A remotely sensed flooding indicator associated with cattle and buffalo leptospirosis cases in Thailand 2011-2013.

BACKGROUND: Leptospirosis is an important zoonotic disease worldwide, caused by spirochetes bacteria of the genus Leptospira. In Thailand, cattle and buffalo used in agriculture are in close contact with human beings. During flooding, bacteria can quickly spread throughout an environment, increasing the risk of leptospirosis infection. The aim of this study was to investigate the association of several environmental factors with cattle and buffalo leptospirosis cases in Thailand, with a focus on flooding. METHOD: A total of 3571 urine samples were collected from cattle and buffalo in 107 districts by field veterinarians from January 2011 to February 2013. All samples were examined for the presence of leptospirosis infection by loop-mediated isothermal amplification (LAMP). Environmental data, including rainfall, percentage of flooded area (estimated by remote sensing), average elevation, and human and livestock population density were used to build a generalized linear mixed model. RESULTS: A total of 311 out of 3571 (8.43%) urine samples tested positive by the LAMP technique. Positive samples were recorded in 51 out of 107 districts (47.66%). Results showed a significant association between the percentage of the area flooded at district level and leptospirosis infection in cattle and buffalo (p = 0.023). Using this data, a map with a predicted risk of leptospirosis can be developed to help forecast leptospirosis cases in the field. CONCLUSIONS: Our model allows the identification of areas and periods when the risk of leptospirosis infection is higher in cattle and buffalo, mainly due to a seasonal flooding. The increased risk of leptospirosis infection can also be higher in humans too. These areas and periods should be targeted for leptospirosis surveillance and control in both humans and animals.

Development of an immunochromatographic test with anti-LipL32-coupled gold nanoparticles for Leptospira detection.

Detection of antibody specific to Leptospira by various immunological techniques has been used for leptospirosis diagnosis. However, the sensitivity of antibody detection during the first few days after infection is low. Molecular techniques are suggested to provide earlier diagnosis than antibody detection, but a rapid and easy to perform assay for Leptospira antigen detection would provide an additional useful tool for disease diagnosis. In this study, we coupled gold nanoparticles with antibody to LipL32, a protein commonly found in pathogenic Leptospira. This coupled gold reagent was used in the immunochromatographic strip for Leptospira detection. We demonstrated that the sensitivity of Leptospira detection by this strip was 10(3) ml(-1). There was no positive result detected when strips were tested with non-pathogenic Leptospira, Staphylococcus aureus, Streptococcus group B, Acinetobacter baumannii, Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Enterococcus faecalis or Enterococcus faecium. These data suggest that gold nanoparticles coupled with antibody to LipL32 could be used for Leptospira detection by a rapid test based on an immunochromatographic technique.

Evaluation of the Panbio Leptospira IgM ELISA among Outpatients Attending Primary Care in Southeast Asia.

Despite estimates suggesting Leptospira spp. being endemic in Southeast Asia, evidence remains limited. Diagnostic accuracy evaluations based on Leptospira ELISA mainly rely on hospitalized and severe patients; therefore, studies measuring the pathogen burden may be inaccurate in the community. We evaluated the Panbio Leptospira ELISA IgM among 656 febrile outpatients attending primary care in Chiangrai, Thailand, and Hlaing Tha Yar, Yangon, Myanmar. ELISA demonstrated limited diagnostic accuracy for the detection of acute leptospiral infection using the manufacturer recommended cutoff, with a sensitivity of 71.4% and specificity of 36.4%, and an area under the receiver operator characteristic curve value of 0.65 (95% CI: 0.41-0.89), compared with our reference test, the PCR assay. ELISA also performed poorly as a screening tool for detecting recent exposure to Leptospira spp. compared with the "gold-standard" microscopic agglutination test, with a specificity of 42.7%. We conclude that the utility of the Leptospira IgM ELISA for both serodiagnosis and seroprevalence is limited in our setting.

Environmental and Behavioral Risk Factors for Severe Leptospirosis in Thailand.

A nationwide prevention and control campaign for leptospirosis in Thailand has led to a decreased incidence rate, but the mortality and case fatality rates have remained stable. Regarding the limited knowledge of risk factors, a case-control study of the association between environmental and behavioral exposure with severe leptospirosis was implemented to identify the risk factors among adults in Thailand. The study was conducted in 12 hospital-based sites. Hospitalized patients with suspected clinical symptoms of leptospirosis were tested for leptospirosis by culture, loop mediated isothermal amplification (LAMP), real-time PCR, and the microscopic agglutination test (MAT). All participants answered a standardized questionnaire about potential risk factors. Risk factors were identified by univariable and multivariable logistic regression. Of the 44 confirmed cases, 33 (75.0%) presented with severe illness, as determined by clinical criteria, and were categorized as severe cases. Non-severe cases were defined as patients with non-severe symptoms of leptospirosis. Living nearby a rubber tree plantation (adjusted OR 11.65, 95% CI 1.08-125.53) and bathing in natural bodies of water (adjusted OR 10.45, 95% CI 1.17-93.35) were both significantly associated with an increased risk of severe leptospirosis. We recommend designating rubber plantations in Thailand as high-risk zones and closely monitoring hospitalized patients in those areas.

Comparison of a slide agglutination test, LeptoTek Dri-Dot, and IgM-ELISA with microscopic agglutination test for Leptospira antibody detection.

A slide agglutination test (SAT), LeptoTek Dri-Dot and IgM-ELISA were compared with a microscopic agglutination test (MAT) for the detection of Leptospira antibodies. Paired sera from 10 patients whose leptospirosis was clinically suspected and diagnosed by MAT, were evaluated in this study. Our data, especially from acute samples, demonstrate the SAT and Dri-Dot were more sensitive as initial screening tests than MAT. IgM-ELISA has an advantage over MAT, SAT, and Dri-Dot since the results can be interpreted from a single serum testing if the results of the test are positive. Eight of the ten cases could be diagnosed by IgM-ELISA. Our data suggest that IgM-ELISA may be used for the diagnosis of leptospirosis. However, the agglutination test is useful for screening and for secondary infection cases for which IgM antibodies may be undetectable. MAT can be performed as a reference test and when information regarding the causative serovar is required.

Investigation on predominant Leptospira serovars and its distribution in humans and livestock in Thailand, 2010-2015.

BACKGROUND: Leptospirosis is a worldwide zoonotic bacterial disease caused by infection with leptospires. Leptospirosis in humans and livestock is an endemic and epidemic disease in Thailand. Livestock may act as reservoirs for leptospires and source for human infection. METHODOLOGY/PRINCIPAL FINDINGS: Data on leptospirosis infection in humans and livestock (Buffaloes, Cattle, and Pigs) species during 2010 to 2015 were analyzed. Serum samples were examined using Microscopic Agglutination Test (MAT) to identify antibodies against Leptospira serovars using a cut-off titer ≥ 1:100. The seroprevalence was 23.7% in humans, 24.8% in buffaloes, 28.1% in cattle, and 11.3% in pigs. Region specific prevalence among humans and livestock was found in a wide range. The most predominant serovars were Shermani, followed by Bratislava, Panama, and Sejroe in human, Shermani, Ranarum, and Tarassovi in buffaloes, and Shermani and Ranarum in cattle and pigs. Equally highest MAT titers against multiple serovars per one sample were found mainly in buffaloes and cattle showing equally titers against Ranarum and Shermani. The correlations of distribution of serovars across Thailand's regions were found to be similar in pattern for cattle but not for buffaloes. In humans, the serovar distribution in the south differed from other regions. By logistic regression, the results indicated that livestock is more susceptible to infection by serovar Shermani when compared to humans. CONCLUSIONS/SIGNIFICANCE: This study gives a detailed picture of the predominance of Leptospira serovars in relation to region, humans and typical livestock. The broad spatial distribution of seroprevalence was analyzed across and within species as well as regions in Thailand. Our finding may guide public health policy makers to implement appropriate control measures and help to reduce the impact of leptospirosis in Thailand.

Evaluation of loop-mediated isothermal amplification method (LAMP) for pathogenic Leptospira spp. detection with leptospires isolation and real-time PCR.

Leptospirosis has been one of the worldwide zoonotic diseases caused by pathogenic Leptospira spp. Many molecular techniques have consecutively been developed to detect such pathogen including loop-mediated isothermal amplification method (LAMP). The objectives of this study were to evaluate the diagnostic accuracy of LAMP assay and real-time PCR using bacterial culture as the gold standard and to assess the agreement among these three tests using Cohen's kappa statistics. In total, 533 urine samples were collected from 266 beef and 267 dairy cattle reared in central region of Thailand. Sensitivity and specificity of LAMP were 96.8% (95% CI 81.5-99.8) and 97.0% (95% CI 94.9-98.2), respectively. The accuracy of LAMP (97.0%) was significantly higher than that of real-time PCR (91.9%) at 95% CI. With Cohen's kappa statistics, culture method and LAMP were substantially agreed with each other (77.4%), whereas real-time PCR only moderately agreed with culture (47.7%) and LAMP (45.3%), respectively. Consequently, LAMP was more effective than real-time PCR in detecting Leptospira spp. in the urine of cattle. Besides, LAMP had less cost and was simpler than real-time PCR. Thus, LAMP was an excellent alternative for routine surveillance of leptospirosis in cattle.

A nationwide survey of pathogenic leptospires in urine of cattle and buffaloes by Loop-mediated isothermal amplification (LAMP) method in Thailand, 2011-2013.

Leptospirosis is a worldwide distributed zoonosis which has long been endemic in Thailand. Cattle and buffaloes are important livestock species that live in close contact with humans, especially in rural areas. These animals may, therefore, act as long-term carriers of leptospirosis for humans and other livestock species. The present study employed loop-mediated isothermal amplification (LAMP) method to detect pathogenic leptospiral 16S rDNA in the urine of cattle and buffaloes for assessing associations between uroprevalence and species, sex, age and spatial distribution. A total of 3,657 urine samples were collected for laboratory diagnosis, and 312 of which turned positive to the test (true prevalence 5.90%; 95% CI 4.98-6.91). The highest true uroprevalence was found in lower northern region at 19.80% (95% CI 15.83-24.32) followed by upper and lower northeastern regions at 15.22% and 6.25%, respectively. However, the highest true uroprevalence in beef cattle, the majority of cattle in Thailand, was recorded in northeastern region which is the endemic area of human leptospirosis. The uroprevalence was not statistically different among species and types of examined animals. Male animals were over twice more likely to be infected compared to females. Excluding animals younger than one year of age due to small sample size, the uroprevalence upraised with increasing age. A collaborative investigation between veterinary and public health sectors is required to holistically explore the link between leptospirosis in humans and livestock, especially in high prevalent areas.

Interpretation of microscopic agglutination test for leptospirosis diagnosis and seroprevalence.

Determination of antibody titer by microscopic agglutination test (MAT) has been used as a tool for leptospirosis diagnosis. Four fold or greater rise in antibody titers between acute and convalescent sera suggests recent Leptospira infection. In addition, results obtained by MAT have been used to predict infecting serovars. However, cross reactivity among various Leptospira serovars have been reported when patient sera were tested with a battery of Leptospira serovars. This study demonstrates cross- reactivity among several Leptospira serovars when MAT was performed on leptospirosis sera. The data support a role of MAT as a tool for diagnosis. However, for information on infecting serovars, Leptospira isolation and molecular identification should be performed.

In vivo gene expression and immunoreactivity of Leptospira collagenase.

Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Leptospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Hamsters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development.

Detection of Leptospira in urine using anti-Leptospira-coated gold nanoparticles.

Serological assays for antibody detection have been widely used for Leptospirosis diagnosis. However, antibody is usually undetectable during the first week after infection. Detection of Leptospira DNA can be done by PCR but this technique requires special equipments and the cost is still relatively high. Here we demonstrate that gold nanoparticles can be used to facilitate Leptospira detection. Gold nanoparticles were coated with rabbit antibody specific to Leptospira interrogans serovar Bratislava and these coated particles were used to detect Leptospira in urine. Agglutination of gold particles indicated the presence of Leptospira in samples tested. The sensitivity of detection was 10 leptospires/ml. No agglutination was observed when anti-Leptospira-coated particles were tested with urine containing the organisms commonly found in urine such as Klebsiella pneumoniae, Escherichia coli and Enterococcus faecalis. This assay is very easy to perform and results could be observed with the naked eyes. Fresh or frozen urine samples could be used. The stability of antibody-coated particles was at least 2 months when kept at 4°C. In conclusion, we have demonstrated that the technique using antibody-coated gold nanoparticles is a promising tool for further validation as a rapid assay for Leptospirosis diagnosis.

Strategies for diagnosis and treatment of suspected leptospirosis: a cost-benefit analysis.

BACKGROUND: Symptoms and signs of leptospirosis are non-specific. Several diagnostic tests for leptospirosis are available and in some instances are being used prior to treatment of leptospirosis-suspected patients. There is therefore a need to evaluate the cost-effectiveness of the different treatment strategies in order to avoid misuse of scarce resources and ensure best possible health outcomes for patients. METHODS: The study population was adult patients, presented with uncomplicated acute febrile illness, without an obvious focus of infection or malaria or typical dengue infection. We compared the cost and effectiveness of 5 management strategies: 1) no patients tested or given antibiotic treatment; 2) all patients given empirical doxycycline treatment; patients given doxycycline when a patient is tested positive for leptospirosis using: 3) lateral flow; 4) MCAT; 5) latex test. The framework used is a cost-benefit analysis, accounting for all direct medical costs in diagnosing and treating patients suspected of leptospirosis. Outcomes are measured in length of fever after treatment which is then converted to productivity losses to capture the full economic costs. FINDINGS: Empirical doxycycline treatment was the most efficient strategy, being both the least costly alternative and the one that resulted in the shortest duration of fever. The limited sensitivity of all three diagnostic tests implied that their use to guide treatment was not cost-effective. The most influential parameter driving these results was the cost of treating patients with complications for patients who did not receive adequate treatment as a result of incorrect diagnosis or a strategy of no-antibiotic-treatment. CONCLUSIONS: Clinicians should continue treating suspected cases of leptospirosis on an empirical basis. This conclusion holds true as long as policy makers are not prioritizing the reduction of use of antibiotics, in which case the use of the latex test would be the most efficient strategy.

Doxycycline versus azithromycin for treatment of leptospirosis and scrub typhus.

Leptospirosis and scrub typhus are important causes of acute fever in Southeast Asia. Options for empirical therapy include doxycycline and azithromycin, but it is unclear whether their efficacies are equivalent. We conducted a multicenter, open, randomized controlled trial with adult patients presenting with acute fever (<15 days), without an obvious focus of infection, at four hospitals in Thailand between July 2003 and January 2005. Patients were randomly allocated to receive either a 7-day course of doxycycline or a 3-day course of azithromycin. The cure rate, fever clearance time, and adverse drug events were compared between the two study groups. A total of 296 patients were enrolled in the study. The cause of acute fever was determined for 151 patients (51%): 69 patients (23.3%) had leptospirosis; 57 patients (19.3%) had scrub typhus; 14 patients (4.7%) had murine typhus; and 11 patients (3.7%) had evidence of both leptospirosis and a rickettsial infection. The efficacy of azithromycin was not inferior to that of doxycycline for the treatment of both leptospirosis and scrub typhus, with comparable fever clearance times in the two treatment arms. Adverse events occurred more frequently in the doxycycline group than in the azithromycin group (27.6% and 10.6%, respectively; P = 0.02). In conclusion, doxycycline is an affordable and effective choice for the treatment of both leptospirosis and scrub typhus. Azithromycin was better tolerated than doxycycline but is more expensive and less readily available.