Premjit Amornchai

Research Area: Microbiology
Technology Exchange: Mass spectrometry
Scientific Themes: Immunology & Infectious Disease and Clinical Trials & Epidemiology
Keywords: Melioidosis, Leptospirosis, Lab diagnosis, Soil survey and epidemiology, BSL3, Safety
Web Links:
Work with B.pseudomallei under BSL3 facility

Work with B.pseudomallei under BSL3 facility

B.pseudomallei colony on day4

B.pseudomallei colony on day4

Demonstration of Leptospira on LVW agar for visitor

Demonstration of Leptospira on LVW agar for visitor

soil collection in the rice field

soil collection in the rice field

Now I'm being a BSL3 lab manager at MORU and my responsibility is to perform full scale management for BSL3 lab facility included with find out work space, equipment, provide induction safety training, solve problem and find out the solution to make our staff work safety and work happy in the lab.  My research interest is melioidosis and leptospirosis.

Name Department Institution Country
Dr Direk Limmathurotsakul Tropical Medicine Oxford University, Bangkok Thailand
Dr David AB Dance Tropical Medicine Oxford University, Vientiane Laos
Professor Sharon Peacock London School of Hygiene and Tropical Medicine United Kingdom
Professor Paul Turner Tropical Medicine Oxford University, Siem Reap Cambodia
Dr Vanaporn Wuthiekanun Tropical Medicine Oxford University, Bangkok Thailand
Dr Janjira Thaipadungpanit Tropical Medicine Oxford University, Bangkok Thailand
Dr Narisara Chantratita Tropical Medicine Oxford University, Bangkok Thailand
Professor Stuart Blacksell Tropical Medicine Oxford University, Bangkok Thailand
Wuthiekanun V, White NJ, Amornchai P, Day NP, Limmathurotsakul D. 2016. Quality controls for antimicrobial disk diffusion testing on Leptospira Vanaporn Wuthiekanun agar. Trans. R. Soc. Trop. Med. Hyg., 110 (11), pp. 673-675.

Rongkard P, Hantrakun V, Dittrich S, Srilohasin P, Amornchai P, Langla S, Lim C, Day NP, AuCoin D, Wuthiekanun V, Limmathurotsakul D. 2016. Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples. PLoS Negl Trop Dis, 10 (12), pp. e0005204. | Show Abstract

BACKGROUND: Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples. METHODOLOGY/PRINCIPAL FINDINGS: First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei. CONCLUSIONS/SIGNIFICANCE: The LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.

Hantrakun V, Rongkard P, Oyuchua M, Amornchai P, Lim C, Wuthiekanun V, Day NP, Peacock SJ, Limmathurotsakul D. 2016. Soil Nutrient Depletion Is Associated with the Presence of Burkholderia pseudomallei. Appl. Environ. Microbiol., 82 (24), pp. 7086-7092. | Show Abstract

Burkholderia pseudomallei is a soil-dwelling bacterium and the cause of melioidosis, which kills an estimated 89,000 people per year worldwide. Agricultural workers are at high risk of infection due to repeated exposure to the bacterium. Little is known about the soil physicochemical properties associated with the presence or absence of the organism. Here, we evaluated the soil physicochemical properties and presence of B. pseudomallei in 6,100 soil samples collected from 61 rice fields in Thailand. The presence of B. pseudomallei was negatively associated with the proportion of clay, proportion of moisture, level of salinity, percentage of organic matter, presence of cadmium, and nutrient levels (phosphorus, potassium, calcium, magnesium, and iron). The presence of B. pseudomallei was not associated with the level of soil acidity (P = 0.54). In a multivariable logistic regression model, the presence of B. pseudomallei was negatively associated with the percentage of organic matter (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.01 to 0.47; P = 0.007), level of salinity (OR, 0.06; 95% CI, 0.01 to 0.74; P = 0.03), and percentage of soil moisture (OR, 0.81; 95% CI, 0.66 to 1.00; P = 0.05). Our study suggests that B. pseudomallei thrives in rice fields that are nutrient depleted. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas. IMPORTANCE: Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. Humans acquire the disease following skin inoculation, inhalation, or ingestion of the bacterium in the environment. The presence of B. pseudomallei in soil defines geographic regions where humans and livestock are at risk of melioidosis, yet little is known about the soil properties associated with the presence of the organism. We evaluated the soil properties and presence of B. pseudomallei in 61 rice fields in East, Central, and Northeast Thailand. We demonstrated that the organism was more commonly found in soils with lower levels of organic matter and nutrients, including phosphorus, potassium, calcium, magnesium, and iron. We also demonstrated that crop residue burning after harvest, which can reduce soil nutrients, was not uncommon. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected 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. 241-3. | Show Abstract

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.

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. 557-9.

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. 1005-8. | Show Abstract

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.

Thaipadungpanit J, Chierakul W, Pattanaporkrattana W, Phoodaeng A, Wongsuvan G, Huntrakun V, Amornchai P, Chatchen S, Kitphati R, Wuthiekanun V et al. 2014. Burkholderia pseudomallei in water supplies, southern Thailand. Emerging Infect. Dis., 20 (11), pp. 1947-9.

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. 4350-2. | Show Abstract

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.

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. 3418-21. | Show Abstract

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, Day NP, Yingst S et al. 2014. Fatal melioidosis in goats in Bangkok, Thailand. Am. J. Trop. Med. Hyg., 91 (2), pp. 287-90. | Show Abstract

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.

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. 797-9. | Show Abstract

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.

Thaipadungpanit J, Wuthiekanun V, Chantratita N, Yimsamran S, Amornchai P, Boonsilp S, Maneeboonyang W, Tharnpoophasiam P, Saiprom N, Mahakunkijcharoen Y 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. 794-6. | Show Abstract

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.

Chheng K, Carter MJ, Emary K, Chanpheaktra N, Moore CE, Stoesser N, Putchhat H, Sona S, Reaksmey S, Kitsutani P et al. 2013. A prospective study of the causes of febrile illness requiring hospitalization in children in Cambodia. PLoS ONE, 8 (4), pp. e60634. | Show Abstract

BACKGROUND: Febrile illnesses are pre-eminent contributors to morbidity and mortality among children in South-East Asia but the causes are poorly understood. We determined the causes of fever in children hospitalised in Siem Reap province, Cambodia. METHODS AND FINDINGS: A one-year prospective study of febrile children admitted to Angkor Hospital for Children, Siem Reap. Demographic, clinical, laboratory and outcome data were comprehensively analysed. Between October 12(th) 2009 and October 12(th) 2010 there were 1225 episodes of febrile illness in 1180 children. Median (IQR) age was 2.0 (0.8-6.4) years, with 850 (69%) episodes in children <5 years. Common microbiological diagnoses were dengue virus (16.2%), scrub typhus (7.8%), and Japanese encephalitis virus (5.8%). 76 (6.3%) episodes had culture-proven bloodstream infection, including Salmonella enterica serovar Typhi (22 isolates, 1.8%), Streptococcus pneumoniae (13, 1.1%), Escherichia coli (8, 0.7%), Haemophilus influenzae (7, 0.6%), Staphylococcus aureus (6, 0.5%) and Burkholderia pseudomallei (6, 0.5%). There were 69 deaths (5.6%), including those due to clinically diagnosed pneumonia (19), dengue virus (5), and melioidosis (4). 10 of 69 (14.5%) deaths were associated with culture-proven bloodstream infection in logistic regression analyses (odds ratio for mortality 3.4, 95% CI 1.6-6.9). Antimicrobial resistance was prevalent, particularly in S. enterica Typhi, (where 90% of isolates were resistant to ciprofloxacin, and 86% were multi-drug resistant). Comorbid undernutrition was present in 44% of episodes and a major risk factor for acute mortality (OR 2.1, 95% CI 1.1-4.2), as were HIV infection and cardiac disease. CONCLUSION: We identified a microbiological cause of fever in almost 50% of episodes in this large study of community-acquired febrile illness in hospitalized children in Cambodia. The range of pathogens, antimicrobial susceptibility, and co-morbidities associated with mortality described will be of use in the development of rational guidelines for infectious disease treatment and control in Cambodia and South-East Asia.

Boonsilp S, Thaipadungpanit J, Amornchai P, Wuthiekanun V, Bailey MS, Holden MT, Zhang C, Jiang X, Koizumi N, Taylor K et al. 2013. A single multilocus sequence typing (MLST) scheme for seven pathogenic Leptospira species. PLoS Negl Trop Dis, 7 (1), pp. e1954. | Show Abstract

BACKGROUND: The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. METHODOLOGY AND FINDINGS: We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. CONCLUSION: The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis.

Emary K, Moore CE, Chanpheaktra N, An KP, Chheng K, Sona S, Duy PT, Nga TV, Wuthiekanun V, Amornchai P et al. 2012. Enteric fever in Cambodian children is dominated by multidrug-resistant H58 Salmonella enterica serovar Typhi with intermediate susceptibility to ciprofloxacin. Trans. R. Soc. Trop. Med. Hyg., 106 (12), pp. 718-24. | Show Abstract

Infections with Salmonella enterica serovar Typhi isolates that are multidrug resistant (MDR: resistant to chloramphenicol, ampicillin, trimethoprim-sulphamethoxazole) with intermediate ciprofloxacin susceptibility are widespread in Asia but there is little information from Cambodia. We studied invasive salmonellosis in children at a paediatric hospital in Siem Reap, Cambodia. Between 2007 and 2011 Salmonella was isolated from a blood culture in 162 children. There were 151 children with enteric fever, including 148 serovar Typhi and three serovar Paratyphi A infections, and 11 children with a non-typhoidal Salmonella infection. Of the 148 serovar Typhi isolates 126 (85%) were MDR and 133 (90%) had intermediate ciprofloxacin susceptibility. Inpatient antimicrobial treatment was ceftriaxone alone or initial ceftriaxone followed by a step-down to oral ciprofloxacin or azithromycin. Complications developed in 37/128 (29%) children admitted with enteric fever and two (1.6%) died. There was one confirmed relapse. In a sample of 102 serovar Typhi strains genotyped by investigation of a subset of single nucleotide polymorphisms, 98 (96%) were the H58 haplotype, the majority of which had the common serine to phenylalanine substitution at codon 83 in the DNA gyrase. We conclude that antimicrobial-resistant enteric fever is common in Cambodian children and therapeutic options are limited.

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

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.

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 pp. 338. | Show Abstract

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.

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. 876-7. | Show Abstract

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, Rholl DA, Sim B, Wuthiekanun V, Limmathurotsakul D, Amornchai P, Thanwisai A, Chua HH, Ooi WF, Holden MT 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. 17165-70. | Show Abstract

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.

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. 5388-91. | Show Abstract

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.

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. 858-61. | Show Abstract

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.

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. 614-20. | Show Abstract

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.

Nickerson EK, Wuthiekanun V, Kumar V, Amornchai P, Wongdeethai N, Chheng K, Chantratita N, Putchhat H, Thaipadungpanit J, Day NP, Peacock SJ. 2011. Emergence of community-associated methicillin-resistant Staphylococcus aureus carriage in children in Cambodia. Am. J. Trop. Med. Hyg., 84 (2), pp. 313-7. | Show Abstract

We previously described the first reported isolation of methicillin-resistant Staphylococcus aureus (MRSA) (a case series of pediatric community-associated MRSA infections) in Cambodia. We define the rate of pediatric MRSA carriage in the same population and characterize the associated bacterial genotypes by using pulsed-field gel electrophoresis and multilocus sequence typing. A prospective cohort study of MRSA carriage conducted over one month at the Angkor Hospital for Children, Siem Reap, Cambodia, identified MRSA carriage in 87 (3.5%) of 2,485 children who came to the outpatient department, and 6 (4.1%) of 145 inpatients, including at least two with cases of nosocomial acquisition. Genotyping of all 93 MRSA isolates resolved 5 genotypes. Most (91%) isolates were assigned to sequence type 834. Only 28 (32%) of 87 MRSA carriers identified in the outpatient department had no history of recent healthcare contact. The study findings have important implications for healthcare in a setting where diagnostic microbiology and access to antimicrobial drugs with efficacy against MRSA are limited.

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

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.

Rattanavong S, Wuthiekanun V, Langla S, Amornchai P, Sirisouk J, Phetsouvanh R, Moore CE, Peacock SJ, Buisson Y, Newton PN. 2011. Randomized soil survey of the distribution of Burkholderia pseudomallei in rice fields in Laos. Appl. Environ. Microbiol., 77 (2), pp. 532-6. | Show Abstract

Melioidosis is a major cause of morbidity and mortality in Southeast Asia, where the causative organism (Burkholderia pseudomallei) is present in the soil. In the Lao People's Democratic Republic (Laos), B. pseudomallei is a significant cause of sepsis around the capital, Vientiane, and has been isolated in soil near the city, adjacent to the Mekong River. We explored whether B. pseudomallei occurs in Lao soil distant from the Mekong River, drawing three axes across northwest, northeast, and southern Laos to create nine sampling areas in six provinces. Within each sampling area, a random rice field site containing a grid of 100 sampling points each 5 m apart was selected. Soil was obtained from a depth of 30 cm and cultured for B. pseudomallei. Four of nine sites (44%) were positive for B. pseudomallei, including all three sites in Saravane Province, southern Laos. The highest isolation frequency was in east Saravane, where 94% of soil samples were B. pseudomallei positive with a geometric mean concentration of 464 CFU/g soil (95% confidence interval, 372 to 579 CFU/g soil; range, 25 to 10,850 CFU/g soil). At one site in northwest Laos (Luangnamtha), only one sample (1%) was positive for B. pseudomallei, at a concentration of 80 CFU/g soil. Therefore, B. pseudomallei occurs in Lao soils beyond the immediate vicinity of the Mekong River, alerting physicians to the likelihood of melioidosis in these areas. Further studies are needed to investigate potential climatic, soil, and biological determinants of this heterogeneity.

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

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.

Pagnarith Y, Kumar V, Thaipadungpanit J, Wuthiekanun V, Amornchai P, Sin L, Day NP, Peacock SJ. 2010. Emergence of pediatric melioidosis in Siem Reap, Cambodia. Am. J. Trop. Med. Hyg., 82 (6), pp. 1106-12. | Show Abstract

We describe the first cases of pediatric melioidosis in Cambodia. Thirty-nine cases were diagnosed at the Angkor Hospital for Children, Siem Reap, between October 2005 and December 2008 after the introduction of microbiology capabilities. Median age was 7.8 years (range = 1.6-16.2 years), 15 cases were male (38%), and 4 cases had pre-existing conditions that may have pre-disposed the patient to melioidosis. Infection was localized in 27 cases (69%) and disseminated in 12 cases (31%). Eleven cases (28%) were treated as outpatients, and 28 (72%) cases were admitted. Eight children (21%) died a median of 2 days after admission; seven deaths were attributable to melioidosis, all of which occurred in children receiving suboptimal antimicrobial therapy and before bacteriological culture results were available. Our findings indicate the need for heightened awareness of melioidosis in Cambodia, and they have led us to review microbiology procedures and antimicrobial prescribing of suspected and confirmed cases.

Chheng K, Tarquinio S, Wuthiekanun V, Sin L, Thaipadungpanit J, Amornchai P, Chanpheaktra N, Tumapa S, Putchhat H, Day NP, Peacock SJ. 2009. Emergence of community-associated methicillin-resistant Staphylococcus aureus associated with pediatric infection in Cambodia. PLoS ONE, 4 (8), pp. e6630. | Show Abstract

BACKGROUND: The incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infection is rising in the developed world but appears to be rare in developing countries. One explanation for this difference is that resource poor countries lack the diagnostic microbiology facilities necessary to detect the presence of CA-MRSA carriage and infection. METHODOLOGY AND PRINCIPAL FINDINGS: We developed diagnostic microbiology capabilities at the Angkor Hospital for Children, Siem Reap, western Cambodia in January 2006 and in the same month identified a child with severe community-acquired impetigo caused by CA-MRSA. A study was undertaken to identify and describe additional cases presenting between January 2006 and December 2007. Bacterial isolates underwent molecular characterization using multilocus sequence typing, staphylococcal cassette chromosome mec (SCCmec) typing, and PCR for the presence of the genes encoding Panton-Valentine Leukocidin (PVL). Seventeen children were identified with CA-MRSA infection, of which 11 had skin and soft tissue infection and 6 had invasive disease. The majority of cases were unrelated in time or place. Molecular characterization identified two independent MRSA clones; fifteen isolates were sequence type (ST) 834, SCCmec type IV, PVL gene-negative, and two isolates were ST 121, SCCmec type V, PVL gene-positive. CONCLUSIONS: This represents the first ever report of MRSA in Cambodia, spread of which would pose a significant threat to public health. The finding that cases were mostly unrelated in time or place suggests that these were sporadic infections in persons who were CA-MRSA carriers or contacts of carriers, rather than arising in the context of an outbreak.

Nickerson EK, Wuthiekanun V, Wongsuvan G, Limmathurosakul D, Srisamang P, Mahavanakul W, Thaipadungpanit J, Shah KR, Arayawichanont A, Amornchai P et al. 2009. Factors predicting and reducing mortality in patients with invasive Staphylococcus aureus disease in a developing country. PLoS ONE, 4 (8), pp. e6512. | Show Abstract

BACKGROUND: Invasive Staphylococcus aureus infection is increasingly recognised as an important cause of serious sepsis across the developing world, with mortality rates higher than those in the developed world. The factors determining mortality in developing countries have not been identified. METHODS: A prospective, observational study of invasive S. aureus disease was conducted at a provincial hospital in northeast Thailand over a 1-year period. All-cause and S. aureus-attributable mortality rates were determined, and the relationship was assessed between death and patient characteristics, clinical presentations, antibiotic therapy and resistance, drainage of pus and carriage of genes encoding Panton-Valentine Leukocidin (PVL). PRINCIPAL FINDINGS: A total of 270 patients with invasive S. aureus infection were recruited. The range of clinical manifestations was broad and comparable to that described in developed countries. All-cause and S. aureus-attributable mortality rates were 26% and 20%, respectively. Early antibiotic therapy and drainage of pus were associated with a survival advantage (both p<0.001) on univariate analysis. Patients infected by a PVL gene-positive isolate (122/248 tested, 49%) had a strong survival advantage compared with patients infected by a PVL gene-negative isolate (all-cause mortality 11% versus 39% respectively, p<0.001). Multiple logistic regression analysis using all variables significant on univariate analysis revealed that age, underlying cardiac disease and respiratory infection were risk factors for all-cause and S. aureus-attributable mortality, while one or more abscesses as the presenting clinical feature and procedures for infectious source control were associated with survival. CONCLUSIONS: Drainage of pus and timely antibiotic therapy are key to the successful management of S. aureus infection in the developing world. Defining the presence of genes encoding PVL provides no practical bedside information and draws attention away from identifying verified clinical risk factors and those interventions that save lives.

Cheng AC, McBryde ES, Wuthiekanun V, Chierakul W, Amornchai P, Day NP, White NJ, Peacock SJ. 2009. Dosing regimens of cotrimoxazole (trimethoprim-sulfamethoxazole) for melioidosis. Antimicrob. Agents Chemother., 53 (10), pp. 4193-9. | Show Abstract

Melioidosis is an infectious disease with a propensity for relapse, despite prolonged antibiotic eradication therapy for 12 to 20 weeks. A pharmacokinetic (PK) simulation study was performed to determine the optimal dosing of cotrimoxazole (trimethoprim-sulfamethoxazole [TMP-SMX]) used in current eradication regimens in Thailand and Australia. Data for bioavailability, protein binding, and coefficients of absorption and elimination were taken from published literature. Apparent volumes of distribution were correlated with body mass and were estimated separately for Thai and Australian populations. In vitro experiments demonstrated concentration-dependent killing. In Australia, the currently used eradication regimen (320 [TMP]/1,600 [SMX] mg every 12 h [q12h]) was predicted to achieve the PK-pharmacodynamic (PD) target (an area under the concentration-time curve from 0 to 24 h/MIC ratio of >25 for both TMP and SMX) for strains with the MIC90 of Australian strains (< or = 1/19 mg/liter). In Thailand, the former regimen of 160/800 mg q12h would not be expected to attain the target for strains with an MIC of > or = 1/19 mg/liter, but the recently implemented weight-based regimen (<40 kg [body weight], 160/800 mg q12h; 40 to 60 kg, 240/1,200 mg q12h; >60 kg, 320/1,600 mg q12h) would be expected to achieve adequate concentrations for strains with an MIC of < or = 1/19 mg/liter. The results were sensitive to the variance of the PK parameters. Prospective PK-PD studies of Asian populations are needed to optimize TMP-SMX dosing in melioidosis.

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

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.

Amornchai P, Chierakul W, Wuthiekanun V, Mahakhunkijcharoen Y, Phetsouvanh R, Currie BJ, Newton PN, van Vinh Chau N, Wongratanacheewin S, Day NP, Peacock SJ. 2007. Accuracy of Burkholderia pseudomallei identification using the API 20NE system and a latex agglutination test. J. Clin. Microbiol., 45 (11), pp. 3774-6. | Show Abstract

In an evaluation of the API 20NE for the identification of Burkholderia spp., 792/800 (99%) Burkholderia pseudomallei and 17/19 (89%) B. cepacia isolates were correctly identified but 10 B. mallei and 98 B. thailandensis isolates were not correctly identified. A latex agglutination test was positive for 796/800 (99.5%) B. pseudomallei isolates and negative for 120 other oxidase-positive gram-negative bacilli.

Chierakul W, Wuthiekanun V, Chaowagul W, Amornchai P, Cheng AC, White NJ, Day NP, Peacock SJ. 2005. Short report: disease severity and outcome of melioidosis in HIV coinfected individuals. Am. J. Trop. Med. Hyg., 73 (6), pp. 1165-6. | Show Abstract

This study examined whether coinfection with HIV and Burkholderia pseudomallei leads to altered disease severity or outcome associated with melioidosis. Coinfection was detected in only 8 of 524 (1.5%) adults with melioidosis in northeast Thailand. Clinical presentation and acute outcome were similar in HIV-positive and HIV-negative patients.

Peacock SJ, Chieng G, Cheng AC, Dance DA, Amornchai P, Wongsuvan G, Teerawattanasook N, Chierakul W, Day NP, Wuthiekanun V. 2005. Comparison of Ashdown's medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomallei. J. Clin. Microbiol., 43 (10), pp. 5359-61. | Show Abstract

Ashdown's medium, Burkholderia pseudomallei selective agar (BPSA), and a commercial Burkholderia cepacia medium were compared for their abilities to grow B. pseudomallei from 155 clinical specimens that proved positive for this organism. The sensitivity of each was equivalent; the selectivity of BPSA was lower than that of Ashdown's or B. cepacia medium.

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. 64-7. | Show Abstract

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.

Wuthiekanun V, Cheng AC, Chierakul W, Amornchai P, Limmathurotsakul D, Chaowagul W, Simpson AJ, Short JM, Wongsuvan G, Maharjan B et al. 2005. Trimethoprim/sulfamethoxazole resistance in clinical isolates of Burkholderia pseudomallei. J. Antimicrob. Chemother., 55 (6), pp. 1029-31. | Show Abstract

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.

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. 555-6. | Show Abstract

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.

Wuthiekanun V, Amornchai P, Chierakul W, Cheng AC, White NJ, Peacock SJ, Day NP. 2004. Evaluation of immunoglobulin M (IgM) and IgG rapid cassette test kits for diagnosis of melioidosis in an area of endemicity. J. Clin. Microbiol., 42 (8), pp. 3435-7. | Show Abstract

An enzyme-linked immunosorbent assay-based rapid cassette immunoglobulin G (IgG) and IgM immunochromogenic test kit was compared to the indirect hemagglutination test (IHA) for the diagnosis of acute melioidosis in northeastern Thailand. Admission sera from 70 culture-confirmed septicemic melioidosis patients and 30 patients with localized infections were tested. As a control group, 80 patients with other acute febrile illnesses (other bacterial infections, leptospirosis, or scrub typhus) and 119 healthy individuals were tested. The diagnostic sensitivity of the IgG and IgM tests and the IHA test were 79, 67, and 72%, respectively, with corresponding specificities of 90, 80, and 68%. This kit represents an improvement over IHA for the diagnosis of melioidosis an area of endemicity although, as with other serological tests, it has reduced diagnostic utility in a population with high background seropositivity.

Wuthiekanun V, White NJ, Amornchai P, Day NP, Limmathurotsakul D. 2016. Quality controls for antimicrobial disk diffusion testing on Leptospira Vanaporn Wuthiekanun agar. Trans. R. Soc. Trop. Med. Hyg., 110 (11), pp. 673-675.

Rongkard P, Hantrakun V, Dittrich S, Srilohasin P, Amornchai P, Langla S, Lim C, Day NP, AuCoin D, Wuthiekanun V, Limmathurotsakul D. 2016. Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples. PLoS Negl Trop Dis, 10 (12), pp. e0005204. | Show Abstract

BACKGROUND: Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples. METHODOLOGY/PRINCIPAL FINDINGS: First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei. CONCLUSIONS/SIGNIFICANCE: The LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.

Hantrakun V, Rongkard P, Oyuchua M, Amornchai P, Lim C, Wuthiekanun V, Day NP, Peacock SJ, Limmathurotsakul D. 2016. Soil Nutrient Depletion Is Associated with the Presence of Burkholderia pseudomallei. Appl. Environ. Microbiol., 82 (24), pp. 7086-7092. | Show Abstract

Burkholderia pseudomallei is a soil-dwelling bacterium and the cause of melioidosis, which kills an estimated 89,000 people per year worldwide. Agricultural workers are at high risk of infection due to repeated exposure to the bacterium. Little is known about the soil physicochemical properties associated with the presence or absence of the organism. Here, we evaluated the soil physicochemical properties and presence of B. pseudomallei in 6,100 soil samples collected from 61 rice fields in Thailand. The presence of B. pseudomallei was negatively associated with the proportion of clay, proportion of moisture, level of salinity, percentage of organic matter, presence of cadmium, and nutrient levels (phosphorus, potassium, calcium, magnesium, and iron). The presence of B. pseudomallei was not associated with the level of soil acidity (P = 0.54). In a multivariable logistic regression model, the presence of B. pseudomallei was negatively associated with the percentage of organic matter (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.01 to 0.47; P = 0.007), level of salinity (OR, 0.06; 95% CI, 0.01 to 0.74; P = 0.03), and percentage of soil moisture (OR, 0.81; 95% CI, 0.66 to 1.00; P = 0.05). Our study suggests that B. pseudomallei thrives in rice fields that are nutrient depleted. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas. IMPORTANCE: Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. Humans acquire the disease following skin inoculation, inhalation, or ingestion of the bacterium in the environment. The presence of B. pseudomallei in soil defines geographic regions where humans and livestock are at risk of melioidosis, yet little is known about the soil properties associated with the presence of the organism. We evaluated the soil properties and presence of B. pseudomallei in 61 rice fields in East, Central, and Northeast Thailand. We demonstrated that the organism was more commonly found in soils with lower levels of organic matter and nutrients, including phosphorus, potassium, calcium, magnesium, and iron. We also demonstrated that crop residue burning after harvest, which can reduce soil nutrients, was not uncommon. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected 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. 241-3. | Show Abstract

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.

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. 557-9.

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. 1005-8. | Show Abstract

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.

Thaipadungpanit J, Chierakul W, Pattanaporkrattana W, Phoodaeng A, Wongsuvan G, Huntrakun V, Amornchai P, Chatchen S, Kitphati R, Wuthiekanun V et al. 2014. Burkholderia pseudomallei in water supplies, southern Thailand. Emerging Infect. Dis., 20 (11), pp. 1947-9.

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. 4350-2. | Show Abstract

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.

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. 3418-21. | Show Abstract

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, Day NP, Yingst S et al. 2014. Fatal melioidosis in goats in Bangkok, Thailand. Am. J. Trop. Med. Hyg., 91 (2), pp. 287-90. | Show Abstract

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.

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. 797-9. | Show Abstract

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.

Thaipadungpanit J, Wuthiekanun V, Chantratita N, Yimsamran S, Amornchai P, Boonsilp S, Maneeboonyang W, Tharnpoophasiam P, Saiprom N, Mahakunkijcharoen Y 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. 794-6. | Show Abstract

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.

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