Evaluation of a genus-specific rGroEL1-524 IgM-ELISA and commercial ELISA kits during the course of leptospirosis in Thailand.

In the present study, we developed a genus-specific rGroEL1-524 IgM-ELISA assay for use in screening diagnosis of suspected leptospirosis among acute undifferentiated febrile illness patients during acute fever. The diagnostic accuracies of the rGroEL1-524 IgM-ELISA, commercial Panbio IgM-ELISA, and Virion-Serion Classic IgG-ELISA were evaluated using 133 Thai leptospirosis sera and 210 controls. Sensitivities were 91.7%, 59.6%, and 17.7% for acute infection, and the specificities were 92.6%, 90.2%, and 88.3% for the non-leptospirosis control, respectively. The rGroEL1-524 IgM-ELISA had high sensitivity, at 92.3% and 91.7%, among culture-positive and MAT-negative cases at 1-3 days post-onset of symptoms (DPO1-3), respectively. Impaired specificity on scrub typhus was found, possibly from antibody cross-reaction to ortholog GroEL. Commercial Panbio IgM-ELISA had sensitivities at DPO1-3 of 30.8% and 41.7% for culture-positive and MAT-negative cases whereas Virion-Serion IgG-ELISA showed sensitivities of 5.9% and 13.3%, respectively. The rGroEL1-524 IgM-ELISA could be useful as a screening test for early diagnosis. The performance of the commercial ELISA suggests the applicability of IgM-ELISA for diagnosis, while IgG-ELISA is useful for seroprevalence surveys. However, confirmation by reference tests is recommended.

Immunodominance of LipL3293-272 peptides revealed by leptospirosis sera and therapeutic monoclonal antibodies.

BACKGROUND/PURPOSE: Leptospirosis is a neglected zoonosis, imposing significant human and veterinary public health burdens. In this study, recombinant LipL3293-147 and LipL32148-184 middle domain of LipL3293-184, and LipL32171-214, and LipL32215-272 of c-terminal LipL32171-272 truncations were defined for immunodominance of the molecule during Leptospira infections revealed by leptospirosis sera. RESULTS: IgM-dominant was directed to highly surface accessible LipL32148-184 and Lipl32171-214. IgG dominance of LipL32148-184 revealed by rabbit anti-Leptospira sera and convalescent leptospirosis paired sera were mapped to highly accessible surface of middle LipL32148-184 truncation whereas two LipL32148-184 and LipL32215-272 truncations were IgG-dominant when revealed by single leptospirosis sera. The IgM-dominant of LipL32148-214 and IgG-dominant LipL32148-184 peptides have highly conserved amino acids of 70% identity among pathogenic and intermediate Leptospira species and were mapped to the highly surface accessible area of LipL32 molecule that mediated interaction of host components. IgG dominance of two therapeutic epitopes located at LipL32243-253 and LipL32122-130 of mAbLPF1 and mAbLPF2, respectively has been shown less IgG-dominant (<30%), located outside IgG-dominant regions characterized by leptospirosis paired sera. CONCLUSION: The IgM- and IgG-dominant LipL32 could be further perspectives for immunodominant LipL32-based serodiagnosis and LipL32 epitope-based vaccine.

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.

Development and evaluation of an immunochromatographic assay to detect serum anti-leptospiral lipopolysaccharide IgM in acute leptospirosis.

Leptospirosis is a common life-threatening disease worldwide. However, its diagnosis is frequently ineffective because the gold standard bacterial culture and microscopic agglutination test (MAT) are usually positive 1-2 weeks after the disease onset. We thus developed an immunochromatographic assay (LEPkit) to detect serum anti-leptospiral lipopolysaccharide (LPS) IgM for rapid diagnosis of acute leptospirosis. Using referenced sera of 77 leptospirosis and 91 non-leptospirosis cases, LEPkit yielded 97.4% sensitivity, 94.5% specificity, 93.8 positive predictive value (PPV), 97.7% negative predictive value (NPV), and 95.8% accuracy. The stability of this kit stored for up to 18 months and its reproducibility were confirmed. Testing in 74 new cases using samples at admission-phase and subsequent paired samples (total n = 135), overall sensitivity was 98.5%, whereas that of culture and single MAT (≥1:400) was 15.6% and 35.6%, respectively. When only the samples at admission-phase were used (n = 74), the sensitivity remained at 98.7%, whereas that of culture and single MAT (≥1:400) was 28.4% and 13.5%, respectively. In summary, our LEPkit was far more effective than any conventional methods for the diagnosis of acute leptospirosis, especially within the first few days after the disease onset. The ease of use, stability and reproducibility further enhance its feasibility for clinical use on-site.

Association between Opisthorchis viverrini and Leptospira spp. infection in endemic Northeast Thailand.

Opisthorchiasis caused by Opisthorchis viverrini is an important foodborne trematodiasis in Thailand, Laos and Cambodia. Interestingly, the opisthorchiasis endemic region overlaps with an area of leptospirosis emergence. Here we report an association between opisthorchiasis and leptospirosis in Thailand. Of 280 sera collected from villagers living around the Lawa wetland complex in Khon Kaen province, 199 (71%) were seropositive for leptospirosis by immunochromatography. Individuals with O. viverrini infection had a significantly higher rate of leptospirosis than those without (P=0.001). Significant higher leptospirosis prevalence was found in males than females (P=0.002). However, females but not males with O. viverrini infection showed a significantly higher seroprevalence of leptospirosis. Twenty-one of 35 environmental samples from the lake (water, mud and fish skin mucus) were positive for Leptospira spp. DNA sequencing, sequence alignment, and phylogenetic analysis of some positive nested PCR products revealed both pathogenic and intermediate pathogenic strains of Leptospira in the samples. Strikingly, O. viverrini metacercariae from the fish were positive for L. interrogans. These results suggest a close association between opisthorchiasis and leptospirosis. Contact with water, mud or eating raw fish harboring liver fluke metacercariae may be risk factors for Leptospira infection.

Leptospirosis: current situation and trends of specific laboratory tests.

Leptospirosis is re-emerging as a worldwide zoonosis and is caused by bacteria of the genus Leptospira. Human leptospirosis is associated with high temperature and humidity. Laboratory tests are indispensible for the early diagnosis and proper disease management. The demand for suitable leptospirosis point-of-care diagnostic tests grows with the awareness and number of incidences. Confirmation is achieved by the microscopic agglutination test, bacterial cultivation, PCR or histopathologic methods. However, high costs, poor standardization and/or elaborate sample preparation prevent routine use at the point of care. Cost-efficient, but insensitive serological methods dominate the diagnostic landscape and, likewise, urgently need improvement toward greater compliance with some of the point-of-care criteria. Combined application of antigen and antibody detection methods increases accuracy, but also new development or transfer of diagnostic technologies should be considered useful. Nano- and microparticle technology may play a key role in improving future antigen detection methods.

Development of a magnetic bead fluorescence microscopy immunoassay to detect and quantify Leptospira in environmental water samples.

Climate change, world population growth, and poverty have led to an increase in the incidence of leptospirosis. Leptospirosis is caused by pathogenic spirochaete bacteria that belong to the genus Leptospira. The bacteria are maintained in the renal tubules of the reservoir hosts (typically a rodent), then shed into the environment via the urine. Water is key for environmental survival and transmission, as leptospires can survive for several weeks in a moist environment. Therefore, environmental epidemiological studies are needed to study the contamination of environmental water sources. However, few such studies have been performed using cultivation of the isolates and PCR assays. But, leptospira cultivation can be easily contaminated by other organisms and takes usually several weeks. Moreover, PCR is a complex and costly analysis for the underdeveloped countries that have the highest incidence of leptospirosis. In this study, we describe two modifications of a fluorescence microscopy assay based on immuno-magnetic separation (IMS) to detect leptospires in environmental water samples that mainly differ in fluorescent dye staining. The first type uses acridine orange fluorescent dye staining combined with multiplexed IMS for sample screening. The detection limit ranged from 10(2) to 10(3) organisms per mL and largely depended on the capture efficiency (CE) of the immuno-magnetic particles. The second type uses serogroup-specific immuno-particles and direct fluorescence antibody staining (DFA) to detect leptospires; the detection limit of this second assay was approximately 10(1) cells per mL. Both assay types were applied to natural and experimentally infected water samples, which were also analysed with DFM and real-time PCR. Our data show that the fluorescent microscopy immunoassay successfully identified experimental leptospire contamination and was as sensitive as PCR. This modified immune-fluorescence assay may therefore enable epidemiological studies of leptospirosis.

Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires.

Leptospirosis caused by Leptospira interrogans is the most widespread zoonosis and a major public health problem worldwide. Based on light-scattering and absorption, quantification of leptospires using UV-VIS spectroscopy was used as an indirect counting technique by measuring the optical density and comparing this to automated direct counting using a counting chamber in combination with imaging and analyzing software. Two serovars, Bangkok and Copenhagenii, from log-phase growth were used for the establishment of standard curves. They were found to be linear and slightly different in gradient for each serovar. The ease, rapidity, and reliability of these two adapted and optimized counting techniques may provide a useful alternative enumeration technique for leptospirosis research.

Leptospirosis research: fast, easy and reliable enumeration of mobile leptospires

Leptospirosis caused by Leptospira interrogans is the most widespread zoonosis and a major public health problem worldwide. Based on light-scattering and absorption, quantification of leptospires using UV-VIS spectroscopy was used as an indirect counting technique by measuring the optical density and comparing this to automated direct counting using a counting chamber in combination with imaging and analyzing software. Two serovars, Bangkok and Copenhagenii, from log-phase growth were used for the establishment of standard curves. They were found to be linear and slightly different in gradient for each serovar. The ease, rapidity, and reliability of these two adapted and optimized counting techniques may provide a useful alternative enumeration technique for leptospirosis research.

Early diagnosis of leptospirosis by immunoglobulin M immunoblot testing.

There is an urgent need for the development of serodiagnostic approaches with improved sensitivity for patients with acute leptospirosis. Immunoblots were performed on 188 sera collected from 74 patients with laboratory-confirmed early leptospiral infection to detect immunoglobulin M (IgM) antibodies to antigens pooled from 10 leptospiral strains prevalent in Thailand. Sera from patients with other febrile diseases served as controls. IgM reactivity to seven distinct antigens, with apparent molecular masses of 14 to 18, 19 to 23, 24 to 30, 32, 35/36, 37, and 41/42 kDa, was observed. The low-molecular-mass 14- to 18-kDa band was the most frequently detected antigen, being recognized in sera from 82.4% of patients during the first 3 days after the onset of symptoms. We evaluated the accuracy of the IgM immunoblot (IgM-IB) test by using reactivity to the 14- to 18-kDa band and/or at least two bands among the 19- to 23-, 24- to 30-, 32-, 35/36-, 37-, and 41/42-kDa antigens as the diagnostic criterion. The sensitivities of the IgM-IB test and the microscopic agglutination test (MAT) were 88.2% and 2.0%, respectively, with sera from patients 1 to 3 days after the onset of symptoms. In contrast, the IgM-IB test was positive with only 2/48 (4.2%) sera from patients with other febrile illnesses. The high sensitivity and specificity of the IgM-IB test for acute leptospirosis would provide greatly improved diagnostic accuracy for identification of patients who would benefit from early antibiotic intervention. In addition, the antigens identified by the IgM-IB test may serve as components of a rapid, accurate, point-of-care diagnostic test for early leptospirosis.

Use of immunoblotting as an alternative method for serogrouping Leptospira.

Leptospirosis is a worldwide zoonotic disease caused by a spirochaete bacterium, Leptospira. Serological detection of this micro-organism basically relies on a conventional microscopic agglutination test (MAT), which has some limitations and disadvantages. In the present study, immunoblotting has been applied as an alternative method for differentiating serogroups and serovars of leptospires. Leptospiral whole-cell lysates from a total of 26 serovars were subjected to immunoblotting using rabbit antisera against individual serovars. The findings clearly demonstrated that the pattern of immunoreactive bands could be used to differentiate between leptospires of different serogroups, consistent with MAT results. There was a multi-band pattern that was unique for the pathogenic Leptospira antigens and was not observed in the non-pathogenic Leptospira biflexa and non-leptospiral bacteria (i.e. Escherichia coli, Burkholderia pseudomallei and Helicobacter pylori). For pathogenic Leptospira species, a prominent smear-like band at approximately 19-30 kDa was present when the antigens were probed with the homologous antisera. The molecular size of the prominent band, although it showed a cross-reaction between members within the same serogroup, differed among different serovars. The results obtained from polyclonal antibodies (antisera) were confirmed using mAb. With its simplicity and safety of experimental procedures, it is proposed that immunoblotting may potentially be useful as an alternative method for differentiating between serogroups of leptospires.

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery.

Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non-leptospirosis patients with a negative MAT were pooled and used as the negative control. 2-D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI-TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti-human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications.

Western immunoblot analysis using a ten leptospira serovars combined antigen for serodiagnosis of leptospirosis.

Leptospirosis is a major zoonotic disease throughout the world. There are unavailable accuracy diagnostic methods for the acute phase of infection. To demonstrate the advantage of Western immunoblot, a mixed leptospira serovars antigen for the serodiagnosis of leptospirosis was employed. SDS-PAGE and Western immunoblot was performed using a 10 mixed leptospira serovars antigen and stained with 16 reference rabbit anti-leptospirosis antibodies. The result showed different immunoreactive band patterns for each reference serum. The bands with molecular weights of 15-20, 23-24, 41 and 45 kDa were commonly found (88% to 100% of the 16 reference sera). Using combined leptospira antigens in a Western immunoblot technique is an alternative and practical strategy for a more sensitive leptospirosis serodiagnosis.

Survey of leptospirosis of small mammals in Thailand.

During 1999-2000, kidney tissues of approximately 15% of 1310 rodents trapped from northeastern provinces of Thailand were tested for the presence of leptospires. Our direct immunofluorescent assay (DFA) for detection of leptospires showed 100% sensitivity and 94% specificity with the culture data. Both methods identified R. norvegicus as the highest source of infection. Among isolated Leptospira, 137 were serotyped by cross agglutinin absorption and/or a microscopic agglutination, and gave some variations and similarities at the serovar level to the DFA results. DFA data demonstrated over half of the positive animals were infected with several serovars of Leptospira interrogans. A subsequent DFA study in Bangkok in 2002 revealed leptospiral infection in 33% of 42 rats and shrews. The most common infecting serovars were Autumnalis and Canicola identified in rural and urban animals, respectively. This finding suggests that wild small mammals may act as important sources of pathogenic leptospires and warrant active surveillance to understand the epidemiology of transmission and control of carrier animals.

Surveillance of leptospirosis after flooding at Loei Province, Thailand by year 2002.

In this surveillance, suspected leptospirosis patients in Loei Hospital, Loei Province were studied by conventional methods of cultivation and microscopic agglutination test (MAT) during July-October, 2002. It was found that 63% of 64 admitted patients and 35% of 34 outpatients were found positive by leptospire cultivation. Antibodies determined by MAT were positive in 78% of 63 admitted patients. Particularly, the five most common agglutinating antibodies were reactive with serovars bratislava (57%), autumnalis (48%), new (38%), australis (37%) and bangkok (29%). The MAT results of 15 OPD patients were 67% positive with the following five serovars, including bratislava (47%), new (20%), bangkok (7%), ranarum (7%) and australis (7%). Accordingly, preventive strategies against leptospirosis outbreaks after flooding in Thailand should be undertaken, including the prompt treatment of the disease in this endemic area.

Effects of static magnetic field on growth of leptospire, Leptospira interrogans serovar canicola: immunoreactivity and cell division.

The effects of the exposure of the bacterium, Leptospira interrogans serovar canicola to a constant magnetic field with magnetic flux density from a permanent ferrite magnet=140+/-5 mT were studied. Changes in Leptospira cells after their exposure to the field were determined on the basis of changes in their growth behavior and agglutination immunoreactivity with a homologous antiserum using dark-field microscopy together with visual imaging. The data showed that the exposed Leptospira cells have lower densities and lower agglutination immunoreactivity than the unexposed control group. Interestingly, some of the exposed Leptospira cells showed abnormal morphologies such as large lengths. We discussed some of the possible reasons for these observations.

Use of Wild Rodents for Environmental Monitoring - Comparison of Rats in Bangkok and Rural Areas of Thailand.

There is a great deal of concern regarding the hazard potential of human exposure to toxic substances and carcinogens as well as infectious agents in the environment. For monitoring purposes fish are well established with regard to aquatic pollution. However, for the human environment, mammalian species might be considered more relevant. As the various types of rats are one of the most common animals sharing human habitants they are natural candidates. In the present study, numbers of such wild rats were trapped in the metropolis of Bangkok and country regions of Thailand for comparison of lesions in the liver and lung which might provide indicators of carcinogens or other hazardous agents in the environment. Glutathione S transferase P form positive foci could be detected in livers, comparable to the laboratory rat case, but without any significant link to site of capture. In contrast, fatty liver and inflammation/cirrhosis were significantly more frequent in animals from the metropolis. Parasite infection also tended to be more prevalent, along with leptospirosis. Inflammatory change was similarly found in the lungs but without any variation between the city and countryside groups. These results suggest that wild rats could be employed as monitors of environmental agents of toxicological significance.