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1.  Leptospira Species in Floodwater during the 2011 Floods in the Bangkok Metropolitan Region, Thailand 
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.
doi:10.4269/ajtmh.13-0124
PMCID: PMC3795115  PMID: 24002484
2.  Accuracy of Loop-Mediated Isothermal Amplification for Diagnosis of Human Leptospirosis in Thailand 
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.
doi:10.4269/ajtmh.2011.10-0473
PMCID: PMC3062458  PMID: 21460019
3.  A Dominant Clone of Leptospira interrogans Associated with an Outbreak of Human Leptospirosis in Thailand 
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.
Author Summary
A sustained outbreak of human leptospirosis occurred in northeast Thailand between 1999 and 2003, the basis for which was unknown. Leptospirosis is a potentially serious infection cause by bacteria known as Leptospira; infection usually occurs following environmental exposure to pathogenic Leptospira shed in the urine of an infected animal. The purpose of this study was to obtain bacterial isolates from humans with leptospirosis around the time of the Thai outbreak for genotyping, and to relate these to the maintenance host animal. To achieve this, a bacterial typing scheme (multilocus sequence typing, MLST) was developed for L. interrogans, the major cause of human disease. This approach has the advantage over existing typing schemes in that the data generated are amenable to detailed evolutionary analysis, and are readily comparable via the internet. Our results demonstrated the emergence of a dominant clone of L. interrogans serovar Autumnalis; this was the major cause of human disease during the outbreak, and was found in a maintenance host which was defined as the bandicoot rat.
doi:10.1371/journal.pntd.0000056
PMCID: PMC2041815  PMID: 17989782
4.  Development and Validation of a Real-Time PCR for Detection of Pathogenic Leptospira Species in Clinical Materials 
PLoS ONE  2009;4(9):e7093.
Available serological diagnostics do not allow the confirmation of clinically suspected leptospirosis at the early acute phase of illness. Several conventional and real-time PCRs for the early diagnosis of leptospirosis have been described but these have been incompletely evaluated. We developed a SYBR Green-based real-time PCR targeting secY and validated it according to international guidelines. To determine the analytical specificity, DNA from 56 Leptospira strains belonging to pathogenic, non-pathogenic and intermediate Leptospira spp. as well as 46 other micro-organisms was included in this study. All the pathogenic Leptospira gave a positive reaction. We found no cross-reaction with saprophytic Leptospira and other micro-organisms, implying a high analytical specificity. The analytical sensitivity of the PCR was one copy per reaction from cultured homologous strain M 20 and 1.2 and 1.5 copy for heterologous strains 1342 K and Sarmin, respectively. In spiked serum & blood and kidney tissue the sensitivity was 10 and 20 copies for M 20, 15 and 30 copies for 1342 K and 30 and 50 copies for Sarmin. To determine the diagnostic sensitivity (DSe) and specificity (DSp), clinical blood samples from 26 laboratory-confirmed and 107 negative patients suspected of leptospirosis were enrolled as a prospective consecutive cohort. Based on culture as the gold standard, we found a DSe and DSp of 100% and 93%, respectively. All eight PCR positive samples that had a negative culture seroconverted later on, implying a higher actual DSp. When using culture and serology as the gold standard, the DSe was lower (89%) while the DSp was higher (100%). DSe was 100% in samples collected within the first – for treatment important - 4 days after onset of the illness. Reproducibility and repeatability of the assay, determined by blind testing kidney samples from 20 confirmed positive and 20 negative rodents both appeared 100%. In conclusion we have described for the first time the development of a robust SYBR Green real-time PCR for the detection of pathogenic Leptospira combined with a detailed assessment of its clinical accuracy, thus providing a method for the early diagnosis of leptospirosis with a well-defined satisfactory performance.
doi:10.1371/journal.pone.0007093
PMCID: PMC2740861  PMID: 19763264
5.  Rapid Isolation and Susceptibility Testing of Leptospira spp. Using a New Solid Medium, LVW Agar 
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 CO2 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% CO2 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 MIC90 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.
doi:10.1128/AAC.01812-12
PMCID: PMC3535913  PMID: 23114772
6.  Fool's Gold: Why Imperfect Reference Tests Are Undermining the Evaluation of Novel Diagnostics: A Reevaluation of 5 Diagnostic Tests for Leptospirosis 
We hypothesized that the gold standard for diagnosing leptospirosis is imperfect. We used Bayesian latent class models and random-effects meta-analysis to test this hypothesis and to determine the true accuracy of a range of alternative tests for leptospirosis diagnosis.
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.
doi:10.1093/cid/cis403
PMCID: PMC3393707  PMID: 22523263
7.  Rapid Diagnosis of Leptospirosis by Multiplex PCR 
Background:
Traditionally, the most common diagnostic approach used for diagnosing leptospirosis was the demonstration of immune-seroconversion in acute and convalescent patient serum samples. Recently, a variety of molecular techniques, including conventional and real-time polymerase chain reaction (PCR), have been developed for the specific detection of pathogenic bacteria from the genus Leptospira. PCR is a sensitive, specific, and rapid technique that has been successfully used to detect several microorganisms; including those of clinical significance.
Methods:
In this study, we developed a multiplex PCR (mPCR) assay for detecting Leptospira’s DNA. The mPCR assay detects both the 16S rRNA gene and the major outer membrane lipoprotein gene, which is known as LipL32. Representative serovars were tested from 10 species of Leptospira and 23 other species of bacteria.
Results:
A positive result was obtained from all leptospiral serovars. The amplification sensitivity for the multiplex assay was 21.8 pg and 1 × 103 leptospires/ml. This mPCR assay has the potential to facilitate a rapid and sensitive diagnosis for acute leptospirosis.
Conclusion:
The mPCR assay developed in this study can be used for the early detection of leptospirosis. The LipL32 gene could also serve as another target to aid in the efficient detection of leptospiral infection because using 2 sets of primers in mPCR increases the sensitivity and specificity of the test.
PMCID: PMC3629659  PMID: 23610544
16S rRNA; leptospirosis; LipL32; mPCR; nucleic acid amplification test
8.  Development of Immunochromatography-Based Methods for Detection of Leptospiral Lipopolysaccharide Antigen in Urine 
Leptospirosis is an infectious disease caused by the spirochete bacteria Leptospira spp. and is commonly found throughout the world. Diagnosis of leptospirosis performed by culture and microscopic agglutination tests is laborious and time-consuming. Therefore, we aimed to develop a novel immunochromatography (ICG)-based method for detecting Leptospira antigen in the urine of patients and animals. We used the 1H6 monoclonal antibody (MAb), which is specific to the lipopolysaccharide (LPS) that is common among Leptospira spp. The MAb was coupled to 40-nm-diameter colloidal gold, and the amounts of labeled antibody and immobilized antibody were 23 μg and 2 μg per test, respectively. Several strains of Leptospira and other bacterial species were used to evaluate the sensitivities and specificities of the assays we developed. The detection limit of the assays was 106 cells/ml when disrupted whole bacterial cells were used. The assays were Leptospira specific since they did not cross-react with non-Leptospira bacteria used in the study. Application of diagnostic assays was done on the urine samples of 46 Leptospira-infected hamsters, 44 patients with suspected leptospirosis, and 14 healthy individuals. Pretreatment of the urine samples by boiling and centrifugation (for ultrafiltration and concentration) eliminated nonspecific reactions that occurred in the assay. The sensitivity and specificity of the ICG-based lateral flow assay (LFA) were 89% and 87%, respectively, which were higher than those of the dipstick assay, which were 80% and 74%, respectively. In summary, this ICG-based LFA can be used as an alternative diagnostic assay for leptospirosis. Further development is still necessary to improve the assay.
doi:10.1128/CVI.00756-12
PMCID: PMC3647744  PMID: 23467776
9.  Polymerase chain reaction for detection of Leptospira spp. in clinical samples. 
Journal of Clinical Microbiology  1992;30(9):2219-2224.
A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative.
Images
PMCID: PMC265482  PMID: 1400983
10.  Isolation and Characterization of New Leptospira Genotypes from Patients in Mayotte (Indian Ocean) 
Background
Leptospirosis has been implicated as a severe and fatal form of disease in Mayotte, a French-administrated territory located in the Comoros archipelago (southwestern Indian Ocean). To date, Leptospira isolates have never been isolated in this endemic region.
Methods and Findings
Leptospires were isolated from blood samples from 22 patients with febrile illness during a 17-month period after a PCR-based screening test was positive. Strains were typed using hyper-immune antisera raised against the major Leptospira serogroups: 20 of 22 clinical isolates were assigned to serogroup Mini; the other two strains belonged to serogroups Grippotyphosa and Pyrogenes, respectively. These isolates were further characterized using partial sequencing of 16S rRNA and ligB gene, Multi Locus VNTR Analysis (MLVA), and pulsed field gel electrophoresis (PFGE). Of the 22 isolates, 14 were L. borgpetersenii strains, 7 L. kirschneri strains, and 1, belonging to serogoup Pyrogenes, was L. interrogans. Results of the genotyping methods were consistent. MLVA defined five genotypes, whereas PFGE allowed the recognition of additional subgroups within the genotypes. PFGE fingerprint patterns of clinical strains did not match any of the patterns in the reference strains belonging to the same serogroup, suggesting that the strains were novel serovars.
Conclusions
Preliminary PCR screening of blood specimen allowed a high isolation frequency of leptospires among patients with febrile illness. Typing of leptospiral isolates showed that causative agents of leptospirosis in Mayotte have unique molecular features.
Author Summary
Leptospirosis has been recognized as an increasing public health problem affecting poor people from developing countries and tropical regions. However, the epidemiology of leptospirosis remains poorly understood in remote parts of the world. In this study of patients from the island of Mayotte, we isolated 22 strains from the blood of patients during the acute phase of illness. The pathogenic Leptospira strains were characterized by serology and various molecular typing methods. Based on serological data, serogroup Mini appears to be the dominant cause of leptospirosis in Mayotte. Further molecular characterization of these isolates allowed the identification of 10 pathogenic Leptospira genotypes that could correspond to previously unknown serovars. Further progress in our understanding of the epidemiology of Leptospira circulating genotypes in highly endemic regions should contribute to the development of novel strategies for the diagnosis and prevention of this neglected emerging disease.
doi:10.1371/journal.pntd.0000724
PMCID: PMC2889827  PMID: 20582311
11.  Cross-Species Surveillance of Leptospira in Domestic and Peri-Domestic Animals in Mahalla City, Gharbeya Governorate, Egypt 
A survey of 179 animals (black rats, dogs, sheep, buffaloes, cattle, donkeys, weasels, and cats) for Leptospira infection was conducted in Mahalla City (Lower Egypt). Blood, urine, and kidney were collected and tested by culture, microscopic agglutination test (MAT), and/or polymerase chain reaction (PCR). Among rats, 26% were positive by PCR, including 7% that were also positive by culture for L. interrogans serovars Grippotyphosa, Pyrogenes, and Icterohaemorrhagiae. L. borpetersenii serovar Polonica was isolated for the first time in Egypt in three rats. MAT titers ≥ 1:800 were observed in 11% of rats and 12% of dogs. L. interrogans serovar Grippotyphosa was detected in one cat. Sheep and donkeys were negative for leptospirosis by all methods. Buffaloes and cattle were seropositive in 20% and 44% of animals, respectively. Data indicate that several pathogenic serovars are circulating in the animals, which may pose exposure risks and account for high rates of acute febrile illness.
doi:10.4269/ajtmh.2011.10-0393
PMCID: PMC3042818  PMID: 21363980
12.  Accuracy of a Commercial IgM ELISA for the Diagnosis of Human Leptospirosis in Thailand 
The Leptospira immunoglobulin M enzyme-linked immunosorbent assay (IgM ELISA) has been recommended for the rapid diagnosis of leptospirosis in endemic areas. We conducted a retrospective case-control study of 218 patients (109 leptospirosis cases confirmed by Leptospira culture and/or microscopic agglutination test and 109 control patients with acute febrile illness) to evaluate the diagnostic accuracy of a commercial IgM ELISA (Panbio) in northeast Thailand. Paired serum samples taken on admission and at least 10 days after the onset of symptoms were tested. Using the cutoff value recommended by the manufacturer (11 Panbio units), sensitivity and specificity of IgM ELISA on paired sera were 90.8% and 55.1%. A receiver operating characteristic curve was used to determine the optimal cutoff value. This was 20 Panbio units, which gave a sensitivity and specificity of 76.1% and 82.6%, respectively, on paired sera. We conclude that using either cutoff value, the accuracy of IgM ELISA is limited in our setting.
doi:10.4269/ajtmh.2012.11-0423
PMCID: PMC3284374  PMID: 22403329
13.  Optimization of Culture of Leptospira from Humans with Leptospirosis▿  
Journal of Clinical Microbiology  2007;45(4):1363-1365.
A prospective study of 989 patients with acute febrile illness was performed in northeast Thailand to define the yield of Leptospira from four different types of blood sample. Based on a comparison of the yields from whole blood, surface plasma, deposit from spun plasma, and clotted blood samples from 80 patients with culture-proven leptospirosis, we suggest a sampling strategy in which culture is performed using whole blood and deposit from spun plasma.
doi:10.1128/JCM.02430-06
PMCID: PMC1865830  PMID: 17301285
14.  Evaluation of pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR  
Iranian Journal of Microbiology  2011;3(3):135-139.
Background and objectives
Leptospirosis is an important zoonotic disease caused by Leptospira interrogans. Leptospirosis leads to economical losses in dairy farm industry. The objective of this study was to evaluate the pathogenic serovars of Leptospira interrogans in dairy cattle herds of Shahrekord by PCR.
Materials and Methods
Two hundred samples (100 urine and 100 blood) were collected from 100 cows randomly and delivered to the laboratory. Samples were stored at -20 °C. DNA was extracted and purified from the plasma and urine samples and concentrated on diatoms in the presence of guanidine thiocyanate (GuSCN). PCR products were detected and identified as Leptospira by ilumination of the expected size of DNA bands after staining of the agarose gel with ethidium bromide gels. PCR products were purified and sequenced.
Results
The results showed that 28% of urine samples and 23% of plasma samples were contaminated. The major serotypes were Icterohaemorrhagiae (50%) and Pomona (37.5%). The urine samples of 17 cows were positive for Leptospira without positive plasma samples. This indicated that these cows are reservoirs in dairy herds of Shahrekord and dangerous for human health. The plasma samples of twelve cows were positive for Leptospira without positive urine samples.
Conclusions
Leptospira serotypes can be maintained in relatively dry regions and must be considered when dealing with leptospirosis in dairy farms of Shahrekord and human health.
PMCID: PMC3279815  PMID: 22347596
Leptospira; Shahrekord; Cattle; PCR
15.  Effects of Temperature on Gene Expression Patterns in Leptospira interrogans Serovar Lai as Assessed by Whole-Genome Microarrays†  
Infection and Immunity  2006;74(10):5848-5859.
Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20°C, 30°C, 37°C, and 39°C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37°C and 39°C) versus environmental temperatures (20°C and 30°C). Additionally, cultures grown at 30°C then shifted overnight to 37°C were compared with those grown long-term at 30°C and 37°C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.
doi:10.1128/IAI.00755-06
PMCID: PMC1594916  PMID: 16988264
16.  Detection of Leptospira spp. in the Aqueous Humor of Horses with Naturally Acquired Recurrent Uveitis 
Journal of Clinical Microbiology  2000;38(7):2731-2733.
Leptospiral organisms have long been presumed to be associated with the presence of equine recurrent uveitis. This project was undertaken to determine the presence of Leptospira spp. in the aqueous humor of horses with uveitis to determine if there was an association with inflammation. Thirty horses were determined to have recurrent uveitis based on clinical evaluation or history. Sixteen horses were judged clinically and historically to be free of uveitis and were used as controls. Aqueous humor samples were cultured and evaluated by PCR for the presence of Leptospira DNA. Serum was collected and evaluated for the presence of antibodies against five serovars in a leptospirosis panel. Twenty-one of 30 horses with recurrent uveitis and one of 16 uveitis-free horses were positive by PCR for the presence of Leptospira DNA. Six of these 21 horses with uveitis were culture positive for leptospires from the aqueous humor. Serologic results did not correlate well with the presence of Leptospira DNA or organisms in the aqueous humor. Leptospira spp. are present in a high percentage of horses with naturally occurring recurrent uveitis.
PMCID: PMC87011  PMID: 10878072
17.  Molecular Confirmation of Co-Infection by Pathogenic Leptospira spp. and Orientia tsutsugamushi in Patients with Acute Febrile Illness in Thailand 
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.
doi:10.4269/ajtmh.13-0402
PMCID: PMC3795116  PMID: 24002486
18.  Characterization of the Outer Membrane Proteome of Leptospira interrogans Expressed during Acute Lethal Infection▿  
Infection and Immunity  2006;75(2):766-773.
Pathogenic Leptospira species adapt to a wide range of environmental conditions during disease transmission and infection. While the proteome of in vitro cultivated Leptospira has been characterized in several studies to date, relatively little is known of the proteome as expressed by Leptospira during disease processes. Isolates of Leptospira obtained from patients suffering the severe pulmonary form of leptospirosis cause acute lethal infection in guinea pigs and chronic asymptomatic infection in rats. Recent studies have demonstrated that protein and lipopolysaccharide constituents of Leptospira recovered from acutely infected guinea pig tissue differ from that of Leptospira in chronically infected rat tissue and in vitro cultivated Leptospira (J. E. Nally, E. Chow, M. C. Fishbein, D. R. Blanco, and M. A. Lovett, Infect. Immun. 73:3251-3260, 2005). In the current study, the proteome of Leptospira expressed during disease processes was characterized relative to that of in vitro cultivated Leptospira (IVCL) after enrichment for hydrophobic membrane proteins with Triton X-114. Protein samples were separated by two-dimensional gel electrophoresis, and antigens expressed during infection were identified by immunoblotting with monospecific antiserum and convalescent rat serum in addition to mass spectrometry. Results suggest a significant increase in the expression of the outer membrane protein Loa22 during acute infection of guinea pigs relative to other outer membrane proteins, whose expression is generally diminished relative to expression in IVCL. Significant amounts of LipL32 are also expressed by Leptospira during acute infection of guinea pigs.
doi:10.1128/IAI.00741-06
PMCID: PMC1828474  PMID: 17101664
19.  A quantitative PCR (TaqMan) assay for pathogenic Leptospira spp 
Background
Leptospirosis is an emerging infectious disease. The differential diagnosis of leptospirosis is difficult due to the varied and often "flu like" symptoms which may result in a missed or delayed diagnosis. There are over 230 known serovars in the genus Leptospira. Confirmatory serological diagnosis of leptospirosis is usually made using the microscopic agglutination test (MAT) which relies on the use of live cultures as the source of antigen, often performed using a panel of antigens representative of local serovars. Other techniques, such as the enzyme linked immunosorbent assay (ELISA) and slide agglutination test (SAT), can detect different classes of antibody but may be subject to false positive reactions and require confirmation of these results by the MAT.
Methods
The polymerase chain reaction (PCR) has been used to detect a large number of microorganisms, including those of clinical significance. The sensitivity of PCR often precludes the need for isolation and culture, thus making it ideal for the rapid detection of organisms involved in acute infections. We employed real-time (quantitative) PCR using TaqMan chemistry to detect leptospires in clinical and environmental samples.
Results and Conclusions
The PCR assay can be applied to either blood or urine samples and does not rely on the isolation and culture of the organism. Capability exists for automation and high throughput testing in a clinical laboratory. It is specific for Leptospira and may discriminate pathogenic and non-pathogenic species. The limit of detection is as low as two cells.
doi:10.1186/1471-2334-2-13
PMCID: PMC117785  PMID: 12100734
Leptospirosis; TaqMan; real-time PCR; diagnosis
20.  Molecular Epidemiology of Leptospirosis in Northern Iran by Nested Polymerase Chain Reaction/Restriction Fragment Length Polymorphism and Sequencing Methods 
This study was conducted to investigate the prevalence of Leptospira species in Mazandaran Province of Iran by using nested polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) methods and sequencing analysis. Blood samples (n = 119) were collected from humans suspected of having leptospirosis from different parts of the province in 2007. By using an indirect immunofluorescent antibody test (IFAT), we determined that 35 (29.4%) of 119 suspected cases had leptospiral antibody titers ≥ 1:80, which confirmed the diagnosis of leptospirosis. Nested PCR assay also determined that 60 (50.4%) of 119 samples showed Leptospira infection. Furthermore, 44 (73.3%) of 60 confirmed leptospirosis amplified products were subjected to sequencing analysis. Sequence alignment identified L. interrogans, L. kirschneri, and L. wolffii species. All positive cases diagnosed by IFAT or PCR were in patients who reported contact with animals, high-risk occupational activities, and exposure to contaminated water. Therefore, it is important to increase attention about this disease among physicians and to strengthen laboratory capacity for its diagnosis in infected patients in Iran.
doi:10.4269/ajtmh.2010.09-0721
PMCID: PMC2861398  PMID: 20439973
21.  Diagnosis of Canine Leptospirosis by a Highly Sensitive FRET-PCR Targeting the lig Genes 
PLoS ONE  2014;9(2):e89507.
Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis.
doi:10.1371/journal.pone.0089507
PMCID: PMC3933566  PMID: 24586833
22.  Carriage of Leptospira interrogans among domestic rats from an urban setting highly endemic for leptospirosis in Brazil 
Acta tropica  2008;108(1):1-5.
A survey was conducted to identify reservoirs for urban leptospirosis in the city of Salvador, Brazil. Sampling protocols were performed in the vicinity of households of severe leptospirosis cases identified during active hospital-based surveillance. Among a total of 142 captured Rattus norvegicus (Norwegian brown rat), 80.3% had a positive culture isolate from urine or kidney specimens and 68.1% had a positive serum sample by microscopic agglutination test (MAT) titre of ≥1:100. Monoclonal antibody-based typing of isolates identified that the agent carried by rats was L. interrogans serovar Copenhageni, which was the same serovar isolated from patients during hospital-based surveillance. Leptospira spp. were not isolated from 8 captured Didelphis marsupialis (Opossum), while 5/7 had a positive MAT titre against a saprophytic serogroup. R. rattus were not captured during the survey. The study findings indicate that the brown rat is a major rodent reservoir for leptospirosis in this urban setting. Furthermore, the high carriage rates of L. interrogans serovar Copenhageni in captured rats suggest that there is a significant degree of environmental contamination with this agent in the household environment of high risk areas, which in turn is a cause of transmission during urban epidemics.
doi:10.1016/j.actatropica.2008.07.005
PMCID: PMC2596941  PMID: 18721789
Leptospira; Leptospirosis; Rats; Poverty Areas
23.  Clinical Spectrum of Pulmonary Involvement in Leptospirosis in a Region of Endemicity, with Quantification of Leptospiral Burden 
Background
Pulmonary involvement in leptospirosis remains poorly recognized in regions where it is endemic, despite reports of recent outbreaks and epidemic disease.
Methods
A prospective, population-based study was carried out to identify febrile patients exposed to Leptospira in urban and rural contexts in Iquitos, Peru. Evidence of exposure to Leptospira was obtained by serologic testing, and diagnosis of leptospirosis was confirmed in pulmonary cases by culture or quantitative real-time PCR assay.
Results
Of 633 consecutively enrolled febrile patients, 321 (50.7%) had antileptospiral IgM antibodies or high titers of antileptospiral antibodies. Seven patients with histories of only urban exposure to leptospires had severe pulmonary manifestations; of these, 5 patients died; 4 of the deaths were caused by pulmonary hemorrhage, and 1 was caused by acute respiratory distress syndrome and multiorgan failure. Real-time, quantitative PCR assay showed high levels of leptospiremia (≥104 leptospires/mL) in most fatal cases; 1 patient, from whom tissue specimens were obtained at autopsy, had ≥105 leptospires/g of lung, kidney, and muscle tissue.
Discussion
This study demonstrates the underdiagnosis of leptospirosis in a region of high endemicity and the underrecognition of grave pulmonary complications. Pulmonary involvement in leptospirosis was present in urban but not rural areas. Presumptive treatment for leptospirosis should be initiated immediately in the appropriate epidemiological and clinical context.
doi:10.1086/427110
PMCID: PMC2366057  PMID: 15668855
24.  LEPTO dipstick, a dipstick assay for detection of Leptospira-specific immunoglobulin M antibodies in human sera. 
We studied a dipstick assay for the detection of Leptospira-specific immunoglobulin M (IgM) antibodies in human serum samples. A high degree of concordance was observed between the results of the dipstick assay and an IgM enzyme-linked immunosorbent assay (ELISA). Application of the dipstick assay for the detection of acute leptospirosis enabled the accurate identification, early in the disease, of a high proportion of the cases of leptospirosis. Analysis of a second serum sample is recommended, in order to determine seroconversion or increased staining intensity. All serum samples from the patients who were confirmed to be positive for leptospirosis by either a positive microscopic agglutination test or a positive culture but were found to be negative by the dipstick assay were also judged to be negative by the IgM ELISA or revealed borderline titers by the IgM ELISA. Some cross-reactivity was observed for sera from patients with diseases other than leptospirosis, and this should be taken into account in the interpretation of test results. The dipstick assay is easy to perform, can be performed quickly, and requires no electricity or special equipment, and the assay components, a dipstick and a staining reagent, can be stored for a prolonged period without a loss of reactivity, even at elevated temperatures.
PMCID: PMC229517  PMID: 8968886
25.  Determining Risk for Severe Leptospirosis by Molecular Analysis of Environmental Surface Waters for Pathogenic Leptospira 
PLoS Medicine  2006;3(8):e308.
Background
Although previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters.
Methods and Findings
A quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (~103 leptospires/ml versus 0.5 × 102 leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources.
Conclusions
Our findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission. This combined quantitative and molecular taxonomical risk assessment of environmental surface waters is globally applicable for assessing risk for leptospiral infection and severe disease in leptospirosis-endemic regions.
Vinetz and colleagues used a quantitative real time PCR assay combined with molecular taxonomic analysis to quantify Leptospira in environmental surface waters in the Peruvian Amazon region of Iquitos.
Editors' Summary
Background.
Humans catch many diseases from animals—so-called zoonotic infections. Often, these occur in limited regions of the world. However, one—leptospirosis—occurs in temperate and tropical climates, and in urban and rural settings, making it the most widespread zoonotic disease. Leptospirosis is caused by Leptospira, a large group of closely related spiral-shaped bacteria that live in both domestic animals (for example, cattle) and wild animals (particularly rats). Millions of humans become infected each year with leptospires through close contact with water, food, or soil contaminated with the urine of infected animals—swimming or wading in contaminated water is particularly hazardous. Some infected people have no symptoms; others develop a flu-like disease that clears up within a few days. However, in 5%–10% of infected people, the disease progresses to a second, sometimes fatal phase. This is usually characterized by jaundice, kidney problems, and an enlarged spleen (it's then called Weil disease) but can also involve the lungs (pulmonary leptospirosis). Leptospirosis can be successfully treated with antibiotics if treatment is started soon after infection.
Why Was This Study Done?
In a recent study in the Peruvian Amazon, half of the people visiting urban hospitals and rural health posts with acute fever had antibodies in their blood to Leptospira, suggesting that they had acute leptospirosis. However, only patients living in urban areas developed pulmonary leptospirosis. In this study, the researchers tested the hypothesis that this pattern arose because more virulent types of Leptospira were present at higher levels in urban environmental surface water than in rural water sources.
What Did the Researchers Do and Find?
Between June 2003 and March 2004, the researchers isolated strains of Leptospira from patients with acute fever who visited a hospital in the town of Iquitos or clinics in nearby villages. Early in 2004, they also collected a large number of different water samples from an urban slum in Iquitos and from a nearby rural community. They measured the concentrations of Leptospira in these samples by using a molecular technique called real-time PCR (polymerase chain reaction) to detect and quantify a type of RNA found only in disease-causing Leptospira. They also identified which specific Leptospira were present in the water samples and the patient samples by sequencing this RNA. The researchers found that leptospires were present in both urban and rural water samples (particularly in samples from gutters and puddles in the urban slum's market area) but that their concentration in the positive water samples from the urban sites was 20 times that in the positive samples from the rural sites. Furthermore, the distribution of different Leptospira types isolated from the patients mirrored that of the bacteria in the local environment. So, one particular type of Leptospira interrogans known as icterohaemorrhagiae—the leptospire most commonly associated with severe leptospirosis in the patients—was found more often in the urban water samples than in the rural ones. Finally, the researchers discovered a new group of Leptospira in the rural environment. This group may contain one or several new species of Leptospira but whether any of them causes human disease is unknown.
What Do These Findings Mean?
These results support the researchers' hypothesis that pulmonary leptospirosis in urban areas of the Peruvian Amazon is associated with high environmental levels of specific disease-causing leptospires. The researchers were able to discover this link only by using molecular techniques—this sort of study is impossible with traditional bacteriological techniques because Leptospira are hard to grow in the laboratory and cannot be isolated efficiently from environmental water sources. Different types can't be identified using a microscope. The researchers' findings need to be validated in other settings, but they suggest that environmental interventions such as reducing sources of standing water and clearing away garbage in urban areas might reduce the number of cases of severe leptospirosis. The distribution of different Leptospira types also suggests that whereas rats may be the main disease reservoir in towns, cattle, pigs, and bats may be more important in rural settings in Peru and presumably elsewhere. Overall, this new information, together with the availability of molecular methods for rapid clinical diagnosis and environmental risk assessment, should aid attempts to control leptospirosis around the world.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030308.
US Centers for Disease Control and Prevention, information for patients and professionals on leptospirosis
The Leptospirosis Information Center, information and advice on human leptospirosis for the public and medical professionals
MedlinePlus encyclopedia entry on leptospirosis
NHS Direct Online, patient information on leptospirosis from the UK National Health Service online encyclopedia
Wikipedia pages on leptospirosis (note: Wikipedia is a free online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030308
PMCID: PMC1551915  PMID: 16933963

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