Dr Janjira Thaipadungpanit

Research Area: Microbiology
Scientific Themes: Tropical Medicine & Global Health and Genetics & Genomics
Keywords: Molecular epidemiology, Molecular diagnosis, acute febril illness, sepsis, leptospirosis, mellioidosis, rickettsiosis and scrub typhus
Web Links:

Janjira Thaipadungpani is a molecular microbiologist based at Mahidol-Oxford Tropical Medicine research Unit, Thailand. She is Head of Molecular Microbiology, the Mahidol-Oxford Tropical Medical Research Unit (MOMRU).

Her research interests include:

  • Molecular epidemiology of leptospirosis and melioidosis using multicolus sequence typing or genome data;
  • Molecular diagnosis for identification aetiology of acute febrile illness and sepsis patients.

Name Department Institution Country
Dr Direk Limmathurotsakul Tropical Medicine Oxford University, Bangkok Thailand
Professor Sharon Peacock London School of Hygiene and Tropical Medicine United Kingdom
Professor Matthew Holden St Andrews United Kingdom
Dr Claire Chewapreecha Wellcome Trust Sanger Institutes United Kingdom
Professor Stuart Blacksell Tropical Medicine Oxford University, Bangkok Thailand
Professor Yoel Lubell Tropical Medicine Oxford University, Bangkok Thailand
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.

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.

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.

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.

Ahmed A, Thaipadungpanit J, Boonsilp S, Wuthiekanun V, Nalam K, Spratt BG, Aanensen DM, Smythe LD, Ahmed N, Feil EJ et al. 2011. Comparison of two multilocus sequence based genotyping schemes for Leptospira species. PLoS Negl Trop Dis, 5 (11), pp. e1374. | Show Abstract

BACKGROUND: Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results. METHODS AND FINDINGS: A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5-96.5) vs. 93.5 (95% CI 88.6-98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L. CONCLUSIONS: This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.

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.

Thaipadungpanit J, Wuthiekanun V, Chierakul W, Smythe LD, Petkanchanapong W, Limpaiboon R, Apiwatanaporn A, Slack AT, Suputtamongkol Y, White NJ et al. 2007. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand. PLoS Negl Trop Dis, 1 (1), pp. e56. | Show Abstract

BACKGROUND: A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. METHODS AND FINDINGS: A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection. CONCLUSIONS: Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.

Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL et al. 2016. What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira. PLoS Negl Trop Dis, 10 (2), pp. e0004403. | Show Abstract

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

Chantratita N, Wikraiphat C, Tandhavanant S, Wongsuvan G, Ariyaprasert P, Suntornsut P, Thaipadungpanit J, Teerawattanasook N, Jutrakul Y, Srisurat N et al. 2016. Comparison of community-onset Staphylococcus argenteus and Staphylococcus aureus sepsis in Thailand: a prospective multicentre observational study. Clin. Microbiol. Infect., 22 (5), pp. 458.e11-9. | Show Abstract

Staphylococcus argenteus is a globally distributed cause of human infection, but diagnostic laboratories misidentify this as Staphylococcus aureus. We determined whether there is clinical utility in distinguishing between the two. A prospective cohort study of community-onset invasive staphylococcal sepsis was conducted in adults at four hospitals in northeast Thailand between 2010 and 2013. Of 311 patients analysed, 58 (19%) were infected with S. argenteus and 253 (81%) with S. aureus. Most S. argenteus (54/58) were multilocus sequence type 2250. Infection with S. argenteus was more common in males, but rates of bacteraemia and drainage procedures were similar in the two groups. S. argenteus precipitated significantly less respiratory failure than S. aureus (5.2% versus 20.2%, adjusted OR 0.21, 95% CI 0.06-0.74, p 0.015), with a similar but non-significant trend for shock (6.9% versus 12.3%, adjusted OR 0.46, 95% CI 0.15-1.44, p 0.18). This did not translate into a difference in death at 28 days (6.9% versus 8.7%, adjusted OR 0.80, 95% CI 0.24-2.65, p 0.72). S. argenteus was more susceptible to antimicrobial drugs compared with S. aureus, and contained fewer toxin genes although pvl was detected in 16% (9/58). We conclude that clinical differences exist in association with sepsis due to S. argenteus versus S. aureus.

Tong SY, Holden MT, Nickerson EK, Cooper BS, Köser CU, Cori A, Jombart T, Cauchemez S, Fraser C, Wuthiekanun V et al. 2015. Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting. Genome Res., 25 (1), pp. 111-8. | Show Abstract

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.

Stoddard RA, Bui D, Haberling DL, Wuthiekanun V, Thaipadungpanit J, Hoffmaster AR. 2014. Viability of Leptospira isolates from a human outbreak in Thailand in various water types, pH, and temperature conditions. Am. J. Trop. Med. Hyg., 91 (5), pp. 1020-2. | Show Abstract

Leptospira spp. isolated from patients during a multiyear outbreak in Thailand were genotyped using multilocus sequence typing and a majority were identified as ST34, especially in earlier years. We tested whether ST34 isolates were better adapted to survive in various pH levels, temperatures, and water sources. Motility and growth were monitored over a 12-week period. Early year ST34 isolates did not appear to have a significant fitness advantage over non-ST34, however, this may have been because a majority of the isolates survived to the termination of the study, with the exception being at high temperature (37°C) and/or basic pH (8.65). Failure to detect a significant fitness advantage of ST34 may be a result of the length of the study or the small sample size. Lengthening the study and looking at virulence and maintenance in the host could yield additional information about this outbreak.

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.

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. O854-6. | Show Abstract

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.

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. Emerging Infect. Dis., 20 (2), pp. 265-8. | Show Abstract

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.

Chetchotisakd P, Chierakul W, Chaowagul W, Anunnatsiri S, Phimda K, Mootsikapun P, Chaisuksant S, Pilaikul J, Thinkhamrop B, Phiphitaporn S 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. 807-14. | Show Abstract

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.

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.

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. 322-31. | Show Abstract

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.

Agampodi SB, Peacock SJ, Thevanesam V, Nugegoda DB, Smythe L, Thaipadungpanit J, Craig SB, Burns MA, Dohnt M, Boonsilp S et al. 2011. Leptospirosis outbreak in Sri Lanka in 2008: lessons for assessing the global burden of disease. Am. J. Trop. Med. Hyg., 85 (3), pp. 471-8. | Show Abstract

Global leptospirosis disease burden estimates are hampered by the lack of scientifically sound data from countries with probable high endemicity and limited diagnostic capacities. We describe the seroepidemiologic and clinical characteristics of the leptospirosis outbreak in 2008 in Sri Lanka. Definitive/presumptive case definitions proposed by the World Health Organization Leptospirosis Epidemiology Reference Group were used for case confirmation. Of the 404 possible cases, 155 were confirmed to have leptospirosis. Highest titers of patient seum samples reacted with serovars Pyrogenes (28.7%), Hardjo (18.8%), Javanica (11.5%), and Hebdomadis (11.5%). Sequencing of the 16S ribosomal DNA gene identified six infections: five with Leptospira interrogans and one with L. weilli. In this patient population, acute renal failure was the main complication (14.8%), followed by myocarditis (7.1%) and heart failure (3.9%). The case-fatality rate was 1.3%. This report strengthens the urgent need for increasing laboratory diagnostic capabilities to determine the causes of epidemic and endemic infectious diseases in Sri Lanka, a finding relevant to other tropical regions.

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.

Sim BM, Chantratita N, Ooi WF, Nandi T, Tewhey R, Wuthiekanun V, Thaipadungpanit J, Tumapa S, Ariyaratne P, Sung WK et al. 2010. Genomic acquisition of a capsular polysaccharide virulence cluster by non-pathogenic Burkholderia isolates. Genome Biol., 11 (8), pp. R89. | Show Abstract

BACKGROUND: Burkholderia thailandensis is a non-pathogenic environmental saprophyte closely related to Burkholderia pseudomallei, the causative agent of the often fatal animal and human disease melioidosis. To study B. thailandensis genomic variation, we profiled 50 isolates using a pan-genome microarray comprising genomic elements from 28 Burkholderia strains and species. RESULTS: Of 39 genomic regions variably present across the B. thailandensis strains, 13 regions corresponded to known genomic islands, while 26 regions were novel. Variant B. thailandensis isolates exhibited isolated acquisition of a capsular polysaccharide biosynthesis gene cluster (B. pseudomallei-like capsular polysaccharide) closely resembling a similar cluster in B. pseudomallei that is essential for virulence in mammals; presence of this cluster was confirmed by whole genome sequencing of a representative variant strain (B. thailandensis E555). Both whole-genome microarray and multi-locus sequence typing analysis revealed that the variant strains formed part of a phylogenetic subgroup distinct from the ancestral B. thailandensis population and were associated with atypical isolation sources when compared to the majority of previously described B. thailandensis strains. In functional assays, B. thailandensis E555 exhibited several B. pseudomallei-like phenotypes, including colony wrinkling, resistance to human complement binding, and intracellular macrophage survival. However, in murine infection assays, B. thailandensis E555 did not exhibit enhanced virulence relative to other B. thailandensis strains, suggesting that additional factors are required to successfully colonize and infect mammals. CONCLUSIONS: The discovery of such novel variant strains demonstrates how unbiased genomic surveys of non-pathogenic isolates can reveal insights into the development and emergence of new pathogenic species.

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.

Wangroongsarb P, Chanket T, Gunlabun K, Long DH, Satheanmethakul P, Jetanadee S, Thaipadungpanit J, Wuthiekanun V, Peacock SJ, Blacksell SD et al. 2007. Molecular typing of Leptospira spp. based on putative O-antigen polymerase gene (wzy), the benefit over 16S rRNA gene sequence. FEMS Microbiol. Lett., 271 (2), pp. 170-9. | Show Abstract

Molecular typing of leptospiral strains based on variation within putative O-antigen polymerase gene (wzy) was determined among reference strains and those isolated from patients. Using the PCR primers designed from the flanking gene of wzy derived from Leptospira interrogans serovar Copenhageni, all L. interrogans serovars as well as human and rodent leptospiral isolates from Thailand could be amplified. The size of PCR product ranged from 1 to 1.5 kb. The limitation of these primer pairs was the inability to amplify those strains whose sequences differ in the region of the primers, these included Leptospira biflexa (serovar Patoc), Leptospira borgpetersenii (serovar Tarassovi) and Leptospira kirschneri (serovar Bim, Bulgarica, Butembo). Notably, amplification was not limited to L. interrogans as demonstrated by the amplification of some strains from L. kirschneri, Leptospira meyeri, Leptospira noguchii, Leptospira santarosai, L. borgpetersenii and Leptospira weilii. The phylogenetic tree of wzy sequence, inferred by posterior probability of the Bayesian, enabled the categorization of leptospiral serovars into seven genetically related group, of which its differentiation power was better than that of the more highly conserved 16S rRNA gene, which is used extensively for genotyping.

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.

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.

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.

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.

Ahmed A, Thaipadungpanit J, Boonsilp S, Wuthiekanun V, Nalam K, Spratt BG, Aanensen DM, Smythe LD, Ahmed N, Feil EJ et al. 2011. Comparison of two multilocus sequence based genotyping schemes for Leptospira species. PLoS Negl Trop Dis, 5 (11), pp. e1374. | Show Abstract

BACKGROUND: Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results. METHODS AND FINDINGS: A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5-96.5) vs. 93.5 (95% CI 88.6-98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L. CONCLUSIONS: This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.

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.

Thaipadungpanit J, Wuthiekanun V, Chierakul W, Smythe LD, Petkanchanapong W, Limpaiboon R, Apiwatanaporn A, Slack AT, Suputtamongkol Y, White NJ et al. 2007. A dominant clone of Leptospira interrogans associated with an outbreak of human leptospirosis in Thailand. PLoS Negl Trop Dis, 1 (1), pp. e56. | Show Abstract

BACKGROUND: A sustained outbreak of leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. METHODS AND FINDINGS: A prospective study was conducted between 2000 and 2005 to identify patients with leptospirosis presenting to Udon Thani Hospital in northeast Thailand, and to isolate the causative organisms from blood. A multilocus sequence typing scheme was developed to genotype these pathogenic Leptospira. Additional typing was performed for Leptospira isolated from human cases in other Thai provinces over the same period, and from rodents captured in the northeast during 2004. Sequence types (STs) were compared with those of Leptospira drawn from a reference collection. Twelve STs were identified among 101 isolates from patients in Udon Thani. One of these (ST34) accounted for 77 (76%) of isolates. ST34 was Leptospira interrogans, serovar Autumnalis. 86% of human Leptospira isolates from Udon Thani corresponded to ST34 in 2000/2001, but this figure fell to 56% by 2005 as the outbreak waned (p = 0.01). ST34 represented 17/24 (71%) of human isolates from other Thai provinces, and 7/8 (88%) rodent isolates. By contrast, 59 STs were found among 76 reference strains, indicating a much more diverse population genetic structure; ST34 was not identified in this collection. CONCLUSIONS: Development of an MLST scheme for Leptospira interrogans revealed that a single ecologically successful pathogenic clone of L. interrogans predominated in the rodent population, and was associated with a sustained outbreak of human leptospirosis in Thailand.

Fouts DE, Matthias MA, Adhikarla H, Adler B, Amorim-Santos L, Berg DE, Bulach D, Buschiazzo A, Chang YF, Galloway RL et al. 2016. What Makes a Bacterial Species Pathogenic?:Comparative Genomic Analysis of the Genus Leptospira. PLoS Negl Trop Dis, 10 (2), pp. e0004403. | Show Abstract

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonotic disease. While whole genome analysis of individual pathogenic, intermediately pathogenic and saprophytic Leptospira species has been reported, comprehensive cross-species genomic comparison of all known species of infectious and non-infectious Leptospira, with the goal of identifying genes related to pathogenesis and mammalian host adaptation, remains a key gap in the field. Infectious Leptospira, comprised of pathogenic and intermediately pathogenic Leptospira, evolutionarily diverged from non-infectious, saprophytic Leptospira, as demonstrated by the following computational biology analyses: 1) the definitive taxonomy and evolutionary relatedness among all known Leptospira species; 2) genomically-predicted metabolic reconstructions that indicate novel adaptation of infectious Leptospira to mammals, including sialic acid biosynthesis, pathogen-specific porphyrin metabolism and the first-time demonstration of cobalamin (B12) autotrophy as a bacterial virulence factor; 3) CRISPR/Cas systems demonstrated only to be present in pathogenic Leptospira, suggesting a potential mechanism for this clade's refractoriness to gene targeting; 4) finding Leptospira pathogen-specific specialized protein secretion systems; 5) novel virulence-related genes/gene families such as the Virulence Modifying (VM) (PF07598 paralogs) proteins and pathogen-specific adhesins; 6) discovery of novel, pathogen-specific protein modification and secretion mechanisms including unique lipoprotein signal peptide motifs, Sec-independent twin arginine protein secretion motifs, and the absence of certain canonical signal recognition particle proteins from all Leptospira; and 7) and demonstration of infectious Leptospira-specific signal-responsive gene expression, motility and chemotaxis systems. By identifying large scale changes in infectious (pathogenic and intermediately pathogenic) vs. non-infectious Leptospira, this work provides new insights into the evolution of a genus of bacterial pathogens. This work will be a comprehensive roadmap for understanding leptospirosis pathogenesis. More generally, it provides new insights into mechanisms by which bacterial pathogens adapt to mammalian hosts.

Chantratita N, Wikraiphat C, Tandhavanant S, Wongsuvan G, Ariyaprasert P, Suntornsut P, Thaipadungpanit J, Teerawattanasook N, Jutrakul Y, Srisurat N et al. 2016. Comparison of community-onset Staphylococcus argenteus and Staphylococcus aureus sepsis in Thailand: a prospective multicentre observational study. Clin. Microbiol. Infect., 22 (5), pp. 458.e11-9. | Show Abstract

Staphylococcus argenteus is a globally distributed cause of human infection, but diagnostic laboratories misidentify this as Staphylococcus aureus. We determined whether there is clinical utility in distinguishing between the two. A prospective cohort study of community-onset invasive staphylococcal sepsis was conducted in adults at four hospitals in northeast Thailand between 2010 and 2013. Of 311 patients analysed, 58 (19%) were infected with S. argenteus and 253 (81%) with S. aureus. Most S. argenteus (54/58) were multilocus sequence type 2250. Infection with S. argenteus was more common in males, but rates of bacteraemia and drainage procedures were similar in the two groups. S. argenteus precipitated significantly less respiratory failure than S. aureus (5.2% versus 20.2%, adjusted OR 0.21, 95% CI 0.06-0.74, p 0.015), with a similar but non-significant trend for shock (6.9% versus 12.3%, adjusted OR 0.46, 95% CI 0.15-1.44, p 0.18). This did not translate into a difference in death at 28 days (6.9% versus 8.7%, adjusted OR 0.80, 95% CI 0.24-2.65, p 0.72). S. argenteus was more susceptible to antimicrobial drugs compared with S. aureus, and contained fewer toxin genes although pvl was detected in 16% (9/58). We conclude that clinical differences exist in association with sepsis due to S. argenteus versus S. aureus.

Tong SY, Holden MT, Nickerson EK, Cooper BS, Köser CU, Cori A, Jombart T, Cauchemez S, Fraser C, Wuthiekanun V et al. 2015. Genome sequencing defines phylogeny and spread of methicillin-resistant Staphylococcus aureus in a high transmission setting. Genome Res., 25 (1), pp. 111-8. | Show Abstract

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.

Stoddard RA, Bui D, Haberling DL, Wuthiekanun V, Thaipadungpanit J, Hoffmaster AR. 2014. Viability of Leptospira isolates from a human outbreak in Thailand in various water types, pH, and temperature conditions. Am. J. Trop. Med. Hyg., 91 (5), pp. 1020-2. | Show Abstract

Leptospira spp. isolated from patients during a multiyear outbreak in Thailand were genotyped using multilocus sequence typing and a majority were identified as ST34, especially in earlier years. We tested whether ST34 isolates were better adapted to survive in various pH levels, temperatures, and water sources. Motility and growth were monitored over a 12-week period. Early year ST34 isolates did not appear to have a significant fitness advantage over non-ST34, however, this may have been because a majority of the isolates survived to the termination of the study, with the exception being at high temperature (37°C) and/or basic pH (8.65). Failure to detect a significant fitness advantage of ST34 may be a result of the length of the study or the small sample size. Lengthening the study and looking at virulence and maintenance in the host could yield additional information about this outbreak.

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