associated with organ failure and death from sepsis. We hypothesized that TLR1 variants would be associated with outcomes in Thais with melioidosis. We collated the global frequencies of three TLR1 variants that are common in white North American populations: rs5743551 (-7202A/G), rs4833095 (742A/G), and rs5743618 (1804G/T). We noted a reversal of the minor allele from white North American subjects to Asian populations that was particularly pronounced for rs5743618. In the Utah residents of European ancestry, the frequency of the rs5743618 T allele was 17% whereas in Vietnamese subjects the frequency was >99%. We conducted a genetic association study in 427 patients with melioidosis to determine the association of TLR1 variation with organ failure or death. We genotyped rs5743551 and rs4833095. The variants were in high linkage disequilibrium but neither variant was associated with organ failure or in-hospital death. In 300 healthy Thai individuals we further tested the association of TLR1 variation with ex vivo blood responses to Pam3CSK4, a TLR1 agonist. Neither variant was robustly associated with blood cytokine responses induced by Pam3CSK4. We identified additional common variation in TLR1 by searching public databases and the published literature and screened three additional TLR1 variants for associations with Pam3CSK4-induced responses but found none. We conclude that the genetic architecture of TLR1 variation differs substantially in southeast Asians compared to other populations and common variation in TLR1 in Thais is not associated with outcome from melioidosis or with altered blood responses to Pam3CSK4. Our findings highlight the need for additional studies of TLR1 and other innate immune genetic modulators of the inflammatory host response and determinants of sepsis in southeast Asian populations.

Jarvis JN, Bicanic T, Loyse A, Namarika D, Jackson A, Nussbaum JC, Longley N, Muzoora C et al. 2014. Determinants of mortality in a combined cohort of 501 patients with HIV-associated Cryptococcal meningitis: implications for improving outcomes. Clin Infect Dis, 58 (5), pp. 736-745. | Show Abstract | Read more

BACKGROUND:  Cryptococcal meningitis (CM) is a leading cause of death in individuals infected with human immunodeficiency virus (HIV). Identifying factors associated with mortality informs strategies to improve outcomes. METHODS:  Five hundred one patients with HIV-associated CM were followed prospectively for 10 weeks during trials in Thailand, Uganda, Malawi, and South Africa. South African patients (n = 266) were followed for 1 year. Similar inclusion/exclusion criteria were applied at all sites. Logistic regression identified baseline variables independently associated with mortality. RESULTS:  Mortality was 17% at 2 weeks and 34% at 10 weeks. Altered mental status (odds ratio [OR], 3.1; 95% confidence interval [CI], 1.7-5.9), high cerebrospinal fluid (CSF) fungal burden (OR, 1.4 per log10 colony-forming units/mL increase; 95% CI, 1.0-1.8), older age (>50 years; OR, 3.9; 95% CI, 1.4-11.1), high peripheral white blood cell count (>10 × 10(9) cells/L; OR, 8.7; 95% CI, 2.5-30.2), fluconazole-based induction treatment, and slow clearance of CSF infection were independently associated with 2-week mortality. Low body weight, anemia (hemoglobin <7.5 g/dL), and low CSF opening pressure were independently associated with mortality at 10 weeks in addition to altered mental status, high fungal burden, high peripheral white cell count, and older age. In those followed for 1 year, overall mortality was 41%. Immune reconstitution inflammatory syndrome occurred in 13% of patients and was associated with 2-week CSF fungal burden (P = .007), but not with time to initiation of antiretroviral therapy (ART). CONCLUSIONS:  CSF fungal burden, altered mental status, and rate of clearance of infection predict acute mortality in HIV-associated CM. The results suggest that earlier diagnosis, more rapidly fungicidal amphotericin-based regimens, and prompt immune reconstitution with ART are priorities for improving outcomes.

Myers ND, Chantratita N, Berrington WR, Chierakul W, Limmathurotsakul D, Wuthiekanun V, Robertson JD, Liggitt HD, Peacock SJ, Skerrett SJ, West TE. 2014. The role of NOD2 in murine and human melioidosis. J Immunol, 192 (1), pp. 300-307. | Show Abstract | Read more

Nucleotide-binding oligomerization domain 2 (NOD2) is a cytosolic pathogen recognition receptor that regulates susceptibility to a variety of infections and chronic diseases. Burkholderia pseudomallei, a facultative intracellular bacterium, causes the tropical infection melioidosis. We hypothesized that NOD2 may participate in host defense in melioidosis. We performed a series of in vitro assays and in vivo experiments and analyzed the association of human genetic variation with infection to delineate the contribution of NOD2 to the host response to B. pseudomallei. We found that transfection with NOD2 mediated NF-κB activation induced by B. pseudomallei stimulation of HEK293 cells. After low-dose inoculation with aerosolized B. pseudomallei, Nod2-deficient mice showed impaired clinical responses and permitted greater bacterial replication in the lung and dissemination to the spleen compared with wild-type mice. IL-6 and KC levels were higher in the lungs of Nod2-deficient mice. In a cohort of 1562 Thai subjects, a common genetic polymorphism in the NOD2 region, rs7194886, was associated with melioidosis, and this effect was most pronounced in women. rs7194886 was not associated with differences in cytokine production induced by whole-blood stimulation with the NOD2 ligand, muramyl dipeptide, or B. pseudomallei. To our knowledge, these findings are the first to characterize the role of NOD2 in host defense in mammalian melioidosis.

Chetchotisakd P, Chierakul W, Chaowagul W, Anunnatsiri S, Phimda K, Mootsikapun P, Chaisuksant S, Pilaikul J et al. 2014. Trimethoprim-sulfamethoxazole versus trimethoprim-sulfamethoxazole plus doxycycline as oral eradicative treatment for melioidosis (MERTH): a multicentre, double-blind, non-inferiority, randomised controlled trial. Lancet, 383 (9919), pp. 807-814. | Show Abstract | Read more

BACKGROUND: Melioidosis, an infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, is difficult to cure. Antimicrobial treatment comprises intravenous drugs for at least 10 days, followed by oral drugs for at least 12 weeks. The standard oral regimen based on trial evidence is trimethoprim-sulfamethoxaxole (TMP-SMX) plus doxycycline. This regimen is used in Thailand but is associated with side-effects and poor adherence by patients, and TMP-SMX alone is recommended in Australia. We compared the efficacy and side-effects of TMP-SMX with TMP-SMX plus doxycycline for the oral phase of melioidosis treatment. METHODS: For this multi-centre, double-blind, non-inferiority, randomised placebo-controlled trial, we enrolled patients (aged ≥15 years) from five centres in northeast Thailand with culture-confirmed melioidosis who had received a course of parenteral antimicrobial drugs. Using a computer-generated sequence, we randomly assigned patients to receive TMP-SMX plus placebo or TMP-SMX plus doxycycline for 20 weeks (1:1; block size of ten, stratified by study site). We followed patients up every 4 months for 1 year and annually thereafter to the end of the study. The primary endpoint was culture-confirmed recurrent melioidosis, and the non-inferiority margin was a hazard ratio (HR) of 1.7. This study is registered with www.controlled-trials.com, number ISRCTN86140460. FINDINGS: We enrolled and randomly assigned 626 patients: 311 to TMP-SMX plus placebo and 315 to TMP-SMX plus doxycycline. 16 patients (5%) in the TMP-SMX plus placebo group and 21 patients (7%) in the TMP-SMX plus doxycycline group developed culture-confirmed recurrent melioidosis (HR 0.81; 95% CI 0.42-1.55). The criterion for non-inferiority was met (p=0.01). Adverse drug reactions were less common in the TMP-SMX plus placebo group than in the TMP-SMX plus doxycycline group (122 [39%] vs 167 [53%]). INTERPRETATION: Our findings suggest that TMP-SMX is not inferior to TMP-SMX plus doxycycline for the oral phase of melioidosis treatment, and is preferable on the basis of safety and tolerance by patients. FUNDING: Thailand Research Fund, the Melioidosis Research Center, the Center of Excellence in Specific Health Problems in Greater Mekong Sub-region cluster, and the Wellcome Trust.

Lim C, Wannapinij P, White L, Day NPJ, Cooper BS, Peacock SJ, Limmathurotsakul D. 2013. Using a web-based application to define the accuracy of diagnostic tests when the gold standard is imperfect PLoS ONE, 8 (11), | Show Abstract | Read more

Background: Estimates of the sensitivity and specificity for new diagnostic tests based on evaluation against a known gold standard are imprecise when the accuracy of the gold standard is imperfect. Bayesian latent class models (LCMs) can be helpful under these circumstances, but the necessary analysis requires expertise in computational programming. Here, we describe open-access web-based applications that allow non-experts to apply Bayesian LCMs to their own data sets via a user-friendly interface. Methods/Principal Findings: Applications for Bayesian LCMs were constructed on a web server using R and WinBUGS programs. The models provided (http://mice.tropmedres.ac) include two Bayesian LCMs: the two-tests in two-population model (Hui and Walter model) and the three-tests in one-population model (Walter and Irwig model). Both models are available with simplified and advanced interfaces. In the former, all settings for Bayesian statistics are fixed as defaults. Users input their data set into a table provided on the webpage. Disease prevalence and accuracy of diagnostic tests are then estimated using the Bayesian LCM, and provided on the web page within a few minutes. With the advanced interfaces, experienced researchers can modify all settings in the models as needed. These settings include correlation among diagnostic test results and prior distributions for all unknown parameters. The web pages provide worked examples with both models using the original data sets presented by Hui and Walter in 1980, and by Walter and Irwig in 1988. We also illustrate the utility of the advanced interface using the Walter and Irwig model on a data set from a recent melioidosis study. The results obtained from the web-based applications were comparable to those published previously. Conclusions: The newly developed web-based applications are open-access and provide an important new resource for researchers worldwide to evaluate new diagnostic tests. © 2013 Lim et al.

Luangasanatip N, Hongsuwan M, Lubell Y, Limmathurotsakul D, Teparrukkul P, Chaowarat S, Day NP, Graves N, Cooper BS. 2013. Long-term survival after intensive care unit discharge in Thailand: a retrospective study. Crit Care, 17 (5), pp. R219. | Show Abstract | Read more

INTRODUCTION: Economic evaluations of interventions in the hospital setting often rely on the estimated long-term impact on patient survival. Estimates of mortality rates and long-term outcomes among patients discharged alive from the intensive care unit (ICU) are lacking from lower- and middle-income countries. This study aimed to assess the long-term survival and life expectancy (LE) amongst post-ICU patients in Thailand, a middle-income country. METHODS: In this retrospective cohort study, data from a regional tertiary hospital in northeast Thailand and the regional death registry were linked and used to assess patient survival time after ICU discharge. Adult ICU patients aged at least 15 years who had been discharged alive from an ICU between 1 January 2004 and 31 December 2005 were included in the study, and the death registry was used to determine deaths occurring in this cohort up to 31st December 2010. These data were used in conjunction with standard mortality life tables to estimate annual mortality and life expectancy. RESULTS: This analysis included 10,321 ICU patients. During ICU admission, 3,251 patients (31.5%) died. Of 7,070 patients discharged alive, 2,527 (35.7%) were known to have died within the five-year follow-up period, a mortality rate 2.5 times higher than that in the Thai general population (age and sex matched). The mean LE was estimated as 18.3 years compared with 25.2 years in the general population. CONCLUSIONS: Post-ICU patients experienced much higher rates of mortality than members of the general population over the five-year follow-up period, particularly in the first year after discharge. Further work assessing Health Related Quality of Life (HRQOL) in both post-ICU patients and in the general population in developing countries is needed.

Suntornsut P, Kasemsupat K, Silairatana S, Wongsuvan G, Jutrakul Y, Wuthiekanun V, Day NP, Peacock SJ, Limmathurotsakul D. 2013. Prevalence of melioidosis in patients with suspected pulmonary tuberculosis and sputum smear negative for acid-fast bacilli in northeast Thailand. Am J Trop Med Hyg, 89 (5), pp. 983-985. | Show Abstract | Read more

The clinical and radiological features of pulmonary melioidosis can mimic tuberculosis. We prospectively evaluated 118 patients with suspected pulmonary tuberculosis who were acid-fast bacilli (AFB) smear negative at Udon Thani Hospital, northeast Thailand. Culture of residual sputum from AFB testing was positive for Burkholderia pseudomallei in three patients (2.5%; 95% confidence interval [CI] 0.5-7.3%). We propose that in melioidosis-endemic areas, residual sputum from AFB testing should be routinely cultured for B. pseudomallei.

Chantratita N, Tandhavanant S, Wongsuvan G, Wuthiekanun V, Teerawattanasook N, Day NP, Limmathurotsakul D, Peacock SJ. 2013. Rapid detection of Burkholderia pseudomallei in blood cultures using a monoclonal antibody-based immunofluorescent assay. Am J Trop Med Hyg, 89 (5), pp. 971-972. | Show Abstract | Read more

Melioidosis is a severe bacterial infection caused by Burkholderia pseudomallei. Rapid antimicrobial therapy is necessary to improve patient outcome, which is aided by direct detection of B. pseudomallei in clinical samples. A drawback for all antigen assays is that the number of B. pseudomallei in blood usually falls below the achievable level of detection. We performed a prospective cohort study of 461 patients with 541 blood cultures to evaluate the utility of a pre-incubation step prior to detection of B. pseudomallei using a monoclonal antibody-based immunofluorescent assay (Mab-IFA). The Mab-IFA was positive in 74 of 76 patients with melioidosis (sensitivity = 97.4%), and negative in 385 patients who did not have blood cultures containing B. pseudomallei (specificity = 100%). The Mab-IFA could be a valuable supplementary tool for rapid detection. We recommend the use of the Mab-IFA to test blood cultures that flag positive in regions where melioidosis is endemic.

Thaipadungpanit J, Wuthiekanun V, Chantratita N, Yimsamran S, Amornchai P, Boonsilp S, Maneeboonyang W, Tharnpoophasiam P et al. 2013. Leptospira species in floodwater during the 2011 floods in the Bangkok Metropolitan Region, Thailand. Am J Trop Med Hyg, 89 (4), pp. 794-796. | Show Abstract | Read more

Floodwater samples (N = 110) collected during the 2011 Bangkok floods were tested for Leptospira using culture and polymerase chain reaction (PCR); 65 samples were PCR-positive for putatively non-pathogenic Leptospira species, 1 sample contained a putatively pathogenic Leptospira, and 6 samples contained Leptospira clustering phylogenetically with the intermediate group. The low prevalence of pathogenic and intermediate Leptospira in floodwater was consistent with the low number of human leptospirosis cases reported to the Bureau of Epidemiology in Thailand. This study provides baseline information on environmental Leptospira in Bangkok together with a set of laboratory tests that could be readily deployed in the event of future flooding.

Sonthayanon P, Chierakul W, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Smythe LD, Day NP, Peacock SJ. 2013. Molecular confirmation of co-infection by pathogenic Leptospira spp. and Orientia tsutsugamushi in patients with acute febrile illness in Thailand. Am J Trop Med Hyg, 89 (4), pp. 797-799. | Show Abstract | Read more

Leptospirosis and scrub typhus are major causes of acute febrile illness in rural Asia, where co-infection is reported to occur based on serologic evidence. We re-examined whether co-infection occurs by using a molecular approach. A duplex real-time polymerase chain reaction was developed that targeted a specific 16S ribosomal RNA gene of pathogenic Leptospira spp. and Orientia tsutsugamushi. Of 82 patients with an acute febrile illness who had dual infection on the basis of serologic tests, 5 (6%) had polymerase chain reaction results positive for both pathogens. We conclude that dual infection occurs, but that serologic tests may overestimate the frequency of co-infections.

Goehler A, Trung TT, Hopf V, Kohler A, Wuthiekanun V, Peacock S, Limmathurotsakul D, Steinmetz I. 2013. Environmental presence of Burkholderia pseudomallei in rice fields as determined by direct PCR from soil samples INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, 303 pp. 75-75.

Tandhavanant S, Wongsuvan G, Wuthiekanun V, Teerawattanasook N, Day NP, Limmathurotsakul D, Peacock SJ, Chantratita N. 2013. Monoclonal antibody-based immunofluorescence microscopy for the rapid identification of Burkholderia pseudomallei in clinical specimens. Am J Trop Med Hyg, 89 (1), pp. 165-168. | Show Abstract | Read more

The diagnosis of melioidosis depends on the culture of Burkholderia pseudomallei, which takes at least 48 hours. We used a polyclonal-FITC-based immunofluorescence microscopic assay (Pab-IFA) on clinical samples to provide a rapid presumptive diagnosis. This has limitations including photobleaching and batch-to-batch variability. This study evaluated an IFA based on a monoclonal antibody specific to B. pseudomallei (Mab-IFA) and Alexa Fluor 488. A diagnostic evaluation was performed on a prospective cohort of 951 consecutive patients with suspected melioidosis. A total of 1,407 samples were tested. Test accuracy was defined against culture as the gold standard, and was also compared against Pab-IFA. A total of 88 samples from 64 patients were culture positive for B. pseudomallei. The diagnostic sensitivity and specificity of the Mab-IFA was comparable to the Pab-IFA (48.4% versus 45.3% for sensitivity, and 99.8% versus 98.8% for specificity). We have incorporated the Mab-IFA into our routine practice.

Limmathurotsakul D, Dance DA, Wuthiekanun V, Kaestli M, Mayo M, Warner J, Wagner DM, Tuanyok A et al. 2013. Systematic review and consensus guidelines for environmental sampling of Burkholderia pseudomallei. PLoS Negl Trop Dis, 7 (3), pp. e2105. | Show Abstract | Read more

BACKGROUND: Burkholderia pseudomallei, a Tier 1 Select Agent and the cause of melioidosis, is a Gram-negative bacillus present in the environment in many tropical countries. Defining the global pattern of B. pseudomallei distribution underpins efforts to prevent infection, and is dependent upon robust environmental sampling methodology. Our objective was to review the literature on the detection of environmental B. pseudomallei, update the risk map for melioidosis, and propose international consensus guidelines for soil sampling. METHODS/PRINCIPAL FINDINGS: An international working party (Detection of Environmental Burkholderia pseudomallei Working Party (DEBWorP)) was formed during the VIth World Melioidosis Congress in 2010. PubMed (January 1912 to December 2011) was searched using the following MeSH terms: pseudomallei or melioidosis. Bibliographies were hand-searched for secondary references. The reported geographical distribution of B. pseudomallei in the environment was mapped and categorized as definite, probable, or possible. The methodology used for detecting environmental B. pseudomallei was extracted and collated. We found that global coverage was patchy, with a lack of studies in many areas where melioidosis is suspected to occur. The sampling strategies and bacterial identification methods used were highly variable, and not all were robust. We developed consensus guidelines with the goals of reducing the probability of false-negative results, and the provision of affordable and 'low-tech' methodology that is applicable in both developed and developing countries. CONCLUSIONS/SIGNIFICANCE: The proposed consensus guidelines provide the basis for the development of an accurate and comprehensive global map of environmental B. pseudomallei.

Cheng AC, Currie BJ, Dance DA, Funnell SG, Limmathurotsakul D, Simpson AJ, Peacock SJ. 2013. Clinical definitions of melioidosis. Am J Trop Med Hyg, 88 (3), pp. 411-413. | Show Abstract | Read more

Clinical definitions of melioidosis and inhalation-acquired melioidosis (Burkholderia pseudomallei infection) are described together with the evidence used to develop these definitions. Such definitions support accurate public health reporting, preparedness planning for deliberate B. pseudomallei release, design of experimental models, and categorization of naturally acquired melioidosis.

West TE, Chantratita N, Chierakul W, Limmathurotsakul D, Wuthiekanun V, Myers ND, Emond MJ, Wurfel MM, Hawn TR, Peacock SJ, Skerrett SJ. 2013. Impaired TLR5 functionality is associated with survival in melioidosis. J Immunol, 190 (7), pp. 3373-3379. | Show Abstract | Read more

Melioidosis is infection caused by the flagellated saprophyte Burkholderia pseudomallei. TLR5 is a pathogen recognition receptor activated by bacterial flagellin. We studied a genetic variant that encodes a defective TLR5 protein, TLR5(1174C)>T, to elucidate the role of TLR5 in melioidosis. We measured NF-κB activation induced by B. pseudomallei in human embryonic kidney-293 cells transfected with TLR5 and found that B. pseudomallei induced TLR5(1174C)- but not TLR5(1174T)-dependent activation of NF-κB. We tested the association of TLR5(1174C)>T with outcome in 600 Thai subjects with melioidosis. In a dominant model, TLR5(1174C)>T was associated with protection against in-hospital death (adjusted odds ratio: 0.20; 95% confidence interval: 0.08-0.50; p = 0.001) and organ failure (adjusted odds ratio: 0.37; 95% confidence interval: 0.19-0.71; p = 0.003). We analyzed blood cytokine production induced by flagellin or heat-killed B. pseudomallei by TLR5(1174C)>T genotype in healthy subjects. Flagellin induced lower monocyte-normalized levels of IL-6, IL-8, TNF-α, IL-10, MCP-1, IL-1ra, G-CSF, and IL-1β in carriers of TLR5(1174T) compared with carriers of TLR5(1174C). B. pseudomallei induced lower monocyte-normalized levels of IL-10 in carriers of TLR5(1174T). We conclude that the hypofunctional genetic variant TLR5(1174C)>T is associated with reduced organ failure and improved survival in melioidosis. This conclusion suggests a deleterious immunoregulatory effect of TLR5 that may be mediated by IL-10 and identifies this receptor as a potential therapeutic target in melioidosis.

Stoesser N, Emary K, Soklin S, Peng An K, Sophal S, Chhomrath S, Day NP, Limmathurotsakul D et al. 2013. The value of intermittent point-prevalence surveys of healthcare-associated infections for evaluating infection control interventions at Angkor Hospital for Children, Siem Reap, Cambodia. Trans R Soc Trop Med Hyg, 107 (4), pp. 248-253. | Show Abstract | Read more

BACKGROUND: There are limited data on the epidemiology of paediatric healthcare-associated infection (HCAI) and infection control in low-income countries. We describe the value of intermittent point-prevalence surveys for monitoring HCAI and evaluating infection control interventions in a Cambodian paediatric hospital. METHODS: Hospital-wide, point-prevalence surveys were performed monthly in 2011. Infection control interventions introduced during this period included a hand hygiene programme and a ventilator-associated pneumonia (VAP) care bundle. RESULTS: Overall HCAI prevalence was 13.8/100 patients at-risk, with a significant decline over time. The highest HCAI rates (50%) were observed in critical care; the majority of HCAIs were respiratory (61%). Klebsiella pneumoniae was most commonly isolated and antimicrobial resistance was widespread. Hand hygiene compliance doubled to 51.6%, and total VAP cases/1000 patient-ventilator days fell from 30 to 10. CONCLUSION: Rates of HCAI were substantial in our institution, and antimicrobial resistance a major concern. Point-prevalence surveys are effective for HCAI surveillance, and in monitoring trends in response to infection control interventions.

Peacock SJ, Limmathurotsakul D, Lubell Y, Koh GC, White LJ, Day NP, Titball RW. 2013. Correction: Melioidosis Vaccines: A Systematic Review and Appraisal of the Potential to Exploit Biodefense Vaccines for Public Health Purposes. PLoS Negl Trop Dis, 7 (2), | Show Abstract | Read more

[This corrects the article on p. e1488 in vol. 6.].

Limmathurotsakul D, Kanoksil M, Wuthiekanun V, Kitphati R, deStavola B, Day NP, Peacock SJ. 2013. Activities of daily living associated with acquisition of melioidosis in northeast Thailand: a matched case-control study. PLoS Negl Trop Dis, 7 (2), pp. e2072. | Show Abstract | Read more

BACKGROUND: Melioidosis is a serious infectious disease caused by the Category B select agent and environmental saprophyte, Burkholderia pseudomallei. Most cases of naturally acquired infection are assumed to result from skin inoculation after exposure to soil or water. The aim of this study was to provide evidence for inoculation, inhalation and ingestion as routes of infection, and develop preventive guidelines based on this evidence. METHODS/PRINCIPAL FINDINGS: A prospective hospital-based 1∶2 matched case-control study was conducted in Northeast Thailand. Cases were patients with culture-confirmed melioidosis, and controls were patients admitted with non-infectious conditions during the same period, matched for gender, age, and diabetes mellitus. Activities of daily living were recorded for the 30-day period before onset of symptoms, and home visits were performed to obtain drinking water and culture this for B. pseudomallei. Multivariable conditional logistic regression analysis based on 286 cases and 512 controls showed that activities associated with a risk of melioidosis included working in a rice field (conditional odds ratio [cOR] = 2.1; 95% confidence interval [CI] 1.4-3.3), other activities associated with exposure to soil or water (cOR = 1.4; 95%CI 0.8-2.6), an open wound (cOR = 2.0; 95%CI 1.2-3.3), eating food contaminated with soil or dust (cOR = 1.5; 95%CI 1.0-2.2), drinking untreated water (cOR = 1.7; 95%CI 1.1-2.6), outdoor exposure to rain (cOR = 2.1; 95%CI 1.4-3.2), water inhalation (cOR = 2.4; 95%CI 1.5-3.9), current smoking (cOR = 1.5; 95%CI 1.0-2.3) and steroid intake (cOR = 3.1; 95%CI 1.4-6.9). B. pseudomallei was detected in water source(s) consumed by 7% of cases and 3% of controls (cOR = 2.2; 95%CI 0.8-5.8). CONCLUSIONS/SIGNIFICANCE: We used these findings to develop the first evidence-based guidelines for the prevention of melioidosis. These are suitable for people in melioidosis-endemic areas, travelers and military personnel. Public health campaigns based on our recommendations are under development in Thailand.

Luangasanatip N, Hongsuwan M, Lubell Y, Limmathurotsakul D, Teparrukkul P, Chaowarat S, Day NPJ, Graves N, Cooper BS. 2013. Long-term survival after intensive care unit discharge in Thailand: a retrospective study CRITICAL CARE, 17 (5), | Read more

Lim C, Wannapinij P, White L, Day NP, Cooper BS, Peacock SJ, Limmathurotsakul D. 2013. Using a web-based application to define the accuracy of diagnostic tests when the gold standard is imperfect. PLoS One, 8 (11), pp. e79489. | Show Abstract | Read more

BACKGROUND: Estimates of the sensitivity and specificity for new diagnostic tests based on evaluation against a known gold standard are imprecise when the accuracy of the gold standard is imperfect. Bayesian latent class models (LCMs) can be helpful under these circumstances, but the necessary analysis requires expertise in computational programming. Here, we describe open-access web-based applications that allow non-experts to apply Bayesian LCMs to their own data sets via a user-friendly interface. METHODS/PRINCIPAL FINDINGS: Applications for Bayesian LCMs were constructed on a web server using R and WinBUGS programs. The models provided (http://mice.tropmedres.ac) include two Bayesian LCMs: the two-tests in two-population model (Hui and Walter model) and the three-tests in one-population model (Walter and Irwig model). Both models are available with simplified and advanced interfaces. In the former, all settings for Bayesian statistics are fixed as defaults. Users input their data set into a table provided on the webpage. Disease prevalence and accuracy of diagnostic tests are then estimated using the Bayesian LCM, and provided on the web page within a few minutes. With the advanced interfaces, experienced researchers can modify all settings in the models as needed. These settings include correlation among diagnostic test results and prior distributions for all unknown parameters. The web pages provide worked examples with both models using the original data sets presented by Hui and Walter in 1980, and by Walter and Irwig in 1988. We also illustrate the utility of the advanced interface using the Walter and Irwig model on a data set from a recent melioidosis study. The results obtained from the web-based applications were comparable to those published previously. CONCLUSIONS: The newly developed web-based applications are open-access and provide an important new resource for researchers worldwide to evaluate new diagnostic tests.

Kanoksil M, Jatapai A, Peacock SJ, Limmathurotsakul D. 2013. Correction: Epidemiology, Microbiology and Mortality Associated with Community-Acquired Bacteremia in Northeast Thailand: A Multicenter Surveillance Study. PLoS One, 8 (10), | Show Abstract | Read more

[This corrects the article on p. e54714 in vol. 8.].

Kurt K, Rasigade JP, Laurent F, Goering RV, Žemličková H, Machova I, Struelens MJ, Zautner AE et al. 2013. Subpopulations of Staphylococcus aureus clonal complex 121 are associated with distinct clinical entities. PLoS One, 8 (3), pp. e58155. | Show Abstract | Read more

We investigated the population structure of Staphylococcus aureus clonal complex CC121 by mutation discovery at 115 genetic housekeeping loci from each of 154 isolates, sampled on five continents between 1953 and 2009. In addition, we pyro-sequenced the genomes from ten representative isolates. The genome-wide SNPs that were ascertained revealed the evolutionary history of CC121, indicating at least six major clades (A to F) within the clonal complex and dating its most recent common ancestor to the pre-antibiotic era. The toxin gene complement of CC121 isolates was correlated with their SNP-based phylogeny. Moreover, we found a highly significant association of clinical phenotypes with phylogenetic affiliations, which is unusual for S. aureus. All isolates evidently sampled from superficial infections (including staphylococcal scalded skin syndrome, bullous impetigo, exfoliative dermatitis, conjunctivitis) clustered in clade F, which included the European epidemic fusidic-acid resistant impetigo clone (EEFIC). In comparison, isolates from deep-seated infections (abscess, furuncle, pyomyositis, necrotizing pneumonia) were disseminated in several clades, but not in clade F. Our results demonstrate that phylogenetic lineages with distinct clinical properties exist within an S. aureus clonal complex, and that SNPs serve as powerful discriminatory markers, able to identify these lineages. All CC121 genomes harboured a 41-kilobase prophage that was dissimilar to S. aureus phages sequenced previously. Community-associated MRSA and MSSA from Cambodia were extremely closely related, suggesting this MRSA arose in the region.

Koh GC, Schreiber MF, Bautista R, Maude RR, Dunachie S, Limmathurotsakul D, Day NP, Dougan G, Peacock SJ. 2013. Host responses to melioidosis and tuberculosis are both dominated by interferon-mediated signaling. PLoS One, 8 (1), pp. e54961. | Show Abstract | Read more

Melioidosis (Burkholderia pseudomallei infection) is a common cause of community-acquired sepsis in Northeast Thailand and northern Australia. B. pseudomallei is a soil saprophyte endemic to Southeast Asia and northern Australia. The clinical presentation of melioidosis may mimic tuberculosis (both cause chronic suppurative lesions unresponsive to conventional antibiotics and both commonly affect the lungs). The two diseases have overlapping risk profiles (e.g., diabetes, corticosteroid use), and both B. pseudomallei and Mycobacterium tuberculosis are intracellular pathogens. There are however important differences: the majority of melioidosis cases are acute, not chronic, and present with severe sepsis and a mortality rate that approaches 50% despite appropriate antimicrobial therapy. By contrast, tuberculosis is characteristically a chronic illness with mortality <2% with appropriate antimicrobial chemotherapy. We examined the gene expression profiles of total peripheral leukocytes in two cohorts of patients, one with acute melioidosis (30 patients and 30 controls) and another with tuberculosis (20 patients and 24 controls). Interferon-mediated responses dominate the host response to both infections, and both type 1 and type 2 interferon responses are important. An 86-gene signature previously thought to be specific for tuberculosis is also found in melioidosis. We conclude that the host responses to melioidosis and to tuberculosis are similar: both are dominated by interferon-signalling pathways and this similarity means gene expression signatures from whole blood do not distinguish between these two diseases.

Kanoksil M, Jatapai A, Peacock SJ, Limmathurotsakul D. 2013. Epidemiology, microbiology and mortality associated with community-acquired bacteremia in northeast Thailand: a multicenter surveillance study. PLoS One, 8 (1), pp. e54714. | Show Abstract | Read more

BACKGROUND: National statistics in developing countries are likely to underestimate deaths due to bacterial infections. Here, we calculated mortality associated with community-acquired bacteremia (CAB) in a developing country using routinely available databases. METHODS/PRINCIPAL FINDINGS: Information was obtained from the microbiology and hospital database of 10 provincial hospitals in northeast Thailand, and compared with the national death registry from the Ministry of Interior, Thailand for the period between 2004 and 2010. CAB was defined in patients who had pathogenic organisms isolated from blood taken within 2 days of hospital admission without a prior inpatient episode in the preceding 30 days. A total of 15,251 CAB patients identified, of which 5,722 (37.5%) died within 30 days of admission. The incidence rate of CAB between 2004 and 2010 increased from 16.7 to 38.1 per 100,000 people per year, and the mortality rate associated with CAB increased from 6.9 to 13.7 per 100,000 people per year. In 2010, the mortality rate associated with CAB was lower than that from respiratory tract infection, but higher than HIV disease or tuberculosis. The most common causes of CAB were Escherichia coli (23.1%), Burkholderia pseudomallei (19.3%), and Staphylococcus aureus (8.2%). There was an increase in the proportion of Extended-Spectrum Beta-Lactamases (ESBL) producing E. coli and Klebsiella pneumoniae over time. CONCLUSIONS: This study has demonstrated that national statistics on causes of death in developing countries could be improved by integrating information from readily available databases. CAB is neglected as an important cause of death, and specific prevention and intervention is urgently required to reduce its incidence and mortality.

Pan-ngum W, Blacksell SD, Lubell Y, Pukrittayakamee S, Bailey MS, de Silva HJ, Lalloo DG, Day NP, White LJ, Limmathurotsakul D. 2013. Estimating the true accuracy of diagnostic tests for dengue infection using bayesian latent class models. PLoS One, 8 (1), pp. e50765. | Show Abstract | Read more

BACKGROUND: Accuracy of rapid diagnostic tests for dengue infection has been repeatedly estimated by comparing those tests with reference assays. We hypothesized that those estimates might be inaccurate if the accuracy of the reference assays is not perfect. Here, we investigated this using statistical modeling. METHODS/PRINCIPAL FINDINGS: Data from a cohort study of 549 patients suspected of dengue infection presenting at Colombo North Teaching Hospital, Ragama, Sri Lanka, that described the application of our reference assay (a combination of Dengue IgM antibody capture ELISA and IgG antibody capture ELISA) and of three rapid diagnostic tests (Panbio NS1 antigen, IgM antibody and IgG antibody rapid immunochromatographic cassette tests) were re-evaluated using bayesian latent class models (LCMs). The estimated sensitivity and specificity of the reference assay were 62.0% and 99.6%, respectively. Prevalence of dengue infection (24.3%), and sensitivities and specificities of the Panbio NS1 (45.9% and 97.9%), IgM (54.5% and 95.5%) and IgG (62.1% and 84.5%) estimated by bayesian LCMs were significantly different from those estimated by assuming that the reference assay was perfect. Sensitivity, specificity, PPV and NPV for a combination of NS1, IgM and IgG cassette tests on admission samples were 87.0%, 82.8%, 62.0% and 95.2%, respectively. CONCLUSIONS: Our reference assay is an imperfect gold standard. In our setting, the combination of NS1, IgM and IgG rapid diagnostic tests could be used on admission to rule out dengue infection with a high level of accuracy (NPV 95.2%). Further evaluation of rapid diagnostic tests for dengue infection should include the use of appropriate statistical models.

Lipsitz R, Garges S, Aurigemma R, Baccam P, Blaney DD, Cheng AC, Currie BJ, Dance D et al. 2012. Workshop on treatment of and postexposure prophylaxis for Burkholderia pseudomallei and B. mallei Infection, 2010. Emerg Infect Dis, 18 (12), pp. e2. | Show Abstract | Read more

The US Public Health Emergency Medical Countermeasures Enterprise convened subject matter experts at the 2010 HHS Burkholderia Workshop to develop consensus recommendations for postexposure prophylaxis against and treatment for Burkholderia pseudomallei and B. mallei infections, which cause melioidosis and glanders, respectively. Drugs recommended by consensus of the participants are ceftazidime or meropenem for initial intensive therapy, and trimethoprim/sulfamethoxazole or amoxicillin/clavulanic acid for eradication therapy. For postexposure prophylaxis, recommended drugs are trimethoprim/sulfamethoxazole or co-amoxiclav. To improve the timely diagnosis of melioidosis and glanders, further development and wide distribution of rapid diagnostic assays were also recommended. Standardized animal models and B. pseudomallei strains are needed for further development of therapeutic options. Training for laboratory technicians and physicians would facilitate better diagnosis and treatment options.

Wuthiekanun V, Amornchai P, Paris DH, Langla S, Thaipadunpanit J, Chierakul W, Smythe LD, White NJ, Day NP, Limmathurotsakul D, Peacock SJ. 2013. Rapid isolation and susceptibility testing of Leptospira spp. using a new solid medium, LVW agar. Antimicrob Agents Chemother, 57 (1), pp. 297-302. | Show Abstract | Read more

Pathogenic Leptospira spp., the causative agents of leptospirosis, are slow-growing Gram-negative spirochetes. Isolation of Leptospira from clinical samples and testing of antimicrobial susceptibility are difficult and time-consuming. Here, we describe the development of a new solid medium that facilitates more-rapid growth of Leptospira spp. and the use of this medium to evaluate the Etest's performance in determining antimicrobial MICs to drugs in common use for leptospirosis. The medium was developed by evaluating the effects of numerous factors on the growth rate of Leptospira interrogans strain NR-20157. These included the type of base agar, the concentration of rabbit serum (RS), and the concentration and duration of CO(2) incubation during the initial period of culture. The highest growth rate of NR-20157 was achieved using a Noble agar base supplemented with 10% RS (named LVW agar), with an initial incubation at 30°C in 5% CO(2) for 2 days prior to continuous culture in air at 30°C. These conditions were used to develop the Etest for three species, L. interrogans (NR-20161), L. kirschnerii (NR-20327), and L. borgpetersenii (NR-20151). The MICs were read on day 7 for all samples. The Etest was then performed on 109 isolates of pathogenic Leptospira spp. The MIC(90) values for penicillin G, doxycycline, cefotaxime, ceftriaxone, and chloramphenicol were 0.64 units/ml and 0.19, 0.047, 0.5, and 2 μg/ml, respectively. The use of LVW agar, which enables rapid growth, isolation of single colonies, and simple antimicrobial susceptibility testing for Leptospira spp., provides an opportunity for new areas of fundamental and applied research.

Stoesser N, Pocock J, Moore CE, Soeng S, Hor P, Sar P, Limmathurotsakul D, Day N et al. 2013. The epidemiology of pediatric bone and joint infections in Cambodia, 2007-11. J Trop Pediatr, 59 (1), pp. 36-42. | Show Abstract | Read more

There are limited data on osteoarticular infections from resource-limited settings in Asia. A retrospective study of patients presenting to the Angkor Hospital for Children, Cambodia, January 2007-July 2011, identified 81 cases (28% monoarticular septic arthritis, 51% single-limb osteomyelitis and 15% multisite infections). The incidence was 13.8/100 000 hospital attendances. The median age was 7.3 years, with a male/female ratio of 1.9:1; 35% presented within 5 days of symptom onset (median 7 days). Staphylococcus aureus was cultured in 29 (36%) cases (52% of culture-positive cases); one isolate was methicillin-resistant (MRSA). Median duration of antimicrobial treatment was 29 days (interquartile range 21-43); rates of surgical intervention were 96%, and 46% of children had sequelae, with one fatality. In this setting osteoarticular infections are relatively common with high rates of surgical intervention and sequelae. Staphylococcus aureus is the commonest culturable cause, but methicillin-resistant S. aureus is not a major problem, unlike in other Asian centers.

Maude RR, Vatcharapreechasakul T, Ariyaprasert P, Maude RJ, Hongsuwan M, Yuentrakul P, Limmathurotsakul D, Koh GC, Chaowagul W, Day NP, Peacock SJ. 2012. Prospective observational study of the frequency and features of intra-abdominal abscesses in patients with melioidosis in northeast Thailand. Trans R Soc Trop Med Hyg, 106 (10), pp. 629-631. | Show Abstract | Read more

Retrospective case series from Thailand have reported the presence of intra-abdominal abscesses in around half of patients with melioidosis, a much higher rate than our clinical experience would suggest. We performed a prospective, observational study of 230 adult patients with culture-confirmed melioidosis in which all patients underwent abdominal ultrasound. One or more abscesses were detected in the liver and/or spleen in 77 (33%) cases. These were often multiple (70%, 31/44 in hepatic abscesses and 88%, 50/57 in splenic abscesses) and clinically silent (27% of cases with abscesses presenting with abdominal pain). The mortality rate at 4 weeks post-discharge was lower in patients who were abscess-positive vs abscess-negative (10%, 8/77 vs 20%, 31/153).

Luangasanatip N, Hongsuwan M, Lubell Y, Srisamang P, Limmathurotsakul D, Cooper B. 2012. Excess length of stay due to hospital-associated infections in Thailand: 8 years retrospective data INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E378-E379. | Read more

Kanoksil M, Jatapai A, Peacock S, Limmathurotsakul D. 2012. Epidemiology, microbiology and mortality of community-acquired bacteremia in northeast Thailand: a multicenter population-based study INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E132-E132. | Read more

Hongsuwan M, Srisamang P, Luangasanatip N, Kanoksil M, Day N, Cooper B, Limmathurotsakul D. 2012. A retrospective study to define the incidence and associated mortality of hospital-acquired bacteraemia at a regional hospital in northeast Thailand INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E385-E385. | Read more

Hongsuwan M, Srisamang P, Luangasanatip N, Kanoksil M, Day N, Limmathurotsakul D, Cooper B. 2012. A qualitative exploration into infection control practices and obstacles to improvements amongst health care workers at a regional hospital INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E373-E373. | Read more

Limmathurotsakul D, Wuthiekanun V, Kanoksil M, deStavola B, Day N, Peacock S. 2012. A matched case-control study identifies activities of daily living associated with acquisition of melioidosis in northeast Thailand INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E350-E351. | Read more

Limmathurotsakul D, Turner E, Lim C, Day N, Cooper B, Peacock S. 2012. Defining the true accuracy of diagnostic tests when the gold standard is imperfect using web-based application INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E398-E398. | Read more

Limmathurotsakul D, Thammasart S, Warrasuth N, Thapanagulsak P, Jatapai A, Pengreungrojanachai V, Anun S, Joraka W et al. 2012. Animal melioidosis in Thailand INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E453-E453. | Read more

Stoesser N, Pocock J, Moore CE, Soeng S, Chhat HP, Sar P, Limmathurotsakul D, Day N, Thy V, Sar V, Parry CM. 2012. Pediatric suppurative parotitis in Cambodia between 2007 and 2011. Pediatr Infect Dis J, 31 (8), pp. 865-868. | Show Abstract | Read more

The causes of suppurative parotitis in Cambodian children are not known. We describe 39 cases at the Angkor Hospital for Children, Siem Reap, between January 2007 and July 2011 (0.07/1000 hospital attendances). The median age was 5.7 years with no neonates affected. Burkholderia pseudomallei was cultured in 29 (74%) cases. No deaths occurred; 1 child developed facial nerve palsy.

Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NP, Cooper B, Peacock SJ. 2012. Fool's gold: Why imperfect reference tests are undermining the evaluation of novel diagnostics: a reevaluation of 5 diagnostic tests for leptospirosis. Clin Infect Dis, 55 (3), pp. 322-331. | Show Abstract | Read more

BACKGROUND: We observed that some patients with clinical leptospirosis supported by positive results of rapid tests were negative for leptospirosis on the basis of our diagnostic gold standard, which involves isolation of Leptospira species from blood culture and/or a positive result of a microscopic agglutination test (MAT). We hypothesized that our reference standard was imperfect and used statistical modeling to investigate this hypothesis. METHODS: Data for 1652 patients with suspected leptospirosis recruited during three observational studies and one randomized control trial that described the application of culture, MAT, immunofluorescence assay (IFA), lateral flow (LF) and/or PCR targeting the 16S rRNA gene were reevaluated using Bayesian latent class models and random-effects meta-analysis. RESULTS: The estimated sensitivities of culture alone, MAT alone, and culture plus MAT (for which the result was considered positive if one or both tests had a positive result) were 10.5% (95% credible interval [CrI], 2.7%-27.5%), 49.8% (95% CrI, 37.6%-60.8%), and 55.5% (95% CrI, 42.9%-67.7%), respectively. These low sensitivities were present across all 4 studies. The estimated specificity of MAT alone (and of culture plus MAT) was 98.8% (95% CrI, 92.8%-100.0%). The estimated sensitivities and specificities of PCR (52.7% [95% CrI, 45.2%-60.6%] and 97.2% [95% CrI, 92.0%-99.8%], respectively), lateral flow test (85.6% [95% CrI, 77.5%-93.2%] and 96.2% [95% CrI, 87.7%-99.8%], respectively), and immunofluorescence assay (45.5% [95% CrI, 33.3%-60.9%] and 96.8% [95% CrI, 92.8%-99.8%], respectively) were considerably different from estimates in which culture plus MAT was considered a perfect gold standard test. CONCLUSIONS: Our findings show that culture plus MAT is an imperfect gold standard against which to compare alterative tests for the diagnosis of leptospirosis. Rapid point-of-care tests for this infection would bring an important improvement in patient care, but their future evaluation will require careful consideration of the reference test(s) used and the inclusion of appropriate statistical models.

Desakorn V, Wuthiekanun V, Thanachartwet V, Sahassananda D, Chierakul W, Apiwattanaporn A, Day NP, Limmathurotsakul D, Peacock SJ. 2012. Accuracy of a commercial IgM ELISA for the diagnosis of human leptospirosis in Thailand. Am J Trop Med Hyg, 86 (3), pp. 524-527. | Show Abstract | Read more

The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) has been recommended for the rapid diagnosis of leptospirosis in endemic areas. We conducted a retrospective case-control study of 218 patients (109 leptospirosis cases confirmed by Leptospira culture and/or microscopic agglutination test and 109 control patients with acute febrile illness) to evaluate the diagnostic accuracy of a commercial IgM ELISA (Panbio) in northeast Thailand. Paired serum samples taken on admission and at least 10 days after the onset of symptoms were tested. Using the cutoff value recommended by the manufacturer (11 Panbio units), sensitivity and specificity of IgM ELISA on paired sera were 90.8% and 55.1%. A receiver operating characteristic curve was used to determine the optimal cutoff value. This was 20 Panbio units, which gave a sensitivity and specificity of 76.1% and 82.6%, respectively, on paired sera. We conclude that using either cutoff value, the accuracy of IgM ELISA is limited in our setting.

Limmathurotsakul D, Thammasart S, Warrasuth N, Thapanagulsak P, Jatapai A, Pengreungrojanachai V, Anun S, Joraka W et al. 2012. Melioidosis in animals, Thailand, 2006-2010. Emerg Infect Dis, 18 (2), pp. 325-327. | Show Abstract | Read more

We retrospectively estimated the incidence of culture-proven melioidosis in animals in Thailand during 2006-2010. The highest incidence was in goats (1.63/100,000/year), followed by incidence in pigs and cattle. The estimated incidence of melioidosis in humans in a given region paralleled that of melioidosis in goats.

Maude RR, Vatcharapreechasakul T, Ariyaprasert P, Maude RJ, Hongsuwan M, Yuentrakul P, Limmathurotsakul D, Koh GCKW, Chaowagul W, Day NPJ, Peacock SJ. 2012. Prospective observational study of the frequency and features of intra-abdominal abscesses in patients with melioidosis in northeast Thailand Transactions of the Royal Society of Tropical Medicine and Hygiene, 106 (10), pp. 629-631. | Show Abstract | Read more

Retrospective case series from Thailand have reported the presence of intra-abdominal abscesses in around half of patients with melioidosis, a much higher rate than our clinical experience would suggest. We performed a prospective, observational study of 230 adult patients with culture-confirmed melioidosis in which all patients underwent abdominal ultrasound. One or more abscesses were detected in the liver and/or spleen in 77 (33%) cases. These were often multiple (70%, 31/44 in hepatic abscesses and 88%, 50/57 in splenic abscesses) and clinically silent (27% of cases with abscesses presenting with abdominal pain). The mortality rate at 4 weeks post-discharge was lower in patients who were abscess-positive vs abscess-negative (10%, 8/77 vs 20%, 31/153). © 2012 Royal Society of Tropical Medicine and Hygiene.

Sarovich DS, Price EP, Limmathurotsakul D, Cook JM, Von Schulze AT, Wolken SR, Keim P, Peacock SJ, Pearson T. 2012. Development of ceftazidime resistance in an acute Burkholderia pseudomallei infection. Infect Drug Resist, 5 (1), pp. 129-132. | Show Abstract | Read more

Burkholderia pseudomallei, a bacterium that causes the disease melioidosis, is intrinsically resistant to many antibiotics. First-line antibiotic therapy for treating melioidosis is usually the synthetic β-lactam, ceftazidime (CAZ), as almost all B. pseudomallei strains are susceptible to this drug. However, acquired CAZ resistance can develop in vivo during treatment with CAZ, which can lead to mortality if therapy is not switched to a different drug in a timely manner. Serial B. pseudomallei isolates obtained from an acute Thai melioidosis patient infected by a CAZ susceptible strain, who ultimately succumbed to infection despite being on CAZ therapy for the duration of their infection, were analyzed. Isolates that developed CAZ resistance due to a proline to serine change at position 167 in the β-lactamase PenA were identified. Importantly, these CAZ resistant isolates remained sensitive to the alternative melioidosis treatments; namely, amoxicillin-clavulanate, imipenem, and meropenem. Lastly, real-time polymerase chain reaction-based assays capable of rapidly identifying CAZ resistance in B. pseudomallei isolates at the position 167 mutation site were developed. The ability to rapidly identify the emergence of CAZ resistant B. pseudomallei populations in melioidosis patients will allow timely alterations in treatment strategies, thereby improving patient outcomes for this serious disease.

Mahavanakul W, Nickerson EK, Srisomang P, Teparrukkul P, Lorvinitnun P, Wongyingsinn M, Chierakul W, Hongsuwan M et al. 2012. Correction: Feasibility of Modified Surviving Sepsis Campaign Guidelines in a Resource-Restricted Setting Based on a Cohort Study of Severe S. Aureus Sepsis. PLoS One, 7 (5), | Show Abstract | Read more

[This corrects the article on p. e29858 in vol. 7.].

Stoesser N, Pocock J, Moore CE, Soeng S, Chhat HP, Sar P, Limmathurotsakul D, Day N, Thy V, Sar V, Parry CM. 2012. Pediatric suppurative parotitis in cambodia between 2007 and 2011 Pediatric Infectious Disease Journal, 31 (8), pp. 865-868. | Show Abstract | Read more

The causes of suppurative parotitis in Cambodian children are not known. We describe 39 cases at the Angkor Hospital for Children, Siem Reap, between January 2007 and July 2011 (0.07/1000 hospital attendances). The median age was 5.7 years with no neonates affected. Burkholderia pseudomallei was cultured in 29 (74%) cases. No deaths occurred; 1 child developed facial nerve palsy. © 2012 by Lippincott Williams & Wilkins.

Price EP, Dale JL, Cook JM, Sarovich DS, Seymour ML, Ginther JL, Kaufman EL, Beckstrom-Sternberg SM et al. 2012. Development and validation of Burkholderia pseudomallei-specific real-time PCR assays for clinical, environmental or forensic detection applications. PLoS One, 7 (5), pp. e37723. | Show Abstract | Read more

The bacterium Burkholderia pseudomallei causes melioidosis, a rare but serious illness that can be fatal if untreated or misdiagnosed. Species-specific PCR assays provide a technically simple method for differentiating B. pseudomallei from near-neighbor species. However, substantial genetic diversity and high levels of recombination within this species reduce the likelihood that molecular signatures will differentiate all B. pseudomallei from other Burkholderiaceae. Currently available molecular assays for B. pseudomallei detection lack rigorous validation across large in silico datasets and isolate collections to test for specificity, and none have been subjected to stringent quality control criteria (accuracy, precision, selectivity, limit of quantitation (LoQ), limit of detection (LoD), linearity, ruggedness and robustness) to determine their suitability for environmental, clinical or forensic investigations. In this study, we developed two novel B. pseudomallei specific assays, 122018 and 266152, using a dual-probe approach to differentiate B. pseudomallei from B. thailandensis, B. oklahomensis and B. thailandensis-like species; other species failed to amplify. Species specificity was validated across a large DNA panel (>2,300 samples) comprising Burkholderia spp. and non-Burkholderia bacterial and fungal species of clinical and environmental relevance. Comparison of assay specificity to two previously published B. pseudomallei-specific assays, BurkDiff and TTS1, demonstrated comparable performance of all assays, providing between 99.7 and 100% specificity against our isolate panel. Last, we subjected 122018 and 266152 to rigorous quality control analyses, thus providing quantitative limits of assay performance. Using B. pseudomallei as a model, our study provides a framework for comprehensive quantitative validation of molecular assays and provides additional, highly validated B. pseudomallei assays for the scientific research community.

Mahavanakul W, Nickerson EK, Srisomang P, Teparrukkul P, Lorvinitnun P, Wongyingsinn M, Chierakul W, Hongsuwan M et al. 2012. Feasibility of modified surviving sepsis campaign guidelines in a resource-restricted setting based on a cohort study of severe S. aureus sepsis [corrected]. PLoS One, 7 (2), pp. e29858. | Show Abstract | Read more

BACKGROUND: The Surviving Sepsis Campaign (SSC) guidelines describe best practice for the management of severe sepsis and septic shock in developed countries, but most deaths from sepsis occur where healthcare is not sufficiently resourced to implement them. Our objective was to define the feasibility and basis for modified guidelines in a resource-restricted setting. METHODS AND FINDINGS: We undertook a detailed assessment of sepsis management in a prospective cohort of patients with severe sepsis caused by a single pathogen in a 1,100-bed hospital in lower-middle income Thailand. We compared their management with the SSC guidelines to identify care bundles based on existing capabilities or additional activities that could be undertaken at zero or low cost. We identified 72 patients with severe sepsis or septic shock associated with S. aureus bacteraemia, 38 (53%) of who died within 28 days. One third of patients were treated in intensive care units (ICUs). Numerous interventions described by the SSC guidelines fell within existing capabilities, but their implementation was highly variable. Care available to patients on general wards covered the fundamental principles of sepsis management, including non-invasive patient monitoring, antimicrobial administration and intravenous fluid resuscitation. We described two additive care bundles, one for general wards and the second for ICUs, that if consistently performed would be predicted to improve outcome from severe sepsis. CONCLUSION: It is feasible to implement modified sepsis guidelines that are scaled to resource availability, and that could save lives prior to the publication of international guidelines for developing countries.

Peacock SJ, Limmathurotsakul D, Lubell Y, Koh GC, White LJ, Day NP, Titball RW. 2012. Melioidosis vaccines: a systematic review and appraisal of the potential to exploit biodefense vaccines for public health purposes. PLoS Negl Trop Dis, 6 (1), pp. e1488. | Show Abstract | Read more

BACKGROUND: Burkholderia pseudomallei is a Category B select agent and the cause of melioidosis. Research funding for vaccine development has largely considered protection within the biothreat context, but the resulting vaccines could be applicable to populations who are at risk of naturally acquired melioidosis. Here, we discuss target populations for vaccination, consider the cost-benefit of different vaccination strategies and review potential vaccine candidates. METHODS AND FINDINGS: Melioidosis is highly endemic in Thailand and northern Australia, where a biodefense vaccine might be adopted for public health purposes. A cost-effectiveness analysis model was developed, which showed that a vaccine could be a cost-effective intervention in Thailand, particularly if used in high-risk populations such as diabetics. Cost-effectiveness was observed in a model in which only partial immunity was assumed. The review systematically summarized all melioidosis vaccine candidates and studies in animal models that had evaluated their protectiveness. Possible candidates included live attenuated, whole cell killed, sub-unit, plasmid DNA and dendritic cell vaccines. Live attenuated vaccines were not considered favorably because of possible reversion to virulence and hypothetical risk of latent infection, while the other candidates need further development and evaluation. Melioidosis is acquired by skin inoculation, inhalation and ingestion, but routes of animal inoculation in most published studies to date do not reflect all of this. We found a lack of studies using diabetic models, which will be central to any evaluation of a melioidosis vaccine for natural infection since diabetes is the most important risk factor. CONCLUSION: Vaccines could represent one strand of a public health initiative to reduce the global incidence of melioidosis.

Boonsilp S, Thaipadungpanit J, Amornchai P, Wuthiekanun V, Chierakul W, Limmathurotsakul D, Day NP, Peacock SJ. 2011. Molecular detection and speciation of pathogenic Leptospira spp. in blood from patients with culture-negative leptospirosis. BMC Infect Dis, 11 (1), pp. 338. | Show Abstract | Read more

BACKGROUND: Pathogenic Leptospira spp. present in the blood of patients with leptospirosis during the first week of symptoms can be detected using culture or PCR. A proportion of patients who are positive by PCR are negative by culture. Leptospira spp. are fastidious bacteria, and we hypothesized that a false-negative culture result may represent infection with a distinct bacterial subset that fail to grow in standard culture medium. METHODS: We evaluated our hypothesis during a prospective study of 418 consecutive patients presenting to a hospital in northeast Thailand with an acute febrile illness. Admission blood samples were taken for Leptospira culture and PCR. A single tube nested PCR that amplified a region of the rrs gene was developed and applied, amplicons sequenced and a phylogenetic tree reconstructed. RESULTS: 39/418 (9%) patients were culture-positive for Leptospira spp., and 81/418 (19%) patients were culture-negative but rrs PCR-positive. The species associated with culture-positive leptospirosis (37 L. interrogans and 2 L. borgpetersenii) were comparable to those associated with culture-negative, PCR-positive leptospirosis (76 L. interrogans, 4 L. borgpetersenii, 1 unidentified, possibly new species). CONCLUSION: Molecular speciation failed to identify a unique bacterial subset in patients with culture-negative, PCR-positive leptospirosis. The rate of false-negative culture was high, and we speculate that antibiotic pre-treatment is the most likely explanation for this.

Velapatiño B, Limmathurotsakul D, Peacock SJ, Speert DP. 2012. Identification of differentially expressed proteins from Burkholderia pseudomallei isolated during primary and relapsing melioidosis. Microbes Infect, 14 (4), pp. 335-340. | Show Abstract | Read more

Burkholderia pseudomallei causes septicemic melioidosis with a high rate of relapse, however microbial determinants of relapse are unknown. Proteins were analyzed from sequential B. pseudomallei isolates from primary and relapsing melioidosis. Analysis by isotope tagging for relative and absolute quantitation revealed that factors required for nitric oxide detoxification (HmpA) and necessary for anaerobic growth (ArcA, ArcC and ArcB) were highly expressed in the relapse isolate. Two-dimensional gel electrophoresis revealed up-regulation of a putative hemolysin-coregulated protein in the primary isolate, and flagellin and HSP20/alpha crystalline in the relapse isolate. These observations provide targets for further analysis of latency and virulence of melioidosis.

Limmathurotsakul D, Wuthiekanun V, Amornchai P, Wongsuwan G, Day NP, Peacock SJ. 2012. Effectiveness of a simplified method for isolation of Burkholderia pseudomallei from soil. Appl Environ Microbiol, 78 (3), pp. 876-877. | Show Abstract | Read more

Detection of environmental Burkholderia pseudomallei indicates a risk for melioidosis and is important for the development of a global risk map. We describe a simple method for detecting B. pseudomallei using direct culture of soil in enrichment broth. This gives a rate of positivity comparable to that obtained with a standard method but is cheaper and labor saving.

Chantratita N, Tandhavanant S, Wikraiphat C, Trunck LA, Rholl DA, Thanwisai A, Saiprom N, Limmathurotsakul D et al. 2012. Proteomic analysis of colony morphology variants of Burkholderia pseudomallei defines a role for the arginine deiminase system in bacterial survival. J Proteomics, 75 (3), pp. 1031-1042. | Show Abstract | Read more

Colony morphology variation of Burkholderia pseudomallei is a notable feature of a proportion of primary clinical cultures from patients with melioidosis. Here, we examined the hypothesis that colony morphology switching results in phenotypic changes associated with enhanced survival under adverse conditions. We generated isogenic colony morphology types II and III from B. pseudomallei strain 153 type I, and compared their protein expression profiles using 2D gel electrophoresis. Numerous proteins were differentially expressed, the most prominent of which were flagellin, arginine deiminase (AD) and carbamate kinase (CK), which were over-expressed in isogenic types II and III compared with parental type I. AD and CK (encoded by arcA and arcC) are components of the arginine deiminase system (ADS) which facilitates acid tolerance. Reverse transcriptase PCR of arcA and arcC mRNA expression confirmed the proteomic results. Transcripts of parental type I strain 153 arcA and arcC were increased in the presence of arginine, in a low oxygen concentration and in acid. Comparison of wild type with arcA and arcC defective mutants demonstrated that the B. pseudomallei ADS was associated with survival in acid, but did not appear to play a role in intracellular survival or replication within the mouse macrophage cell line J774A.1. These data provide novel insights into proteomic alterations that occur during the complex process of morphotype switching, and lend support to the idea that this is associated with a fitness advantage in vivo.

Chantratita N, Rholl DA, Sim B, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Thanwisai A, Chua HH et al. 2011. Antimicrobial resistance to ceftazidime involving loss of penicillin-binding protein 3 in Burkholderia pseudomallei. Proc Natl Acad Sci U S A, 108 (41), pp. 17165-17170. | Show Abstract | Read more

Known mechanisms of resistance to β-lactam antibiotics include β-lactamase expression, altered drug target, decreased bacterial permeability, and increased drug efflux. Here, we describe a unique mechanism of β-lactam resistance in the biothreat organism Burkholderia pseudomallei (the cause of melioidosis), associated with treatment failure during prolonged ceftazidime therapy of natural infection. Detailed comparisons of the initial ceftazidime-susceptible infecting isolate and subsequent ceftazidime-resistant variants from six patients led us to identify a common, large-scale genomic loss involving a minimum of 49 genes in all six resistant strains. Mutational analysis of wild-type B. pseudomallei demonstrated that ceftazidime resistance was due to deletion of a gene encoding a penicillin-binding protein 3 (BPSS1219) present within the region of genomic loss. The clinical ceftazidime-resistant variants failed to grow using commonly used laboratory culture media, including commercial blood cultures, rendering the variants almost undetectable in the diagnostic laboratory. Melioidosis is notoriously difficult to cure and clinical treatment failure is common in patients treated with ceftazidime, the drug of first choice across most of Southeast Asia where the majority of cases are reported. The mechanism described here represents an explanation for ceftazidime treatment failure, and may be a frequent but undetected resistance event.

Koh GC, Meijers JC, Maude RR, Limmathurotsakul D, Day NP, Peacock SJ, van der Poll T, Wiersinga WJ. 2011. Diabetes does not influence activation of coagulation, fibrinolysis or anticoagulant pathways in Gram-negative sepsis (melioidosis). Thromb Haemost, 106 (6), pp. 1139-1148. | Show Abstract | Read more

Diabetes is associated with a disturbance of the haemostatic balance and is an important risk factor for sepsis, but the influence of diabetes on the pathogenesis of sepsis remains unclear. Melioidosis ( Burkholderia pseudomallei infection) is a common cause of community-acquired sepsis in Southeast Asia and northern Australia. We sought to investigate the impact of pre-existing diabetes on the coagulation and fibrinolytic systems during sepsis caused by B.pseudomallei . We recruited a cohort of 44 patients (34 with diabetes and 10 without diabetes) with culture-proven melioidosis. Diabetes was defined as a pre-admission diagnosis of diabetes or an HbA₁c>7.8% at enrolment. Thirty healthy blood donors and 52 otherwise healthy diabetes patients served as controls. Citrated plasma was collected from all subjects; additionally in melioidosis patients follow-up specimens were collected seven and ≥ 28 days after enrolment where possible. Relative to uninfected healthy controls, diabetes per se (i.e. in the absence of infection) was characterised by a procoagulant effect. Melioidosis was associated with activation of coagulation (thrombin-antithrombin complexes (TAT), prothrombin fragment F₁+₂ and fibrinogen concentrations were elevated; PT and PTT prolonged), suppression of anti-coagulation (antithrombin, protein C, total and free protein S levels were depressed) and abnormalities of fibrinolysis (D-dimer and plasmin-antiplasmin complex [PAP] were elevated). Remarkably, none of these haemostatic alterations were influenced by pre-existing diabetes. In conclusion, although diabetes is associated with multiple abnormalities of coagulation, anticoagulation and fibrinolysis, these changes are not detectable when superimposed on the background of larger abnormalities attributable to B. pseudomallei sepsis.

Cited:

92

Scopus

Limmathurotsakul D, Peacock SJ. 2011. Melioidosis: a clinical overview BRITISH MEDICAL BULLETIN, 99 (1), pp. 125-139. | Show Abstract | Read more

Introduction: melioidosis, an infection caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, has emerged as an important cause of morbidity and mortality in Southeast Asia and northern Australia. Sources of data: a review of the literature using PubMed. Areas of agreement: approaches to diagnosis and antimicrobial therapy. Areas of controversy: whether seroconversion signals the presence of a quiescent bacterial focus and an increase in long-term risk of melioidosis. Areas timely for developing research: melioidosis is potentially preventable, but there is a striking lack of evidence on which to base an effective prevention programme. An accurate map defining the global distribution of B. pseudomallei is needed, together with studies on the relative importance of different routes of infection. There is a marked difference in mortality from melioidosis in high-income versus lower income countries, and affordable strategies that reduce death from severe sepsis (from any cause) in resource-restricted settings are needed. © The Author 2010. Published by Oxford University Press. All rights reserved.

Trung TT, Hetzer A, Goehler A, Topfstedt E, Wuthiekanun V, Limmathurotsakul D, Peacock SJ, Steinmetz I. 2011. Highly Sensitive Direct Detection and Quantification of Burkholderia pseudomallei Bacteria in Environmental Soil Samples by Using Real-Time PCR APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 77 (18), pp. 6486-6494. | Show Abstract | Read more

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10 4 genome equivalents (range, 3.65 × 10 2 to 7.85 × 10 5) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei. © 2011, American Society for Microbiology.

Wuthiekanun V, Amornchai P, Saiprom N, Chantratita N, Chierakul W, Koh GC, Chaowagul W, Day NP, Limmathurotsakul D, Peacock SJ. 2011. Survey of antimicrobial resistance in clinical Burkholderia pseudomallei isolates over two decades in Northeast Thailand. Antimicrob Agents Chemother, 55 (11), pp. 5388-5391. | Show Abstract | Read more

A 21-year survey conducted in northeast Thailand of antimicrobial resistance to parenteral antimicrobial drugs used to treat melioidosis identified 24/4,021 (0.6%) patients with one or more isolates resistant to ceftazidime (n = 8), amoxicillin-clavulanic acid (n = 4), or both drugs (n = 12). Two cases were identified at admission, and the remainder were detected a median of 15 days after starting antimicrobial therapy. Resistance to carbapenem drugs was not detected. These findings support the current prescribing recommendations for melioidosis.

Trung TT, Hetzer A, Göhler A, Topfstedt E, Wuthiekanun V, Limmathurotsakul D, Peacock SJ, Steinmetz I. 2011. Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR. Appl Environ Microbiol, 77 (18), pp. 6486-6494. | Show Abstract | Read more

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.

West TE, Chierakul W, Chantratita N, Limmathurotsakul D, Wuthiekanun V, Emond MJ, Hawn TR, Peacock SJ, Skerrett SJ. 2012. Toll-like receptor 4 region genetic variants are associated with susceptibility to melioidosis. Genes Immun, 13 (1), pp. 38-46. | Show Abstract | Read more

Melioidosis is a tropical infection caused by the Gram-negative soil saprophyte Burkholderia pseudomallei. Despite broad exposure of northeastern Thais, disease develops in only a small proportion of individuals. Although diabetes is a risk factor, the mechanisms of host susceptibility to melioidosis are still poorly understood. We postulated that Toll-like receptors (TLRs) regulate host susceptibility to disease, and that genetic variation in TLRs is associated with melioidosis. We analyzed the frequency of eight previously described TLR pathway polymorphisms in 490 cases compared with 950 non-hospitalized controls or 458 hospitalized controls. Based on these results, we then analyzed the frequency of additional TLR4 or TLR6-1-10 region polymorphisms in cases and controls. We found that the TLR4(1196C>T) variant was associated with protection from melioidosis when compared with non-hospitalized controls. The TLR1(742A>G) and TLR1(-7202A>G) variants were associated with melioidosis when compared with hospitalized controls. In further analyses, we found that two additional TLR4 region polymorphisms were associated with disease. In diabetics, three other TLR6-1-10 region polymorphisms were associated with disease when compared with hospitalized controls. We conclude that TLR genetic variants may modulate host susceptibility to melioidosis. Confirmation of these findings and further investigation of the mechanisms are required.

Limmathurotsakul D, Wuthiekanun V, Wongsuvan G, Pangmee S, Amornchai P, Teparrakkul P, Teerawattanasook N, Day NP, Peacock SJ. 2011. Repeat blood culture positive for B. pseudomallei indicates an increased risk of death from melioidosis. Am J Trop Med Hyg, 84 (6), pp. 858-861. | Show Abstract | Read more

Melioidosis, a bacterial infection caused by Burkholderia pseudomallei, is notoriously difficult to cure despite appropriate antimicrobial therapy and has a mortality rate of up to 40%. We demonstrate that a blood culture positive for B. pseudomallei taken at the end of the first and/or second week after hospitalization for melioidosis is a strong prognostic factor for death (adjusted odds ratio = 4.2, 95% confidence interval = 2.1-8.7, P < 0.001 and adjusted odds ratio = 2.6, 95% confidence interval = 1.1-6.0, P = 0.03, respectively). However, repeat cultures of respiratory secretions, urine, throat swabs, or pus/surface swabs provide no prognostic information. This finding highlights the need for follow-up blood cultures in patients with melioidosis.

Limmathurotsakul D, Peacock SJ. 2011. Melioidosis: a clinical overview. Br Med Bull, 99 (1), pp. 125-139. | Show Abstract | Read more

INTRODUCTION: Melioidosis, an infection caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, has emerged as an important cause of morbidity and mortality in Southeast Asia and northern Australia. SOURCES OF DATA: a review of the literature using PubMed. AREAS OF AGREEMENT: Approaches to diagnosis and antimicrobial therapy. AREAS OF CONTROVERSY: Whether seroconversion signals the presence of a quiescent bacterial focus and an increase in long-term risk of melioidosis. AREAS TIMELY FOR DEVELOPING RESEARCH: Melioidosis is potentially preventable, but there is a striking lack of evidence on which to base an effective prevention programme. An accurate map defining the global distribution of B. pseudomallei is needed, together with studies on the relative importance of different routes of infection. There is a marked difference in mortality from melioidosis in high-income versus lower income countries, and affordable strategies that reduce death from severe sepsis (from any cause) in resource-restricted settings are needed.

Trung TT, Hetzer A, Topfstedt E, Göhler A, Limmathurotsakul D, Wuthiekanun V, Peacock SJ, Steinmetz I. 2011. Improved culture-based detection and quantification of Burkholderia pseudomallei from soil. Trans R Soc Trop Med Hyg, 105 (6), pp. 346-351. | Show Abstract | Read more

Environmental surveillance of the Gram-negative soil bacterium Burkholderia pseudomallei, the aetiological agent of melioidosis, is important in order to define human populations and livestock at risk of acquiring the infection. This study aimed to develop a more sensitive method for the detection of B. pseudomallei from soil samples in endemic areas compared with the currently used culture method based on soil dispersion in water. We report the development of a new protocol that involves soil dispersion in a polyethylene glycol (PEG) and sodium deoxycholate (DOC) solution to increase the yield of viable B. pseudomallei from soil samples. Comparative testing of soil samples from Northeast Thailand covering a wide range of B. pseudomallei concentrations demonstrated a significantly higher recovery (P<0.0001) of B. pseudomallei colony-forming units by the new method compared with the conventional method. The data indicate that using the detergents PEG and DOC not only results in a higher recovery of viable B. pseudomallei but also results in a shift in the bacterial species recovered from soil samples. Future studies on the geographical distribution and population structure of B. pseudomallei in soil are likely to benefit from the new protocol described here.

Limmathurotsakul D, Chantratita N, Teerawattanasook N, Piriyagitpaiboon K, Thanwisai A, Wuthiekanun V, Day NP, Cooper B, Peacock SJ. 2011. Enzyme-linked immunosorbent assay for the diagnosis of melioidosis: better than we thought. Clin Infect Dis, 52 (8), pp. 1024-1028. | Show Abstract | Read more

We used Bayesian latent-class models to generate receiver operating characteristic curves and to revise the cutoff values for an enzyme-linked immunosorbent assay that has been developed previously for melioidosis. The new cutoff was unbiased towards misclassification caused by an imperfect gold standard and resulted in an increase in both sensitivity (from 66.4% to 80.2%) and specificity (82.1% and 95.0%).

Sonthayanon P, Chierakul W, Wuthiekanun V, Thaipadungpanit J, Kalambaheti T, Boonsilp S, Amornchai P, Smythe LD, Limmathurotsakul D, Day NP, Peacock SJ. 2011. Accuracy of loop-mediated isothermal amplification for diagnosis of human leptospirosis in Thailand. Am J Trop Med Hyg, 84 (4), pp. 614-620. | Show Abstract | Read more

There is a lack of diagnostic tests for leptospirosis in technology-restricted settings. We developed loop-mediated isothermal amplification (LAMP) specific for the 16S ribosomal RNA gene (rrs) of pathogenic and intermediate group Leptospira species. The lower limit of detection was 10 genomic equivalents/reaction, and analytical specificity was high; we observed positive reactions for pathogenic/intermediate groups and negative reactions for non-pathogenic Leptospira species and other bacterial species. We evaluated this assay in Thailand by using a case-control study of 133 patients with laboratory-proven leptospirosis and 133 patients with other febrile illnesses. Using admission blood, we found that the rrs LAMP showed positive results in 58 of 133 cases (diagnostic sensitivity = 43.6, 95% confidence interval [CI] = 35.0-52.5) and in 22 of 133 controls (diagnostic specificity = 83.5, 95% CI = 76.0-89.3). Sensitivity was high for 39 patients who were culture positive for Leptospira spp. (84.6, 95% CI = 69.5-94.1). The rrs LAMP can provide an admission diagnosis in approximately half of patients with leptospirosis, but its clinical utility is reduced by a lower specificity.

Suwannasaen D, Mahawantung J, Chaowagul W, Limmathurotsakul D, Felgner PL, Davies H, Bancroft GJ, Titball RW, Lertmemongkolchai G. 2011. Human immune responses to Burkholderia pseudomallei characterized by protein microarray analysis. J Infect Dis, 203 (7), pp. 1002-1011. | Show Abstract | Read more

BACKGROUND: We aimed to determine the antibody and T cell responses to Burkholderia pseudomallei of humans to select candidate vaccine antigens. METHODS: For antibody profiling, a protein microarray of 154 B. pseudomallei proteins was probed with plasma from 108 healthy individuals and 72 recovered patients. Blood from 20 of the healthy and 30 of the recovered individuals was also obtained for T cell assays. RESULTS: Twenty-seven proteins distinctively reacted with human plasma following environmental exposure or clinical melioidosis. We compared the responses according to the patient's history of subsequent relapse, and antibody response to BPSL2765 was higher in plasma from individuals who had only 1 episode of disease than in those with recurrent melioidosis. A comparison of antibody and T cell responses to 5 B. pseudomallei proteins revealed that BimA and flagellin-induced responses were similar but that BPSS0530 could induce T cell responses in healthy controls more than in recovered patients. CONCLUSIONS: By combining large-scale antibody microarrays and assays of T cell-mediated immunity, we identified a panel of novel B. pseudomallei proteins that show distinct patterns of reactivity in different stages of human melioidosis. These proteins may be useful candidates for development of subunit-based vaccines and in monitoring the risks of treatment failure and relapse.

Koh GC, Maude RR, Schreiber MF, Limmathurotsakul D, Wiersinga WJ, Wuthiekanun V, Lee SJ, Mahavanakul W et al. 2011. Glyburide is anti-inflammatory and associated with reduced mortality in melioidosis. Clin Infect Dis, 52 (6), pp. 717-725. | Show Abstract | Read more

BACKGROUND: Patients with diabetes mellitus are more prone to bacterial sepsis, but there are conflicting data on whether outcomes are worse in diabetics after presentation with sepsis. Glyburide is an oral hypoglycemic agent used to treat diabetes mellitus. This K(ATP)-channel blocker and broad-spectrum ATP-binding cassette (ABC) transporter inhibitor has broad-ranging effects on the immune system, including inhibition of inflammasome assembly and would be predicted to influence the host response to infection. METHODS: We studied a cohort of 1160 patients with gram-negative sepsis caused by a single pathogen (Burkholderia pseudomallei), 410 (35%) of whom were known to have diabetes. We subsequently studied prospectively diabetics with B. pseudomallei infection (n = 20) to compare the gene expression profile of peripheral whole blood leukocytes in patients who were taking glyburide against those not taking any sulfonylurea. RESULTS: Survival was greater in diabetics than in nondiabetics (38% vs 45%, respectively, P = .04), but the survival benefit was confined to the patient group taking glyburide (adjusted odds ratio .47, 95% confidence interval .28-.74, P = .005). We identified differential expression of 63 immune-related genes (P = .001) in patients taking glyburide, the sum effect of which we predict to be antiinflammatory in the glyburide group. CONCLUSIONS: We present observational evidence for a glyburide-associated benefit during human melioidosis and correlate this with an anti-inflammatory effect of glyburide on the immune system.

Limmathurotsakul D, Cooper B, Peacock SJ, De Stavola B. 2011. Long-term outcome of Q fever endocarditis. Lancet Infect Dis, 11 (2), pp. 81. | Read more

Thaipadungpanit J, Chierakul W, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Boonslip S, Smythe LD, Limpaiboon R, Hoffmaster AR, Day NP, Peacock SJ. 2011. Diagnostic accuracy of real-time PCR assays targeting 16S rRNA and lipL32 genes for human leptospirosis in Thailand: a case-control study. PLoS One, 6 (1), pp. e16236. | Show Abstract | Read more

BACKGROUND: Rapid PCR-based tests for the diagnosis of leptospirosis can provide information that contributes towards early patient management, but these have not been adopted in Thailand. Here, we compare the diagnostic sensitivity and specificity of two real-time PCR assays targeting rrs or lipL32 for the diagnosis of leptospirosis in northeast Thailand. METHODS/PRINCIPAL FINDINGS: A case-control study of 266 patients (133 cases of leptospirosis and 133 controls) was constructed to evaluate the diagnostic sensitivity and specificity (DSe & DSp) of both PCR assays. The median duration of illness prior to admission of cases was 4 days (IQR 2-5 days; range 1-12 days). DSe and DSp were determined using positive culture and/or microscopic agglutination test (MAT) as the gold standard. The DSe was higher for the rrs assay than the lipL32 assay (56%, (95% CI 47-64%) versus 43%, (95% CI 34-52%), p<0.001). No cases were positive for the lipL32 assay alone. There was borderline evidence to suggest that the DSp of the rrs assay was lower than the lipL32 assay (90% (95% CI 83-94%) versus 93%, (95%CI 88-97%), p = 0.06). Nine controls gave positive reactions for both assays and 5 controls gave a positive reaction for the rrs assay alone. The DSe of the rrs and lipL32 assays were high in the subgroup of 39 patients who were culture positive for Leptospira spp. (95% and 87%, respectively, p = 0.25). CONCLUSIONS/SIGNIFICANCE: Early detection of Leptospira using PCR is possible for more than half of patients presenting with leptospirosis and could contribute to individual patient care.

West TE, Myers ND, Limmathurotsakul D, Liggitt HD, Chantratita N, Peacock SJ, Skerrett SJ. 2010. Pathogenicity of high-dose enteral inoculation of Burkholderia pseudomallei to mice. Am J Trop Med Hyg, 83 (5), pp. 1066-1069. | Show Abstract | Read more

Melioidosis is a frequently lethal tropical infection caused by the environmental saprophyte Burkholderia pseudomallei. Although transcutaneous inoculation and inhalation are considered the primary routes of infection, suggestive clinical evidence implicates ingestion as a possible alternative route. We show that in BALB/c and C57BL/6 mice, direct gastric inoculation of high doses of B. pseudomallei causes systemic infection that may be lethal or cause chronic disseminated infection. Mice may shed bacteria in the stool for weeks after infection, and high titers of B. pseudomallei-specific IgG are detectable. This report of enteric murine melioidosis supports further consideration of this route of infection.

Koh GC, Limmathurotsakul D. 2010. Gamma interferon supplementation for melioidosis. Antimicrob Agents Chemother, 54 (10), pp. 4520. | Read more

Limmathurotsakul D, Jamsen K, Arayawichanont A, Simpson JA, White LJ, Lee SJ, Wuthiekanun V, Chantratita N et al. 2010. Defining the true sensitivity of culture for the diagnosis of melioidosis using Bayesian latent class models. PLoS One, 5 (8), pp. e12485. | Show Abstract | Read more

BACKGROUND: Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests. METHODS/PRINCIPAL FINDINGS: Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard. CONCLUSIONS/SIGNIFICANCE: Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections.

Limmathurotsakul D, Wuthiekanun V, Chantratita N, Wongsuvan G, Amornchai P, Day NP, Peacock SJ. 2010. Burkholderia pseudomallei is spatially distributed in soil in northeast Thailand. PLoS Negl Trop Dis, 4 (6), pp. e694. | Show Abstract | Read more

BACKGROUND: Melioidosis is a frequently fatal infectious disease caused by the soil dwelling Gram-negative bacterium Burkholderia pseudomallei. Environmental sampling is important to identify geographical distribution of the organism and related risk of infection to humans and livestock. The aim of this study was to evaluate spatial distribution of B. pseudomallei in soil and consider the implications of this for soil sampling strategies. METHODS AND FINDINGS: A fixed-interval sampling strategy was used as the basis for detection and quantitation by culture of B. pseudomallei in soil in two environmental sites (disused land covered with low-lying scrub and rice field) in northeast Thailand. Semivariogram and indicator semivariogram were used to evaluate the distribution of B. pseudomallei and its relationship with range between sampling points. B. pseudomallei was present on culture of 80/100 sampling points taken from the disused land and 28/100 sampling points from the rice field. The median B. pseudomallei cfu/gram from positive sampling points was 378 and 700 for the disused land and the rice field, respectively (p = 0.17). Spatial autocorrelation of B. pseudomallei was present, in that samples taken from areas adjacent to sampling points that were culture positive (negative) for B. pseudomallei were also likely to be culture positive (negative), and samples taken from areas adjacent to sampling points with a high (low) B. pseudomallei count were also likely to yield a high (low) count. Ranges of spatial autocorrelation in quantitative B. pseudomallei count were 11.4 meters in the disused land and 7.6 meters in the rice field. CONCLUSIONS: We discuss the implications of the uneven distribution of B. pseudomallei in soil for future environmental studies, and describe a range of established geostatistical sampling approaches that would be suitable for the study of B. pseudomallei that take account of our findings.

Limmathurotsakul D, Wongratanacheewin S, Teerawattanasook N, Wongsuvan G, Chaisuksant S, Chetchotisakd P, Chaowagul W, Day NP, Peacock SJ. 2010. Increasing incidence of human melioidosis in Northeast Thailand. Am J Trop Med Hyg, 82 (6), pp. 1113-1117. | Show Abstract | Read more

Melioidosis is a serious community-acquired infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei. A prospective cohort study identified 2,243 patients admitted to Sappasithiprasong Hospital in northeast Thailand with culture-confirmed melioidosis between 1997 and 2006. These data were used to calculate an average incidence rate for the province of 12.7 cases of melioidosis per 100,000 people per year. Incidence increased incrementally from 8.0 (95% confidence interval [CI] = 7.2-10.0) in 2000 to 21.3 (95% CI = 19.2-23.6) in 2006 (P < 0.001; chi(2) test for trend). Male sex, age >/= 45 years, and either known or undiagnosed diabetes were independent risk factors for melioidosis. The average mortality rate from melioidosis over the study period was 42.6%. The minimum estimated population mortality rate from melioidosis in 2006 was 8.63 per 100,000 people (95% CI = 7.33-10.11), the third most common cause of death from infectious diseases in northeast Thailand after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and tuberculosis.

Price EP, Hornstra HM, Limmathurotsakul D, Max TL, Sarovich DS, Vogler AJ, Dale JL, Ginther JL et al. 2010. Within-host evolution of Burkholderia pseudomallei in four cases of acute melioidosis. PLoS Pathog, 6 (1), pp. e1000725. | Show Abstract | Read more

Little is currently known about bacterial pathogen evolution and adaptation within the host during acute infection. Previous studies of Burkholderia pseudomallei, the etiologic agent of melioidosis, have shown that this opportunistic pathogen mutates rapidly both in vitro and in vivo at tandemly repeated loci, making this organism a relevant model for studying short-term evolution. In the current study, B. pseudomallei isolates cultured from multiple body sites from four Thai patients with disseminated melioidosis were subjected to fine-scale genotyping using multilocus variable-number tandem repeat analysis (MLVA). In order to understand and model the in vivo variable-number tandem repeat (VNTR) mutational process, we characterized the patterns and rates of mutations in vitro through parallel serial passage experiments of B. pseudomallei. Despite the short period of infection, substantial divergence from the putative founder genotype was observed in all four melioidosis cases. This study presents a paradigm for examining bacterial evolution over the short timescale of an acute infection. Further studies are required to determine whether the mutational process leads to phenotypic alterations that impact upon bacterial fitness in vivo. Our findings have important implications for future sampling strategies, since colonies in a single clinical sample may be genetically heterogeneous, and organisms in a culture taken late in the infective process may have undergone considerable genetic change compared with the founder inoculum.

Tandhavanant S, Thanwisai A, Limmathurotsakul D, Korbsrisate S, Day NP, Peacock SJ, Chantratita N. 2010. Effect of colony morphology variation of Burkholderia pseudomallei on intracellular survival and resistance to antimicrobial environments in human macrophages in vitro. BMC Microbiol, 10 (1), pp. 303. | Show Abstract | Read more

BACKGROUND: Primary diagnostic cultures from patients with melioidosis demonstrate variation in colony morphology of the causative organism, Burkholderia pseudomallei. Variable morphology is associated with changes in the expression of a range of putative virulence factors. This study investigated the effect of B. pseudomallei colony variation on survival in the human macrophage cell line U937 and under laboratory conditions simulating conditions within the macrophage milieu. Isogenic colony morphology types II and III were generated from 5 parental type I B. pseudomallei isolates using nutritional limitation. Survival of types II and III were compared with type I for all assays. RESULTS: Morphotype was associated with survival in the presence of H2O2 and antimicrobial peptide LL-37, but not with susceptibility to acid, acidified sodium nitrite, or resistance to lysozyme, lactoferrin, human neutrophil peptide-1 or human beta defensin-2. Incubation under anaerobic conditions was a strong driver for switching of type III to an alternative morphotype. Differences were noted in the survival and replication of the three types following uptake by human macrophages, but marked strain-to strain-variability was observed. Uptake of type III alone was associated with colony morphology switching. CONCLUSIONS: Morphotype is associated with phenotypes that alter the ability of B. pseudomallei to survive in adverse environmental conditions.

Bicanic T, Muzoora C, Brouwer AE, Meintjes G, Longley N, Taseera K, Rebe K, Loyse A et al. 2009. Independent association between rate of clearance of infection and clinical outcome of HIV-associated cryptococcal meningitis: analysis of a combined cohort of 262 patients. Clin Infect Dis, 49 (5), pp. 702-709. | Show Abstract | Read more

BACKGROUND: Progress in therapy for cryptococcal meningitis has been slow because of the lack of a suitable marker of treatment response. Previously, we demonstrated the statistical power of a novel endpoint, the rate of clearance of infection, based on serial quantitative cultures of cerebrospinal fluid, to differentiate the fungicidal activity of alternative antifungal drug regimens. We hypothesized that the rate of clearance of infection should also be a clinically meaningful endpoint. METHODS: We combined data from cohorts of patients with human immunodeficiency virus-associated cryptococcal meningitis from Thailand, South Africa, and Uganda, for whom the rate of clearance of infection was determined, and clinical and laboratory data prospectively collected, and explored the association between the rate of clearance of infection and mortality by Cox survival analyses. RESULTS: The combined cohort comprised 262 subjects. Altered mental status at presentation, a high baseline organism load, and a slow rate of clearance of infection were independently associated with increased mortality at 2 and 10 weeks. Rate of clearance of infection was associated with antifungal drug regimen and baseline cerebrospinal fluid interferon-gamma levels. CONCLUSIONS: The results support the use of the rate of clearance of infection or early fungicidal activity as a means to explore antifungal drug dosages and combinations in phase II studies. An increased understanding of how the factors determining outcome interrelate may help clarify opportunities for intervention.

Limmathurotsakul D, Chaowagul W, Day NPJ, Peacock SJ. 2009. Short Report: Patterns of Organ Involvement in Recurrent Melioidosis AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 81 (2), pp. 335-337. | Show Abstract

Recurrent melioidosis can be caused by two different mechanisms: relapse or re-infection. We examined the pattern of organ involvement in the first and second episodes in individual patients. Evaluation of 140 patients with recurrence showed that similar patterns of disease occurred during the first and second episode, independent of whether this was caused by relapse or re-infection. Copyright © 2009 by The American Society of Tropical Medicine and Hygiene.

Limmathurotsakul D, Chaowagul W, Day NP, Peacock SJ. 2009. Patterns of organ involvement in recurrent melioidosis. Am J Trop Med Hyg, 81 (2), pp. 335-337. | Show Abstract

Recurrent melioidosis can be caused by two different mechanisms: relapse or re-infection. We examined the pattern of organ involvement in the first and second episodes in individual patients. Evaluation of 140 patients with recurrence showed that similar patterns of disease occurred during the first and second episode, independent of whether this was caused by relapse or re-infection.

Wongsuvan G, Limmathurotsakul D, Wannapasni S, Chierakul W, Teerawattanasook N, Wuthiekanun V. 2009. Lack of correlation of Burkholderia pseudomallei quantities in blood, urine, sputum and pus. Southeast Asian J Trop Med Public Health, 40 (4), pp. 781-784. | Show Abstract

We evaluated the correlation of Burkholderia pseudomallei quantities in blood versus urine, sputum or pus. Correlations between bacterial counts in blood and other samples were not found. It is likely that an initial seeding event to extracellular organs is followed by independent growth of B. pseudomallei, and that bacteria in the urine were not passively filtered from the bloodstream.

Wiersinga WJ, Limmathurotsakul D, Cheng AC. 2009. Turning green with shock. Neth J Med, 67 (7), pp. 291-294.

Tippayawat P, Saenwongsa W, Mahawantung J, Suwannasaen D, Chetchotisakd P, Limmathurotsakul D, Peacock SJ, Felgner PL et al. 2009. Phenotypic and functional characterization of human memory T cell responses to Burkholderia pseudomallei. PLoS Negl Trop Dis, 3 (4), pp. e407. | Show Abstract | Read more

BACKGROUND: Infection with the Gram-negative bacterium Burkholderia pseudomallei is an important cause of community-acquired lethal sepsis in endemic regions in southeast Asia and northern Australia and is increasingly reported in other tropical areas. In animal models, production of interferon-gamma (IFN-gamma) is critical for resistance, but in humans the characteristics of IFN-gamma production and the bacterial antigens that are recognized by the cell-mediated immune response have not been defined. METHODS: Peripheral blood from 133 healthy individuals who lived in the endemic area and had no history of melioidosis, 60 patients who had recovered from melioidosis, and 31 other patient control subjects were stimulated by whole bacteria or purified bacterial proteins in vitro, and IFN-gamma responses were analyzed by ELISPOT and flow cytometry. FINDINGS: B. pseudomallei was a potent activator of human peripheral blood NK cells for innate production of IFN-gamma. In addition, healthy individuals with serological evidence of exposure to B. pseudomallei and patients recovered from active melioidosis developed CD4(+) (and CD8(+)) T cells that recognized whole bacteria and purified proteins LolC, OppA, and PotF, members of the B. pseudomallei ABC transporter family. This response was primarily mediated by terminally differentiated T cells of the effector-memory (T(EMRA)) phenotype and correlated with the titer of anti-B. pseudomallei antibodies in the serum. CONCLUSIONS: Individuals living in a melioidosis-endemic region show clear evidence of T cell priming for the ability to make IFN-gamma that correlates with their serological status. The ability to detect T cell responses to defined B. pseudomallei proteins in large numbers of individuals now provides the opportunity to screen candidate antigens for inclusion in protein or polysaccharide-conjugate subunit vaccines against this important but neglected disease.

Bicanic T, Brouwer AE, Meintjes G, Rebe K, Limmathurotsakul D, Chierakul W, Teparrakkul P, Loyse A et al. 2009. Relationship of cerebrospinal fluid pressure, fungal burden and outcome in patients with cryptococcal meningitis undergoing serial lumbar punctures. AIDS, 23 (6), pp. 701-706. | Show Abstract | Read more

OBJECTIVES: To assess impact of serial lumbar punctures on association between cerebrospinal fluid (CSF) opening pressure and prognosis in HIV-associated cryptococcal meningitis; to explore time course and relationship of opening pressure with neurological findings, CSF fungal burden, immune response, and CD4 cell count. DESIGN: Evaluation of 163 HIV-positive ART-naive patients enrolled in three trials of amphotericin B-based therapy for cryptococcal meningitis in Thailand and South Africa. METHODS: Study protocols required four lumbar punctures with measurements of opening pressure over the first 2 weeks of treatment and additional lumbar punctures if opening pressure raised. Fungal burden and clearance, CSF immune parameters, CD4 cell count, neurological symptoms and signs, and outcome at 2 and 10 weeks were compared between groups categorized by opening pressure at cryptococcal meningitis diagnosis. RESULTS: Patients with higher baseline fungal burden had higher baseline opening pressure. High fungal burden appeared necessary but not sufficient for development of high pressure. Baseline opening pressure was not associated with CD4 cell count, CSF pro-inflammatory cytokines, or altered mental status. Day 14 opening pressure was associated with day 14 fungal burden. Overall mortality was 12% (20/162) at 2 weeks and 26% (42/160) at 10 weeks, with no significant differences between opening pressure groups. CONCLUSION: Studies are needed to define factors, in addition to fungal burden, associated with raised opening pressure. Aggressive management of raised opening pressure through repeated CSF drainage appeared to prevent any adverse impact of raised opening pressure on outcome in patients with cryptococcal meningitis. The results support increasing access to manometers in resource-poor settings and routine management of opening pressure in patients with cryptococcal meningitis.

Nickerson EK, Hongsuwan M, Limmathurotsakul D, Wuthiekanun V, Shah KR, Srisomang P, Mahavanakul W, Wacharaprechasgul T et al. 2009. Staphylococcus aureus bacteraemia in a tropical setting: patient outcome and impact of antibiotic resistance. PLoS One, 4 (1), pp. e4308. | Show Abstract | Read more

BACKGROUND: Most information on invasive Staphylococcus aureus infections comes from temperate countries. There are considerable knowledge gaps in epidemiology, treatment, drug resistance and outcome of invasive S. aureus infection in the tropics. METHODS: A prospective, observational study of S. aureus bacteraemia was conducted in a 1000-bed regional hospital in northeast Thailand over 1 year. Detailed clinical data were collected and final outcomes determined at 12 weeks, and correlated with antimicrobial susceptibility profiles of infecting isolates. PRINCIPAL FINDINGS: Ninety-eight patients with S. aureus bacteraemia were recruited. The range of clinical manifestations was similar to that reported from temperate countries. The prevalence of endocarditis was 14%. The disease burden was highest at both extremes of age, whilst mortality increased with age. The all-cause mortality rate was 52%, with a mortality attributable to S. aureus of 44%. Methicillin-resistant S. aureus (MRSA) was responsible for 28% of infections, all of which were healthcare-associated. Mortality rates for MRSA and methicillin-susceptible S. aureus (MSSA) were 67% (18/27) and 46% (33/71), respectively (p = 0.11). MRSA isolates were multidrug resistant. Only vancomycin or fusidic acid would be suitable as empirical treatment options for suspected MRSA infection. CONCLUSIONS: S. aureus is a significant pathogen in northeast Thailand, with comparable clinical manifestations and a similar endocarditis prevalence but higher mortality than industrialised countries. S. aureus bacteraemia is frequently associated with exposure to healthcare settings with MRSA causing a considerable burden of disease. Further studies are required to define setting-specific strategies to reduce mortality from S. aureus bacteraemia, prevent MRSA transmission, and to define the burden of S. aureus disease and emergence of drug resistance throughout the developing world.

Wuthiekanun V, Limmathurotsakul D, Chantratita N, Feil EJ, Day NP, Peacock SJ. 2009. Burkholderia Pseudomallei is genetically diverse in agricultural land in Northeast Thailand. PLoS Negl Trop Dis, 3 (8), pp. e496. | Show Abstract | Read more

BACKGROUND: The soil-dwelling Gram-negative bacterium Burkholderia pseudomallei is the cause of melioidosis. Extreme structuring of genotype and genotypic frequency has been demonstrated for B. pseudomallei in uncultivated land, but its distribution and genetic diversity in agricultural land where most human infections are probably acquired is not well defined. METHODS: Fixed-interval soil sampling was performed in a rice paddy in northeast Thailand in which 100 grams of soil was sampled at a depth of 30 cm from 10x10 sampling points each measuring 2.5 m by 2.5 m. Soil was cultured for the presence of B. pseudomallei and genotyping of colonies present on primary culture plates was performed using a combination of pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). PRINCIPAL FINDINGS: B. pseudomallei was cultured from 28/100 samples. Genotyping of 630 primary colonies drawn from 11 sampling points demonstrated 10 PFGE banding pattern types, which on MLST were resolved into 7 sequence types (ST). Overlap of genotypes was observed more often between sampling points that were closely positioned. Two sampling points contained mixed B. pseudomallei genotypes, each with a numerically dominant genotype and one or more additional genotypes present as minority populations. CONCLUSIONS: Genetic diversity and structuring of B. pseudomallei exists despite the effects of flooding and the physical and chemical processes associated with farming. These findings form an important baseline for future studies of environmental B. pseudomallei.

Cheng AC, Wuthiekanun V, Limmathurotsakul D, Chierakul W, Peacock SJ. 2008. Intensity of exposure and incidence of melioidosis in Thai children. Trans R Soc Trop Med Hyg, 102 Suppl 1 (SUPPL. 1), pp. S37-S39. | Show Abstract | Read more

There is a high background seroprevalence of antibodies to Burkholderia pseudomallei in Thailand that limits its use as a diagnostic tool. It is believed that this results from childhood exposure to the bacterium in mud and surface water. The increasing prevalence of antibodies with age is a marker of the intensity of exposure. A susceptible-infected-susceptible (SIS) model was calibrated with data on seroprevalence in children (<15 years) in Udon Thani and Ubon Ratchathani (n=2214). In this mathematical model, children were assumed to gain antibodies at a constant rate related to exposure events, and waning antibody response occurred at a constant rate. The intensity of exposure appeared to be higher in Udon Thani than in Ubon Ratchathani, with 24% vs. 11% of patients becoming seropositive each year. In Udon Thani children, antibodies appeared to be long-lasting, compared with those in Ubon Ratchathani, where the mean duration was 5.2 years. Based on an estimated paediatric disease incidence in Ubon Ratchathani of 4.15 per 100,000 population, it is estimated that approximately 1 in 4600 antibody-producing exposures results in clinical infection. Childhood seroprevalence can be used as a marker of intensity of exposure. Further work to separate the effect of exposure to B. thailandensis and cross-reactivity to B. pseudomallei is proposed.

Cheng AC, West TE, Limmathurotsakul D, Peacock SJ. 2008. Strategies to reduce mortality from bacterial sepsis in adults in developing countries. PLoS Med, 5 (8), pp. e175. | Read more

Tumapa S, Holden MT, Vesaratchavest M, Wuthiekanun V, Limmathurotsakul D, Chierakul W, Feil EJ, Currie BJ, Day NP, Nierman WC, Peacock SJ. 2008. Burkholderia pseudomallei genome plasticity associated with genomic island variation. BMC Genomics, 9 (1), pp. 190. | Show Abstract | Read more

BACKGROUND: Burkholderia pseudomallei is a soil-dwelling saprophyte and the cause of melioidosis. Horizontal gene transfer contributes to the genetic diversity of this pathogen and may be an important determinant of virulence potential. The genome contains genomic island (GI) regions that encode a broad array of functions. Although there is some evidence for the variable distribution of genomic islands in B. pseudomallei isolates, little is known about the extent of variation between related strains or their association with disease or environmental survival. RESULTS: Five islands from B. pseudomallei strain K96243 were chosen as representatives of different types of genomic islands present in this strain, and their presence investigated in other B. pseudomallei. In silico analysis of 10 B. pseudomallei genome sequences provided evidence for the variable presence of these regions, together with micro-evolutionary changes that generate GI diversity. The diversity of GIs in 186 isolates from NE Thailand (83 environmental and 103 clinical isolates) was investigated using multiplex PCR screening. The proportion of all isolates positive by PCR ranged from 12% for a prophage-like island (GI 9), to 76% for a metabolic island (GI 16). The presence of each of the five GIs did not differ between environmental and disease-associated isolates (p > 0.05 for all five islands). The cumulative number of GIs per isolate for the 186 isolates ranged from 0 to 5 (median 2, IQR 1 to 3). The distribution of cumulative GI number did not differ between environmental and disease-associated isolates (p = 0.27). The presence of GIs was defined for the three largest clones in this collection (each defined as a single sequence type, ST, by multilocus sequence typing); these were ST 70 (n = 15 isolates), ST 54 (n = 11), and ST 167 (n = 9). The rapid loss and/or acquisition of gene islands was observed within individual clones. Comparisons were drawn between isolates obtained from the environment and from patients with melioidosis in order to examine the role of genomic islands in virulence and clinical associations. There was no reproducible association between the individual or cumulative presence of five GIs and a range of clinical features in 103 patients with melioidosis. CONCLUSION: Horizontal gene transfer of mobile genetic elements can rapidly alter the gene repertoire of B. pseudomallei. This study confirms the utility of a range of approaches in defining the presence and significance of genomic variation in natural populations of B. pseudomallei.

Cheng AC, Chierakul W, Chaowagul W, Chetchotisakd P, Limmathurotsakul D, Dance DA, Peacock SJ, Currie BJ. 2008. Consensus guidelines for dosing of amoxicillin-clavulanate in melioidosis. Am J Trop Med Hyg, 78 (2), pp. 208-209. | Show Abstract

Melioidosis is an infectious disease endemic to northern Australia and Southeast Asia. In response to clinical confusion regarding the appropriate dose of amoxicillin-clavulanate, we have developed guidelines for the appropriate dosing of this second-line agent. For eradication therapy for melioidosis, we recommend 20/5 mg/kg orally, three times daily.

Chantratita N, Meumann E, Thanwisai A, Limmathurotsakul D, Wuthiekanun V, Wannapasni S, Tumapa S, Day NP, Peacock SJ. 2008. Loop-mediated isothermal amplification method targeting the TTS1 gene cluster for detection of Burkholderia pseudomallei and diagnosis of melioidosis. J Clin Microbiol, 46 (2), pp. 568-573. | Show Abstract | Read more

Melioidosis is a severe infection caused by Burkholderia pseudomallei. The timely implementation of effective antimicrobial treatment requires rapid diagnosis. Loop-mediated isothermal amplification (LAMP) targeting the TTS1 gene cluster was developed for the detection of B. pseudomallei. LAMP was sensitive and specific for the laboratory detection of this organism. The lower limit of detection was 38 genomic copies per reaction, and LAMP was positive for 10 clinical B. pseudomallei isolates but negative for 5 B. thailandensis and 5 B. mallei isolates. A clinical evaluation was conducted in northeast Thailand to compare LAMP to an established real-time PCR assay targeting the same TTS1 gene cluster. A total of 846 samples were obtained from 383 patients with suspected melioidosis, 77 of whom were subsequently diagnosed with culture-confirmed melioidosis. Of these 77 patients, a positive result was obtained from one or more specimens by PCR in 26 cases (sensitivity, 34%; 95% confidence interval [CI], 23.4 to 45.4%) and by LAMP in 34 cases (sensitivity, 44%; 95% CI, 32.8 to 55.9%) (P = 0.02). All samples from 306 patients that were culture negative for B. pseudomallei were negative by PCR (specificity, 100%; 95% CI, 98.8 to 100%), but 5 of 306 patients (1.6%) were positive by LAMP (specificity, 98.4%; 95% CI, 96.2 to 99.5%) (P = 0.03). The diagnostic accuracies of PCR and LAMP were 86.7% (95% CI, 82.9 to 89.9%) and 87.5% (95% CI, 83.7 to 90.6%), respectively (P = 0.47). Both assays were very insensitive when applied to blood samples; PCR and LAMP were positive for 0 and 1 of 44 positive blood cultures, respectively. The PCR and LAMP assays evaluated here are not sufficiently sensitive to replace culture in our clinical setting.

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Cheng AC, Chierakul W, Chaowagul W, Chetchotisakd P, Limmathurotsakul D, Dance DAB, Peacock SJ, Currie BJ. 2008. Short report: Consensus guidelines for dosing of amoxicillin-clavulanate in melioidosis AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 78 (2), pp. 208-209. | Show Abstract

Melioidosis is an infectious disease endemic to northern Australia and Southeast Asia. In response to clinical confusion regarding the appropriate dose of amoxicillin-clavulanate, we have developed guidelines for the appropriate dosing of this second-line agent. For eradication therapy for melioidosis, we recommend 20/5 mg/kg orally, three times daily. Copyright © 2008 by The American Society of Tropical Medicine and Hygiene.

Teparrakkul P, Tsai JJ, Chierakul W, Gerstenmaier JF, Wacharaprechasgu T, Piyaphanee W, Limmathurotsakul D, Chaowagul W, Day NP, Peacock SJ. 2008. Rheumatological manifestations in patients with melioidosis. Southeast Asian J Trop Med Public Health, 39 (4), pp. 649-655. | Show Abstract

Melioidosis, an infection caused by the bacterium Burkholderia pseudomallei, has a wide range of clinical manifestations. Here, we describe rheumatological melioidosis (involving one or more of joint, bone or muscle), and compare features and outcome with patients without rheumatological involvement. A retrospective study of patients with culture-confirmed melioidosis admitted to Sappasithiprasong Hospital, Ubon Ratchathani during 2002 and 2005 identified 679 patients with melioidosis, of whom 98 (14.4%) had rheumatological melioidosis involving joint (n=52), bone (n = 5), or muscle (n = 12), or a combination of these (n=29). Females were over-represented in the rheumatological group, and diabetes and thalassemia were independent risk factors for rheumatological involvement (OR; 2.49 and 9.56, respectively). Patients with rheumatological involvement had a more chronic course, as reflected by a longer fever clearance time (13 vs 7 days, p = 0.06) and hospitalization (22 vs 14 days, p < 0.001), but lower mortality (28% vs 44%, p = 0.005). Patients with signs and symptoms of septic arthritis for longer than 2 weeks were more likely to have extensive infection of adjacent bone and muscle, particularly in diabetic patients. Surgical intervention was associated with a survival benefit, bur not a shortening of the course of infection.

Dondorp AM, Ince C, Charunwatthana P, Hanson J, van Kuijen A, Faiz MA, Rahman MR, Hasan M et al. 2008. Direct in vivo assessment of microcirculatory dysfunction in severe falciparum malaria. J Infect Dis, 197 (1), pp. 79-84. | Show Abstract | Read more

BACKGROUND: This study sought to describe and quantify microcirculatory changes in the mucosal surfaces of patients with severe malaria, by direct in vivo observation using orthogonal polarization spectral (OPS) imaging. METHODS: The microcirculation in the rectal mucosa of adult patients with severe malaria was assessed by use of OPS imaging, at admission and then daily. Comparison groups comprised patients with uncomplicated falciparum malaria, patients with bacterial sepsis, and healthy individuals. RESULTS: Erythrocyte velocities were measured directly in 43 adult patients with severe falciparum malaria, of whom 20 died. Microcirculatory blood flow was markedly disturbed, with heterogeneous obstruction that was proportional to severity of disease. Blocked capillaries were found in 29 patients (67%) and were associated with concurrent hyperdynamic blood flow (erythrocyte velocity, >750 mm/s) in adjacent vessels in 27 patients (93%). The proportion of blocked capillaries correlated with the base deficit in plasma and with the concentration of lactate. Abnormalities disappeared when the patients recovered. In healthy individuals and in patients with uncomplicated malaria or sepsis, no stagnant erythrocytes were detected, and, in patients with sepsis, hyperdynamic blood flow was prominent. CONCLUSION: Patients with severe falciparum malaria show extensive microvascular obstruction that is proportional to the severity of the disease. This finding underscores the prominent role that microvascular obstruction plays in the pathophysiology of severe malaria and illustrates the fundamental difference between the microvascular pathophysiology of malaria and that of bacterial sepsis.

Chantratita N, Wuthiekanun V, Limmathurotsakul D, Vesaratchavest M, Thanwisai A, Amornchai P, Tumapa S, Feil EJ, Day NP, Peacock SJ. 2008. Genetic diversity and microevolution of Burkholderia pseudomallei in the environment. PLoS Negl Trop Dis, 2 (2), pp. e182. | Show Abstract | Read more

BACKGROUND: The soil dwelling Gram-negative pathogen Burkholderia pseudomallei is the cause of melioidosis. The diversity and population structure of this organism in the environment is poorly defined. METHODS AND FINDINGS: We undertook a study of B. pseudomallei in soil sampled from 100 equally spaced points within 237.5 m(2) of disused land in northeast Thailand. B. pseudomallei was present on direct culture of 77/100 sampling points. Genotyping of 200 primary plate colonies from three independent sampling points was performed using a combination of pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Twelve PFGE types and nine sequence types (STs) were identified, the majority of which were present at only a single sampling point. Two sampling points contained four STs and the third point contained three STs. Although the distance between the three sampling points was low (7.6, 7.9, and 13.3 meters, respectively), only two STs were present in more than one sampling point. Each of the three samples was characterized by the localized expansion of a single B. pseudomallei clone (corresponding to STs 185, 163, and 93). Comparison of PFGE and MLST results demonstrated that two STs contained strains with variable PFGE banding pattern types, indicating geographic structuring even within a single MLST-defined clone. CONCLUSIONS: We discuss the implications of this extreme structuring of genotype and genotypic frequency in terms of micro-evolutionary dynamics and ecology, and how our results may inform future sampling strategies.

Limmathurotsakul D, Chaowagul W, Chantratita N, Wuthiekanun V, Biaklang M, Tumapa S, White NJ, Day NP, Peacock SJ. 2008. A simple scoring system to differentiate between relapse and re-infection in patients with recurrent melioidosis. PLoS Negl Trop Dis, 2 (10), pp. e327. | Show Abstract | Read more

BACKGROUND: Melioidosis is an important cause of morbidity and mortality in East Asia. Recurrent melioidosis occurs in around 10% of patients following treatment either because of relapse with the same strain or re-infection with a new strain of Burkholderia pseudomallei. Distinguishing between the two is important but requires bacterial genotyping. The aim of this study was to develop a simple scoring system to distinguish re-infection from relapse. METHODS: In a prospective study of 2,804 consecutive adult patients with melioidosis presenting to Sappasithiprasong Hospital, NE Thailand, between 1986 and 2005, there were 141 patients with recurrent melioidosis with paired strains available for genotyping. Of these, 92 patients had relapse and 49 patients had re-infection. Variables associated with relapse or re-infection were identified by multivariable logistic regression and used to develop a predictive model. Performance of the scoring system was quantified with respect to discrimination (area under receiver operating characteristic curves, AUC) and categorization (graphically). Bootstrap resampling was used to internally validate the predictors and adjust for over-optimism. FINDINGS: Duration of oral antimicrobial treatment, interval between the primary episode and recurrence, season, and renal function at recurrence were independent predictors of relapse or re-infection. A score of < 5 correctly identified relapse in 76 of 89 patients (85%), whereas a score > or = 5 correctly identified re-infection in 36 of 52 patients (69%). The scoring index had good discriminative power, with a bootstrap bias-corrected AUC of 0.80 (95%CI: 0.73-0.87). CONCLUSIONS: A simple scoring index to predict the cause of recurrent melioidosis has been developed to provide important bedside information where rapid bacterial genotyping is unavailable.

Druar C, Yu F, Barnes JL, Okinaka RT, Chantratita N, Beg S, Stratilo CW, Olive AJ et al. 2008. Evaluating Burkholderia pseudomallei Bip proteins as vaccines and Bip antibodies as detection agents. FEMS Immunol Med Microbiol, 52 (1), pp. 78-87. | Show Abstract | Read more

Burkholderia pseudomallei is a biothreat agent and an important natural pathogen, causing melioidosis in humans and animals. A type III secretion system (TTSS-3) has been shown to be critical for virulence. Because TTSS components from other pathogens have been used successfully as diagnostic agents and as experimental vaccines, it was investigated whether this was the case for BipB, BipC and BipD, components of B. pseudomallei's TTSS-3. The sequences of BipB, BipC and BipD were found to be highly conserved among B. pseudomallei and B. mallei isolates. A collection of monoclonal antibodies (mAbs) specific for each Bip protein was obtained. Most recognized both native and denatured Bip protein. Burkholderia pseudomallei or B. mallei did not express detectable BipB or BipD under the growth conditions used. However, anti-BipD mAbs did recognize the TTSS needle structures of a Shigella strain engineered to express BipD. The authors did not find that BipB, BipC or BipD are protective antigens because vaccination of mice with any single protein did not result in protection against experimental melioidosis. Enzyme-linked immunosorbent assay (ELISA) studies showed that human melioidosis patients had antibodies to BipB and BipD. However, these ELISAs had low diagnostic accuracy in endemic regions, possibly due to previous patient exposure to B. pseudomallei.

Wuthiekanun V, Limmathurotsakul D, Wongsuvan G, Chierakul W, Teerawattanasook N, Teparrukkul P, Day NP, Peacock SJ. 2007. Short report: Quantitation of B. pseudomallei in clinical samples AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 77 (5), pp. 812-813. | Show Abstract

We undertook a prospective study to quantitate Burkholderia pseudomallei in blood, pus, respiratory secretions, and urine obtained from 414 patients with melioidosis. The median was count 1.1, 1.5 × 104, 1.1 × 105, and 1,1 × 107 CFU/mL in these sample types, respectively. This provides important insights into the likely feasibility of future studies such as expression microarray analysis using clinical material. Copyright © 2007 by The American Society of Tropical Medicine and Hygiene.

Wuthiekanun V, Limmathurotsakul D, Wongsuvan G, Chierakul W, Teerawattanasook N, Teparrukkul P, Day NP, Peacock SJ. 2007. Quantitation of B. Pseudomallei in clinical samples. Am J Trop Med Hyg, 77 (5), pp. 812-813. | Show Abstract

We undertook a prospective study to quantitate Burkholderia pseudomallei in blood, urine, respiratory secretions, and pus [corrected] obtained from 414 patients with melioidosis. The median was count 1.1, 1.5 x 10(4), 1.1 x 10(5), and 1.1 x 10(7) CFU/mL in these sample types, respectively. This provides important insights into the likely feasibility of future studies such as expression microarray analysis using clinical material.

Limmathurotsakul D, Wuthiekanun V, Chantratita N, Wongsuvan G, Thanwisai A, Biaklang M, Tumapa S, Lee S, Day NP, Peacock SJ. 2007. Simultaneous infection with more than one strain of Burkholderia pseudomallei is uncommon in human melioidosis. J Clin Microbiol, 45 (11), pp. 3830-3832. | Show Abstract | Read more

A prospective study was performed to determine the rate at which patients with melioidosis are infected with more than one strain of Burkholderia pseudomallei. Genotyping of 2,058 bacterial colonies isolated from 215 samples taken from 133 patients demonstrated that mixed infection is uncommon (2/133 cases [1.5%; 95% confidence interval, 0.2 to 5.3%]).

Chantratita N, Wuthiekanun V, Limmathurotsakul D, Thanwisai A, Chantratita W, Day NP, Peacock SJ. 2007. Prospective clinical evaluation of the accuracy of 16S rRNA real-time PCR assay for the diagnosis of melioidosis. Am J Trop Med Hyg, 77 (5), pp. 814-817. | Show Abstract

The accuracy of a Burkholderia pseudomallei 16s rRNA real-time PCR assay was evaluated against culture for the diagnosis of melioidosis in Thailand. A total of 846 samples were obtained from 383 patients with suspected melioidosis. One or more specimens were PCR positive for 47 of 77 patients with culture-proven melioidosis (sensitivity 61.0%, 95% CI: 49.2-72.0%). PCR was negative for all 306 patients who were culture negative for B. pseudomallei (specificity 100%, 95% CI: 98.8-100%). Diagnostic sensitivity of PCR was 22.7% for patients who were culture positive for blood only, compared with 79.4% for patients who were culture positive for samples other than blood. The median (interquartile range) B. pseudomallei colony count in blood for 44 of 77 patients with positive blood cultures was 2.4 CFU/ml (0.2-13.5 CFU/ml); this may explain the low sensitivity of PCR for this specimen. The PCR assay described here is not sufficiently sensitive to replace culture in our clinical setting.

Cheng AC, Limmathurotsakul D, Chierakul W, Getchalarat N, Wuthiekanun V, Stephens DP, Day NP, White NJ, Chaowagul W, Currie BJ, Peacock SJ. 2007. A randomized controlled trial of granulocyte colony-stimulating factor for the treatment of severe sepsis due to melioidosis in Thailand. Clin Infect Dis, 45 (3), pp. 308-314. | Show Abstract | Read more

BACKGROUND: Melioidosis is a tropical infectious disease associated with significant mortality. Most deaths occur early and are caused by fulminant sepsis. METHODS: In this randomized, placebo-controlled trial, we assessed the efficacy of lenograstim (granulocyte colony-stimulating factor [G-CSF], 263 mu g per day administered intravenously) in ceftazidime-treated patients with severe sepsis caused by suspected melioidosis in Thailand. RESULTS: Over a 27-month period, 60 patients were enrolled to receive either G-CSF (30 patients, 18 of whom had culture-confirmed melioidosis) or placebo (30 patients, 23 of whom had culture-confirmed melioidosis). Mortality rates were similar in both groups (G-CSF group, 70%; placebo group, 87%; risk ratio, 0.81; 95% confidence interval, 0.61-1.06; P=.2), including among patients with confirmed melioidosis (83% vs. 96%; P=.3). The duration of survival was longer for patients who received G-CSF than for patients who received placebo (33 h vs. 18.6 h; hazard ratio, 0.56; 95% confidence interval, 0.31-1.00; P=.05). CONCLUSIONS: Receipt of G-CSF is associated with a longer duration of survival but is not associated with a mortality benefit in patients with severe sepsis who are suspected of having melioidosis in Thailand. We hypothesize that G-CSF may "buy time" for severely septic patients, but survival is more likely to be improved by management of associated metabolic abnormalities and organ dysfunction associated with severe sepsis.

Limmathurotsakul D, Chaowagul W, Wongsrikaew P, Narmwong A, Day NP, Peacock SJ. 2007. Variable presentation of neurological melioidosis in Northeast Thailand. Am J Trop Med Hyg, 77 (1), pp. 118-120. | Show Abstract

We describe three instructive cases of neurologic melioidosis that demonstrate the variable nature of clinical manifestations and disease pathology. The appropriate duration and choice of parenteral and oral antimicrobial therapy for neurologic melioidosis are also discussed.

Wiersinga WJ, Wieland CW, Dessing MC, Chantratita N, Cheng AC, Limmathurotsakul D, Chierakul W, Leendertse M et al. 2007. Toll-like receptor 2 impairs host defense in gram-negative sepsis caused by Burkholderia pseudomallei (Melioidosis). PLoS Med, 4 (7), pp. e248. | Show Abstract | Read more

BACKGROUND: Toll-like receptors (TLRs) are essential in host defense against pathogens by virtue of their capacity to detect microbes and initiate the immune response. TLR2 is seen as the most important receptor for gram-positive bacteria, while TLR4 is regarded as the gram-negative TLR. Melioidosis is a severe infection caused by the gram-negative bacterium, Burkholderia pseudomallei, that is endemic in Southeast Asia. We aimed to characterize the expression and function of TLRs in septic melioidosis. METHODS AND FINDINGS: Patient studies: 34 patients with melioidosis demonstrated increased expression of CD14, TLR1, TLR2, and TLR4 on the cell surfaces of monocytes and granulocytes, and increased CD14, TLR1, TLR2, TLR4, LY96 (also known as MD-2), TLR5, and TLR10 mRNA levels in purified monocytes and granulocytes when compared with healthy controls. In vitro experiments: Whole-blood and alveolar macrophages obtained from TLR2 and TLR4 knockout (KO) mice were less responsive to B. pseudomallei in vitro, whereas in the reverse experiment, transfection of HEK293 cells with either TLR2 or TLR4 rendered these cells responsive to this bacterium. In addition, the lipopolysaccharide (LPS) of B. pseudomallei signals through TLR2 and not through TLR4. Mouse studies: Surprisingly, TLR4 KO mice were indistinguishable from wild-type mice with respect to bacterial outgrowth and survival in experimentally induced melioidosis. In contrast, TLR2 KO mice displayed a markedly improved host defenses as reflected by a strong survival advantage together with decreased bacterial loads, reduced lung inflammation, and less distant-organ injury. CONCLUSIONS: Patients with melioidosis displayed an up-regulation of multiple TLRs in peripheral blood monocytes and granulocytes. Although both TLR2 and TLR4 contribute to cellular responsiveness to B. pseudomallei in vitro, TLR2 detects the LPS of B. pseudomallei, and only TLR2 impacts on the immune response of the intact host in vivo. Inhibition of TLR2 may be a novel treatment strategy in melioidosis.

Wiersinga WJ, Dessing MC, Kager PA, Cheng AC, Limmathurotsakul D, Day NP, Dondorp AM, van der Poll T, Peacock SJ. 2007. High-throughput mRNA profiling characterizes the expression of inflammatory molecules in sepsis caused by Burkholderia pseudomallei. Infect Immun, 75 (6), pp. 3074-3079. | Show Abstract | Read more

Sepsis is characterized by an uncontrolled inflammatory response to invading microorganisms. We describe the inflammatory mRNA profiles in whole-blood leukocytes, monocytes, and granulocytes using a multigene system for 35 inflammatory markers that included pro- and anti-inflammatory cytokines, chemokines, and signal transduction molecules in a case-control study with 34 patients with sepsis caused by the gram-negative bacterium Burkholderia pseudomallei (the pathogen causing melioidosis) and 32 healthy volunteers. Relative to healthy controls, patients with sepsis showed increased transcription of a whole array of inflammatory genes in peripheral blood leukocytes, granulocytes, and monocytes. Specific monocyte and granulocyte mRNA profiles were identified. Strong correlations were found between inflammatory mRNA expression levels in monocytes and clinical outcome. These data underline the notion that circulating leukocytes are an important source for inflammatory mediators in patients with gram-negative sepsis. Gene profiling such as was done here provides an excellent tool to obtain insight into the extent of inflammation activation in patients with severe infection.

Mahavanakul W, Limmathurotsakul D, Teerawattanasuk N, Peacock SJ. 2007. Invasive Erysipelothrix rhusiopathiae infection in northeast Thailand. Southeast Asian J Trop Med Public Health, 38 (3), pp. 478-481. | Show Abstract

Three cases of invasive Erysipelothrix rhusipathiae infection, which is considered rare, presented to a hospital in Ubon Ratchathani, northeast Thailand during 2006. Patients presented with variable clinical manifestations including diffused cutaneous lesions, bacteremia and endocarditis. Erysipelothrix infection may be an emerging infection in immunocompromized individuals in Thailand.

Wuthiekanun V, Chierakul W, Limmathurotsakul D, Smythe LD, Symonds ML, Dohnt MF, Slack AT, Limpaiboon R et al. 2007. Optimization of culture of Leptospira from humans with leptospirosis. J Clin Microbiol, 45 (4), pp. 1363-1365. | Show Abstract | Read more

A prospective study of 989 patients with acute febrile illness was performed in northeast Thailand to define the yield of Leptospira from four different types of blood sample. Based on a comparison of the yields from whole blood, surface plasma, deposit from spun plasma, and clotted blood samples from 80 patients with culture-proven leptospirosis, we suggest a sampling strategy in which culture is performed using whole blood and deposit from spun plasma.

Chantratita N, Wuthiekanun V, Boonbumrung K, Tiyawisutsri R, Vesaratchavest M, Limmathurotsakul D, Chierakul W, Wongratanacheewin S et al. 2007. Biological relevance of colony morphology and phenotypic switching by Burkholderia pseudomallei. J Bacteriol, 189 (3), pp. 807-817. | Show Abstract | Read more

Melioidosis is a notoriously protracted illness and is difficult to cure. We hypothesize that the causative organism, Burkholderia pseudomallei, undergoes a process of adaptation involving altered expression of surface determinants which facilitates persistence in vivo and that this is reflected by changes in colony morphology. A colony morphotyping scheme and typing algorithm were developed using clinical B. pseudomallei isolates. Morphotypes were divided into seven types (denoted I to VII). Type I gave rise to other morphotypes (most commonly type II or III) by a process of switching in response to environmental stress, including starvation, iron limitation, and growth at 42 degrees C. Switching was associated with complex shifts in phenotype, one of which (type I to type II) was associated with a marked increase in production of factors putatively associated with in vivo concealment. Isogenic types II and III, derived from type I, were examined using several experimental models. Switching between isogenic morphotypes occurred in a mouse model, where type II appeared to become adapted for persistence in a low-virulence state. Isogenic type II demonstrated a significant increase in intracellular replication fitness compared with parental type I after uptake by epithelial cells in vitro. Isogenic type III demonstrated a higher replication fitness following uptake by macrophages in vitro, which was associated with a switch to type II. Mixed B. pseudomallei morphologies were common in individual clinical specimens and were significantly more frequent in samples of blood, pus, and respiratory secretions than in urine and surface swabs. These findings have major implications for therapeutics and vaccine development.

Chantratita N, Wuthiekanun V, Thanwisai A, Limmathurotsakul D, Cheng AC, Chierakul W, Day NP, Peacock SJ. 2007. Accuracy of enzyme-linked immunosorbent assay using crude and purified antigens for serodiagnosis of melioidosis. Clin Vaccine Immunol, 14 (1), pp. 110-113. | Show Abstract | Read more

Five enzyme-linked immunosorbent assays developed to detect antibodies to different Burkholderia pseudomallei antigen preparations were evaluated as diagnostic tests for melioidosis in northeast Thailand. The highest diagnostic indices were observed for an affinity-purified antigen (sensitivity, 82%; specificity, 72%) and crude B. pseudomallei antigen (sensitivity, 81%; specificity, 70%), an improvement over the indirect hemagglutination assay (sensitivity, 73%; specificity, 64%).

Cheng AC, Peacock SJ, Limmathurotsakul D, Wongsuvan G, Chierakul W, Amornchai P, Getchalarat N, Chaowagul W, White NJ, Day NP, Wuthiekanun V. 2006. Prospective evaluation of a rapid immunochromogenic cassette test for the diagnosis of melioidosis in northeast Thailand. Trans R Soc Trop Med Hyg, 100 (1), pp. 64-67. | Show Abstract | Read more

A prospective study was performed to compare a rapid immunochromogenic cassette test (ICT) with the indirect haemagglutination assay (IHA) and clinical rules for the diagnosis of melioidosis in an endemic area. The sensitivity and specificity of the IgG ICT was 86% and 47%, and the IgM ICT was 82% and 47%, respectively. These were similar to the results for IHA (sensitivity 73%, specificity 64%) and clinical rules (73% and 37%). ICT lacks clinical utility as a result of high background rates of positive Burkholderia pseudomallei serology in this population. Low sensitivity and specificity of clinical rules is consistent with the protean manifestations of melioidosis and clinical difficulty in identifying patients with melioidosis.

Chierakul W, Anunnatsiri S, Short JM, Maharjan B, Mootsikapun P, Simpson AJ, Limmathurotsakul D, Cheng AC et al. 2005. Two randomized controlled trials of ceftazidime alone versus ceftazidime in combination with trimethoprim-sulfamethoxazole for the treatment of severe melioidosis. Clin Infect Dis, 41 (8), pp. 1105-1113. | Show Abstract | Read more

BACKGROUND: Two antibiotic regimens are used commonly in Thailand for the initial treatment of severe melioidosis: ceftazidime in combination with trimethoprim-sulfamethoxazole (TMP-SMX) and ceftazidime monotherapy. It is not known whether TMP-SMX provides an additional benefit. METHODS: Two prospective, randomized trials that compared these regimens for patients presenting with acute severe melioidosis were started independently at tertiary care hospitals in Ubon Ratchathani and Khon Kaen (in northeastern Thailand), and the results were analyzed together as a prospective, individual-patient data meta-analysis. The primary end point was in-hospital mortality rate. RESULTS: The in-hospital mortality rate among all enrolled patients (n=449) was not significantly different between those randomized to ceftazidime alone (25.1%; 56 of 223 subjects) and those randomized to ceftazidime with TMP-SMX (26.6%; 60 of 226 subjects; odds ratio [OR], 1.08; 95% confidence interval [CI], 0.7-1.7; stratified P=.73). Of the 241 patients with culture-confirmed melioidosis, 51 (21.2%) died. Of these 241 patients, 31 (12.9%) died > or =48 h after the time of study entry. Among patients with melioidosis, there was no difference in death rate between the 2 treatment groups for either all deaths (OR, 0.88; 95% CI, 0.48-1.6; stratified P=.70) or for deaths that occurred > or =48 h after hospital admission (OR, 0.88; 95% CI, 0.41-1.9; stratified P=.73). Conditional logistic regression analysis revealed that bacteremia, respiratory failure, and renal failure were independently associated with death and treatment failure. Drug regimens were not associated with death or treatment failure in this model. CONCLUSION: We conclude that the addition of TMP-SMX to ceftazidime therapy during initial treatment of severe melioidosis does not reduce the acute mortality rate.

Chaowagul W, Chierakul W, Simpson AJ, Short JM, Stepniewska K, Maharjan B, Rajchanuvong A, Busarawong D et al. 2005. Open-label randomized trial of oral trimethoprim-sulfamethoxazole, doxycycline, and chloramphenicol compared with trimethoprim-sulfamethoxazole and doxycycline for maintenance therapy of melioidosis. Antimicrob Agents Chemother, 49 (10), pp. 4020-4025. | Show Abstract | Read more

Melioidosis (infection caused by Burkholderia pseudomallei) requires a prolonged course of oral antibiotics following initial intravenous therapy to reduce the risk of relapse after cessation of treatment. The current recommendation is a four-drug regimen (trimethoprim [TMP], sulfamethoxazole [SMX], doxycycline, and chloramphenicol) and a total treatment time of 12 to 20 weeks. Drug side effects are common; the aim of this study was to compare the efficacy and tolerance of the four-drug regimen with a three-drug regimen (TMP-SMX and doxycycline). An open-label, randomized trial was conducted in northeast Thailand. A total of 180 adult Thai patients were enrolled, of which 91 were allocated to the four-drug regimen and 89 to the three-drug regimen. The trial was terminated early due to poor drug tolerance, particularly of the four-drug regimen. The culture-confirmed relapse rates at 1 year were 6.6% and 5.6% for the four- and three-drug regimens, respectively (P = 0.79). The three-drug regimen was better tolerated than the four-drug regimen; 36% of patients receiving four drugs and 19% of patients receiving three drugs required a switch in therapy due to side effects (P = 0.01). The duration of oral therapy was significantly associated with relapse; after adjustment for confounders, patients receiving less than 12 weeks of oral therapy had a 5.7-fold increase of relapse or death. A combination of TMP-SMX and doxycycline is as effective as and better tolerated than the conventional four-drug regimen for the oral treatment phase of melioidosis.

Tiyawisutsri R, Peacock SJ, Langa S, Limmathurotsakul D, Cheng AC, Chierakul W, Chaowagul W, Day NP, Wuthiekanun V. 2005. Antibodies from patients with melioidosis recognize Burkholderia mallei but not Burkholderia thailandensis antigens in the indirect hemagglutination assay. J Clin Microbiol, 43 (9), pp. 4872-4874. | Show Abstract | Read more

The indirect hemagglutination assay routinely used to detect antibodies to Burkholderia pseudomallei was modified to detect cross-reactivity of antibodies to B. pseudomallei, B. mallei, and B. thailandensis antigens. We demonstrate a lack of cross-reactivity between B. pseudomallei and B. thailandensis but marked cross-reactivity between B. pseudomallei and B. mallei.

Wuthiekanun V, Cheng AC, Chierakul W, Amornchai P, Limmathurotsakul D, Chaowagul W, Simpson AJ, Short JM et al. 2005. Trimethoprim/sulfamethoxazole resistance in clinical isolates of Burkholderia pseudomallei. J Antimicrob Chemother, 55 (6), pp. 1029-1031. | Show Abstract | Read more

OBJECTIVES: Trimethoprim/sulfamethoxazole is commonly used to treat melioidosis. Antimicrobial susceptibility testing using the disc diffusion method is commonly used in melioidosis-endemic areas, but may overestimate resistance to trimethoprim/sulfamethoxazole. PATIENTS AND METHODS: We performed disc diffusion and Etest on isolates from the first positive culture for all patients presenting to Sappasithiprasong Hospital, Ubon Ratchathani, Thailand, with culture-confirmed melioidosis between 1992 and 2003. RESULTS: The estimated resistance rate for 1976 clinical Burkholderia pseudomallei isolates was 13% by Etest and 71% by disc diffusion. All isolates classed as either susceptible (n=358) or as having intermediate resistance (n=218) on disc diffusion were susceptible by Etest. Only 258 of the 1400 (18%) isolates classed as resistant on disc diffusion were resistant by Etest. CONCLUSIONS: Disc diffusion testing of B. pseudomallei may be useful as a limited screening tool in resource poor settings. Isolates assigned as 'susceptible' or 'intermediate' by disc diffusion may be viewed as 'susceptible'; those assigned as 'resistant' require further evaluation by MIC methodology.

Limmathurotsakul D, Wuthiekanun V, Chierakul W, Cheng AC, Maharjan B, Chaowagul W, White NJ, Day NP, Peacock SJ. 2005. Role and significance of quantitative urine cultures in diagnosis of melioidosis. J Clin Microbiol, 43 (5), pp. 2274-2276. | Show Abstract | Read more

Melioidosis is associated with significant mortality in countries in which it is endemic. Previous studies have demonstrated that quantitative Burkholderia pseudomallei counts in blood are predictive of mortality. Here we examine the relationship between outcomes and quantitative B. pseudomallei counts in urine. A total of 755 patients presenting to Sappasithiprasong Hospital, Ubon Ratchathani, northeast Thailand (in the northeast part of the country), with melioidosis between July 1993 and October 2003 had quantitative urine cultures performed within 72 h of admission. Urine culture results were divided into the following groups: (i) no growth of B. pseudomallei from a neat sample or pellet, (ii) positive result from a centrifuged pellet only (< 10(3) CFU/ml), (iii) detection of between 10(3) CFU/ml and 10(5) CFU/ml from a neat sample, or (iv) detection of > or = 10(5) CFU/ml from a neat sample. The overall in-hospital mortality rate was 45%. Patients with negative urine cultures had the lowest death rate (39%). Mortality rates rose with increasing B. pseudomallei counts in urine, from 58% for those with positive spun pellets only to 61% for those with between 10(3) CFU/ml and 10(5) CFU/ml and 71% for those with > or = 10(5) CFU/ml. This was independent of age, presence of bacteremia, known risk factors for melioidosis such as diabetes, and the prior administration of antibiotics. The presence of B. pseudomallei in urine during systemic infection is associated with a poor prognosis.

Wuthiekanun V, Desakorn V, Wongsuvan G, Amornchai P, Cheng AC, Maharjan B, Limmathurotsakul D, Chierakul W, White NJ, Day NP, Peacock SJ. 2005. Rapid immunofluorescence microscopy for diagnosis of melioidosis. Clin Diagn Lab Immunol, 12 (4), pp. 555-556. | Show Abstract | Read more

An immunofluorescent (IF) method that detects Burkholderia pseudomallei in clinical specimens within 10 min was devised. The results of this rapid method and those of an existing IF method were prospectively compared with the culture results for 776 specimens from patients with suspected melioidosis. The sensitivities of both IF tests were 66%, and the specificities were 99.5 and 99.4%, respectively.

Tauran PM, Sennang N, Rusli B, Wiersinga WJ, Dance D, Arif M, Limmathurotsakul D. 2015. Emergence of Melioidosis in Indonesia. Am J Trop Med Hyg, 93 (6), pp. 1160-1163. | Show Abstract | Read more

Melioidosis is known to be highly endemic in parts of southeast Asia and northern Australia; however, cases are rarely reported in Indonesia. Here we report three cases of melioidosis in Makassar, South Sulawesi, Indonesia occurring between 2013 and 2014. Two patients died and the other was lost to follow-up. Burkholderia pseudomallei isolates from all three cases were identified by the VITEK2 Compact installed in the hospital in 2012. None of the three patients reported received antimicrobials recommended for melioidosis because of the delayed recognition of the organism. We reviewed the literature and found only seven reports of melioidosis in Indonesia. Five were reported before 1960. We suggest that melioidosis is endemic throughout Indonesia but currently under-recognized. Training on how to identify B. pseudomallei accurately and safely in all available microbiological facilities should be provided, and consideration should be given to making melioidosis a notifiable disease in Indonesia.

Chetchotisakd P, Chierakul W, Chaowagul W, Anunnatsiri S, Phimda K, Mootsikapun P, Chaisuksant S, Pilaikul J et al. 2014. Trimethoprim-sulfamethoxazole versus trimethoprim-sulfamethoxazole plus doxycycline as oral eradicative treatment for melioidosis (MERTH): a multicentre, double-blind, non-inferiority, randomised controlled trial. Lancet, 383 (9919), pp. 807-814. | Show Abstract | Read more

BACKGROUND: Melioidosis, an infectious disease caused by the Gram-negative bacillus Burkholderia pseudomallei, is difficult to cure. Antimicrobial treatment comprises intravenous drugs for at least 10 days, followed by oral drugs for at least 12 weeks. The standard oral regimen based on trial evidence is trimethoprim-sulfamethoxaxole (TMP-SMX) plus doxycycline. This regimen is used in Thailand but is associated with side-effects and poor adherence by patients, and TMP-SMX alone is recommended in Australia. We compared the efficacy and side-effects of TMP-SMX with TMP-SMX plus doxycycline for the oral phase of melioidosis treatment. METHODS: For this multi-centre, double-blind, non-inferiority, randomised placebo-controlled trial, we enrolled patients (aged ≥15 years) from five centres in northeast Thailand with culture-confirmed melioidosis who had received a course of parenteral antimicrobial drugs. Using a computer-generated sequence, we randomly assigned patients to receive TMP-SMX plus placebo or TMP-SMX plus doxycycline for 20 weeks (1:1; block size of ten, stratified by study site). We followed patients up every 4 months for 1 year and annually thereafter to the end of the study. The primary endpoint was culture-confirmed recurrent melioidosis, and the non-inferiority margin was a hazard ratio (HR) of 1.7. This study is registered with www.controlled-trials.com, number ISRCTN86140460. FINDINGS: We enrolled and randomly assigned 626 patients: 311 to TMP-SMX plus placebo and 315 to TMP-SMX plus doxycycline. 16 patients (5%) in the TMP-SMX plus placebo group and 21 patients (7%) in the TMP-SMX plus doxycycline group developed culture-confirmed recurrent melioidosis (HR 0.81; 95% CI 0.42-1.55). The criterion for non-inferiority was met (p=0.01). Adverse drug reactions were less common in the TMP-SMX plus placebo group than in the TMP-SMX plus doxycycline group (122 [39%] vs 167 [53%]). INTERPRETATION: Our findings suggest that TMP-SMX is not inferior to TMP-SMX plus doxycycline for the oral phase of melioidosis treatment, and is preferable on the basis of safety and tolerance by patients. FUNDING: Thailand Research Fund, the Melioidosis Research Center, the Center of Excellence in Specific Health Problems in Greater Mekong Sub-region cluster, and the Wellcome Trust.

Lim C, Wannapinij P, White L, Day NPJ, Cooper BS, Peacock SJ, Limmathurotsakul D. 2013. Using a web-based application to define the accuracy of diagnostic tests when the gold standard is imperfect PLoS ONE, 8 (11), | Show Abstract | Read more

Background: Estimates of the sensitivity and specificity for new diagnostic tests based on evaluation against a known gold standard are imprecise when the accuracy of the gold standard is imperfect. Bayesian latent class models (LCMs) can be helpful under these circumstances, but the necessary analysis requires expertise in computational programming. Here, we describe open-access web-based applications that allow non-experts to apply Bayesian LCMs to their own data sets via a user-friendly interface. Methods/Principal Findings: Applications for Bayesian LCMs were constructed on a web server using R and WinBUGS programs. The models provided (http://mice.tropmedres.ac) include two Bayesian LCMs: the two-tests in two-population model (Hui and Walter model) and the three-tests in one-population model (Walter and Irwig model). Both models are available with simplified and advanced interfaces. In the former, all settings for Bayesian statistics are fixed as defaults. Users input their data set into a table provided on the webpage. Disease prevalence and accuracy of diagnostic tests are then estimated using the Bayesian LCM, and provided on the web page within a few minutes. With the advanced interfaces, experienced researchers can modify all settings in the models as needed. These settings include correlation among diagnostic test results and prior distributions for all unknown parameters. The web pages provide worked examples with both models using the original data sets presented by Hui and Walter in 1980, and by Walter and Irwig in 1988. We also illustrate the utility of the advanced interface using the Walter and Irwig model on a data set from a recent melioidosis study. The results obtained from the web-based applications were comparable to those published previously. Conclusions: The newly developed web-based applications are open-access and provide an important new resource for researchers worldwide to evaluate new diagnostic tests. © 2013 Lim et al.

Kanoksil M, Jatapai A, Peacock S, Limmathurotsakul D. 2012. Epidemiology, microbiology and mortality of community-acquired bacteremia in northeast Thailand: a multicenter population-based study INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E132-E132. | Read more

Hongsuwan M, Srisamang P, Luangasanatip N, Kanoksil M, Day N, Cooper B, Limmathurotsakul D. 2012. A retrospective study to define the incidence and associated mortality of hospital-acquired bacteraemia at a regional hospital in northeast Thailand INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E385-E385. | Read more

Limmathurotsakul D, Wuthiekanun V, Amornchai P, Wongsuwan G, Day NP, Peacock SJ. 2012. Effectiveness of a simplified method for isolation of Burkholderia pseudomallei from soil. Appl Environ Microbiol, 78 (3), pp. 876-877. | Show Abstract | Read more

Detection of environmental Burkholderia pseudomallei indicates a risk for melioidosis and is important for the development of a global risk map. We describe a simple method for detecting B. pseudomallei using direct culture of soil in enrichment broth. This gives a rate of positivity comparable to that obtained with a standard method but is cheaper and labor saving.

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Limmathurotsakul D, Peacock SJ. 2011. Melioidosis: a clinical overview BRITISH MEDICAL BULLETIN, 99 (1), pp. 125-139. | Show Abstract | Read more

Introduction: melioidosis, an infection caused by the environmental Gram-negative bacillus Burkholderia pseudomallei, has emerged as an important cause of morbidity and mortality in Southeast Asia and northern Australia. Sources of data: a review of the literature using PubMed. Areas of agreement: approaches to diagnosis and antimicrobial therapy. Areas of controversy: whether seroconversion signals the presence of a quiescent bacterial focus and an increase in long-term risk of melioidosis. Areas timely for developing research: melioidosis is potentially preventable, but there is a striking lack of evidence on which to base an effective prevention programme. An accurate map defining the global distribution of B. pseudomallei is needed, together with studies on the relative importance of different routes of infection. There is a marked difference in mortality from melioidosis in high-income versus lower income countries, and affordable strategies that reduce death from severe sepsis (from any cause) in resource-restricted settings are needed. © The Author 2010. Published by Oxford University Press. All rights reserved.

Limmathurotsakul D, Wuthiekanun V, Wongsuvan G, Pangmee S, Amornchai P, Teparrakkul P, Teerawattanasook N, Day NP, Peacock SJ. 2011. Repeat blood culture positive for B. pseudomallei indicates an increased risk of death from melioidosis. Am J Trop Med Hyg, 84 (6), pp. 858-861. | Show Abstract | Read more

Melioidosis, a bacterial infection caused by Burkholderia pseudomallei, is notoriously difficult to cure despite appropriate antimicrobial therapy and has a mortality rate of up to 40%. We demonstrate that a blood culture positive for B. pseudomallei taken at the end of the first and/or second week after hospitalization for melioidosis is a strong prognostic factor for death (adjusted odds ratio = 4.2, 95% confidence interval = 2.1-8.7, P < 0.001 and adjusted odds ratio = 2.6, 95% confidence interval = 1.1-6.0, P = 0.03, respectively). However, repeat cultures of respiratory secretions, urine, throat swabs, or pus/surface swabs provide no prognostic information. This finding highlights the need for follow-up blood cultures in patients with melioidosis.

Limmathurotsakul D, Chantratita N, Teerawattanasook N, Piriyagitpaiboon K, Thanwisai A, Wuthiekanun V, Day NP, Cooper B, Peacock SJ. 2011. Enzyme-linked immunosorbent assay for the diagnosis of melioidosis: better than we thought. Clin Infect Dis, 52 (8), pp. 1024-1028. | Show Abstract | Read more

We used Bayesian latent-class models to generate receiver operating characteristic curves and to revise the cutoff values for an enzyme-linked immunosorbent assay that has been developed previously for melioidosis. The new cutoff was unbiased towards misclassification caused by an imperfect gold standard and resulted in an increase in both sensitivity (from 66.4% to 80.2%) and specificity (82.1% and 95.0%).

Limmathurotsakul D, Jamsen K, Arayawichanont A, Simpson JA, White LJ, Lee SJ, Wuthiekanun V, Chantratita N et al. 2010. Defining the true sensitivity of culture for the diagnosis of melioidosis using Bayesian latent class models. PLoS One, 5 (8), pp. e12485. | Show Abstract | Read more

BACKGROUND: Culture remains the diagnostic gold standard for many bacterial infections, and the method against which other tests are often evaluated. Specificity of culture is 100% if the pathogenic organism is not found in healthy subjects, but the sensitivity of culture is more difficult to determine and may be low. Here, we apply Bayesian latent class models (LCMs) to data from patients with a single Gram-negative bacterial infection and define the true sensitivity of culture together with the impact of misclassification by culture on the reported accuracy of alternative diagnostic tests. METHODS/PRINCIPAL FINDINGS: Data from published studies describing the application of five diagnostic tests (culture and four serological tests) to a patient cohort with suspected melioidosis were re-analysed using several Bayesian LCMs. Sensitivities, specificities, and positive and negative predictive values (PPVs and NPVs) were calculated. Of 320 patients with suspected melioidosis, 119 (37%) had culture confirmed melioidosis. Using the final model (Bayesian LCM with conditional dependence between serological tests), the sensitivity of culture was estimated to be 60.2%. Prediction accuracy of the final model was assessed using a classification tool to grade patients according to the likelihood of melioidosis, which indicated that an estimated disease prevalence of 61.6% was credible. Estimates of sensitivities, specificities, PPVs and NPVs of four serological tests were significantly different from previously published values in which culture was used as the gold standard. CONCLUSIONS/SIGNIFICANCE: Culture has low sensitivity and low NPV for the diagnosis of melioidosis and is an imperfect gold standard against which to evaluate alternative tests. Models should be used to support the evaluation of diagnostic tests with an imperfect gold standard. It is likely that the poor sensitivity/specificity of culture is not specific for melioidosis, but rather a generic problem for many bacterial and fungal infections.

Limmathurotsakul D, Wuthiekanun V, Chantratita N, Wongsuvan G, Amornchai P, Day NP, Peacock SJ. 2010. Burkholderia pseudomallei is spatially distributed in soil in northeast Thailand. PLoS Negl Trop Dis, 4 (6), pp. e694. | Show Abstract | Read more

BACKGROUND: Melioidosis is a frequently fatal infectious disease caused by the soil dwelling Gram-negative bacterium Burkholderia pseudomallei. Environmental sampling is important to identify geographical distribution of the organism and related risk of infection to humans and livestock. The aim of this study was to evaluate spatial distribution of B. pseudomallei in soil and consider the implications of this for soil sampling strategies. METHODS AND FINDINGS: A fixed-interval sampling strategy was used as the basis for detection and quantitation by culture of B. pseudomallei in soil in two environmental sites (disused land covered with low-lying scrub and rice field) in northeast Thailand. Semivariogram and indicator semivariogram were used to evaluate the distribution of B. pseudomallei and its relationship with range between sampling points. B. pseudomallei was present on culture of 80/100 sampling points taken from the disused land and 28/100 sampling points from the rice field. The median B. pseudomallei cfu/gram from positive sampling points was 378 and 700 for the disused land and the rice field, respectively (p = 0.17). Spatial autocorrelation of B. pseudomallei was present, in that samples taken from areas adjacent to sampling points that were culture positive (negative) for B. pseudomallei were also likely to be culture positive (negative), and samples taken from areas adjacent to sampling points with a high (low) B. pseudomallei count were also likely to yield a high (low) count. Ranges of spatial autocorrelation in quantitative B. pseudomallei count were 11.4 meters in the disused land and 7.6 meters in the rice field. CONCLUSIONS: We discuss the implications of the uneven distribution of B. pseudomallei in soil for future environmental studies, and describe a range of established geostatistical sampling approaches that would be suitable for the study of B. pseudomallei that take account of our findings.

Limmathurotsakul D, Wongratanacheewin S, Teerawattanasook N, Wongsuvan G, Chaisuksant S, Chetchotisakd P, Chaowagul W, Day NP, Peacock SJ. 2010. Increasing incidence of human melioidosis in Northeast Thailand. Am J Trop Med Hyg, 82 (6), pp. 1113-1117. | Show Abstract | Read more

Melioidosis is a serious community-acquired infectious disease caused by the Gram-negative environmental bacterium Burkholderia pseudomallei. A prospective cohort study identified 2,243 patients admitted to Sappasithiprasong Hospital in northeast Thailand with culture-confirmed melioidosis between 1997 and 2006. These data were used to calculate an average incidence rate for the province of 12.7 cases of melioidosis per 100,000 people per year. Incidence increased incrementally from 8.0 (95% confidence interval [CI] = 7.2-10.0) in 2000 to 21.3 (95% CI = 19.2-23.6) in 2006 (P < 0.001; chi(2) test for trend). Male sex, age >/= 45 years, and either known or undiagnosed diabetes were independent risk factors for melioidosis. The average mortality rate from melioidosis over the study period was 42.6%. The minimum estimated population mortality rate from melioidosis in 2006 was 8.63 per 100,000 people (95% CI = 7.33-10.11), the third most common cause of death from infectious diseases in northeast Thailand after human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) and tuberculosis.

Limmathurotsakul D, Chaowagul W, Chantratita N, Wuthiekanun V, Biaklang M, Tumapa S, White NJ, Day NP, Peacock SJ. 2008. A simple scoring system to differentiate between relapse and re-infection in patients with recurrent melioidosis. PLoS Negl Trop Dis, 2 (10), pp. e327. | Show Abstract | Read more

BACKGROUND: Melioidosis is an important cause of morbidity and mortality in East Asia. Recurrent melioidosis occurs in around 10% of patients following treatment either because of relapse with the same strain or re-infection with a new strain of Burkholderia pseudomallei. Distinguishing between the two is important but requires bacterial genotyping. The aim of this study was to develop a simple scoring system to distinguish re-infection from relapse. METHODS: In a prospective study of 2,804 consecutive adult patients with melioidosis presenting to Sappasithiprasong Hospital, NE Thailand, between 1986 and 2005, there were 141 patients with recurrent melioidosis with paired strains available for genotyping. Of these, 92 patients had relapse and 49 patients had re-infection. Variables associated with relapse or re-infection were identified by multivariable logistic regression and used to develop a predictive model. Performance of the scoring system was quantified with respect to discrimination (area under receiver operating characteristic curves, AUC) and categorization (graphically). Bootstrap resampling was used to internally validate the predictors and adjust for over-optimism. FINDINGS: Duration of oral antimicrobial treatment, interval between the primary episode and recurrence, season, and renal function at recurrence were independent predictors of relapse or re-infection. A score of < 5 correctly identified relapse in 76 of 89 patients (85%), whereas a score > or = 5 correctly identified re-infection in 36 of 52 patients (69%). The scoring index had good discriminative power, with a bootstrap bias-corrected AUC of 0.80 (95%CI: 0.73-0.87). CONCLUSIONS: A simple scoring index to predict the cause of recurrent melioidosis has been developed to provide important bedside information where rapid bacterial genotyping is unavailable.

Burden of Antimicrobial Resistance in Southeast Asia

The burden of antimicrobial resistance (AMR) in Southeast Asian (SEA) countries is unclear. The Review on Antimicrobial Resistance compiled by Lord Jim O’Neill estimated that, currently, 700,000 deaths are attributable to AMR yearly worldwide. However, the review pointed out that the number was likely to be an underestimate due to lack of data from low- and middle-income countries (LMICs). Recently, we integrated readily available databases from nine public hospitals in Thailand, and confirmed ...

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