Publications

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    ABSTRACT: 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.
    Expert Review of Clinical Immunology 03/2013; 9(3):263-80. · 2.89 Impact Factor
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    ABSTRACT: 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.
    Acta tropica 04/2012; 122(1):119-25. · 2.79 Impact Factor
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    ABSTRACT: Motivated by the lack of related studies and an insufficient understanding of the response of pathogenic spirochetes, including leptospira to ultraviolet-A (UV-A) (or other stresses), we comparatively studied the effects of UV-A radiation on the Leptospira interrogans serovar Bataviae, Canicola and Pomona. The main purpose of this work was to investigate the effects of UV-A irradiation—both short term (immediate) and long term (post-irradiation)—on leptospires at different UV-A dosages, controlled by the duration of exposure time. It was observed that survival fractions linearly decrease from 100 to about 70, 60 and 50% for serovar Pomona, Bataviae and Canicola, respectively. This indicates that, for different serovars, UV-A irradiation has a quantitatively different effects on growth. Short term effects suggest that Pomona may be more resistant to UV-A than the other serovars. Long term effects show that, when compared with the control group, the treated groups of bacteria re-grow when the exposure time is equal or lesser than 6 h (~ 2 -6), while the groups exposed for 12 h or longer experienced little change or a slight decrease. This may indicate that UV-A radiation is able to inhibit the growth of bacteria, but does not prevent self-defense from taking place. UV-A radiation's effect on antigenic components was also investigated. The immunoblotting method was used and the results are supported by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results. Possible explanations for these results are discussed.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 04/2010; 9:3196-3206. · 0.57 Impact Factor
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    ABSTRACT: Mammary tumors are by far the most common tumors in female dogs and effective treatment relies on prompt and accurate diagnostic procedures. Canine mammary tumors may originate from various cell types, such as luminal epithelial, myoepithelial and stromal cells. This study aimed to differentiate luminal epithelial and myoepithelial lineages, using specific markers including AE1/AE3, Vimentin, and p63. Such data can be useful for prognosis. Canine mammary tumors were collected by surgical resection and tissue samples were investigated using the avidin-biotin-immunoperoxidase method with used primary antibodies against AE1/AE3, vimentin, and p63. Luminal epithelial-origin tumors were found to be immunoreactive with AE1/AE3 and vimentin monoclonal antibody, while myoepithelial-origin tumors were positive for p63 and vimentin . In addition, canine mixed tumors showed reactivity with all three antibodies. In summary, AE1/AE3, p63 and vimentin can be used as specific immunohistochemical markers to distinguish lumino-epithelial and myoepithelial lineages of canine mammary tumors.
    Asian Pacific journal of cancer prevention: APJCP 01/2010; 11(1):227-30. · 1.27 Impact Factor
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    ABSTRACT: The effect of exposure to ultaviolet-A (UVA) radiation was studied on the pathogenic spirochetes Leptospira interrogans serovar Canicola for different time durations. Changes in cell growth and viability due to UVA exposure were determined by using the conventional microscopic agglutination test (MAT), dark-field microscopy and spectrophotometry measurements. Changes in antigens and protein expression in the cells were detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot. Decrease in cell growth and viability was found to be related to the exposure period, or was dose dependent. The growth decreased sharply at a very high rate for the first 24 h of exposure (112.3 J/cm 2); then it reached the minimum within about a 1d of exposure and leveled off for further treatment until the 7d exposure period. Immunoblot revealed the presence of 21 kDa antigenic protein in the unexposed cells, which disappeared after exposure to UVA for 24 h. SDS-PAGE analysis indicated the presence of a 76 kDa protein band in the cells exposed to UVA for 2 to 24 h. Exposure to UVA for more than 24 h decreased this protein, but the proteins of molecular mass between 56 and 70 kDa appeared. This work is the first step toward understanding the effects of UVA on leptospira bacteria. Further investigation of the mechanisms involved in UVAinteraction with leptospira will eventually lead to development of new strategies to control or prevent leptospira in the environment.
    AFRICAN JOURNAL OF BIOTECHNOLOGY 08/2009; 8:3341-3352. · 0.57 Impact Factor
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    ABSTRACT: 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.
    Biological research 02/2009; 42(1):5-12. · 1.13 Impact Factor
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    ABSTRACT: 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.
    Clinical and vaccine Immunology: CVI 04/2008; 15(3):492-8. · 2.60 Impact Factor
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    J Health Res. 01/2008; 22:143-146.
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    ABSTRACT: 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.
    Journal of Medical Microbiology 06/2007; 56(Pt 5):587-92. · 2.30 Impact Factor
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    ABSTRACT: 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.
    PROTEOMICS - CLINICAL APPLICATIONS 03/2007; 1(4):400 - 409. · 1.81 Impact Factor
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    ABSTRACT: Detection of antibodies against Helicobacter pylori by indirect immunofluorescent assay (IFA) in rabbits has been developed as a non-invasive screening method. This study aimed to detect the antibody response by IFA after immunization with Helicobacter pylori in rabbits. Six healthy New Zealand white female rabbits were immunized subcutaneously with Helicobacter pylori, 4 times, on days 0, 14, 28 and 42, were used for IFA evaluation. Blood samples were then collected from each rabbit on day 0 and weekly, for twelve weeks. H. pylori were coated on slides and fixed with absolute methanol and air dried. The coated slides were incubated with sample rabbit sera for 50 minutes at room temperature and washed with PBS. The slides were incubated with goat anti-rabbit IgG antibody conjugated with FITC for 50 minutes at room temperature. The slides were evaluated by fluorescent microscopy. The rabbit sera had evaluated IFA titers starting from week 4 with a peak at week 8. Antibodies were constantly detected until the end of observation (week 12). The means log 2 H. pylori antibody titers were 10.50 ± 1.32, 11.83 ± 1.065, 13.33 ± 0.19, 14.00 ± 0.23, 14.83 ± 0.37, 15.00 ± 0, 15.00 ± 0, 15.00 ± 0, and 14.83 ± 0.90 (mean ± SE, n = 6), respectively. Given the ability to detect H. pylori antibody in rabbits using IFA, the results of this experiment may be useful for evaluating the epidemiology and diagnosis of H. pylori infection in veterinary public health studies.
    01/2007; 240(38).
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    Anuchai Niwetpathomwat, Galayanee Doungchawee
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    ABSTRACT: 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.
    The Southeast Asian journal of tropical medicine and public health 04/2006; 37(2):309-11. · 0.61 Impact Factor
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    ABSTRACT: 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.
    The Southeast Asian journal of tropical medicine and public health 12/2005; 36(6):1516-22. · 0.61 Impact Factor
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    ABSTRACT: 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.
    The Southeast Asian journal of tropical medicine and public health 02/2005; 36 Suppl 4:202-5. · 0.61 Impact Factor
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    ABSTRACT: 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.
    Journal of Bioscience and Bioengineering 02/2004; 98(3):182-6. · 1.74 Impact Factor
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    ABSTRACT: 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.
    Asian Pacific journal of cancer prevention: APJCP 02/2002; 3(4):367-368. · 1.27 Impact Factor
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    ABSTRACT: Antibodies to collagenous and noncollagenous components of glomerular basement membrane (GBM) have been detected by immunoblotting in some sera from patients with various kinds of glomerulonephritis. A half proportion of patients with rapidly progressive glomerulonephritis (RPGN), chronic focal glomerulonephritis (CFGN), idiopathic membranous glomerulonephritis (MGN). IgA nephropathy and lupus nephritis (LE-GN) had IgG antibodies to heterogenous components in acid insoluble fraction of pepsin digested GBM. This acid insoluble fraction represented a complex of collagen and noncollagenous proteins of GBM. Following digestion of acid insoluble fraction with bacterial collagenase, the triple helical collagenous components of GBM were destroyed and released most likely of noncollagenous proteins. Antibodies to this noncollagenous proteins were found in only some patients with chronic glomerulonephritis (17.6%) and lupus nephritis (21.4%). Upon reaction with human placenta derived type IV collagen, different frequencies of antibody response were found in patients of different groups. However, all these reactive sera showed a similar immunoblotting pattern. The relationship between antibody response to antigenic components from human GBM or human placenta and pathogenesis of renal disease is unclear. However, the occurrence of spontaneous autoantibody response to some exposed GBM self antigens may mediate further renal destruction resulting in chronic ongoing stage of the disease.
    Journal of the Medical Association of Thailand = Chotmaihet thangphaet 02/1992; 75 Suppl 1:25-31.
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    ABSTRACT: Renal disease associated with Opisthorchis viverrini infection was investigated in Syrian golden hamsters. On the fourth week after infection with 100 viable metacercariae; anti-tegumental membrane antibodies were detected in the sera by immunofluorescence antibody technic and by enzyme-linked immunosorbent assay. Six weeks after infection tegumental and anti-tegumental membrane immune-complex and amyloid fibrils were found in the glomeruli. Amyloid was characterized to be AA protein. Acute proliferative glomerulonephritis associated with the brightest immune-complex deposits developed in week 8 after infection. Intensity of immune-complexes in all glomeruli were reduce gradually thereafter and replaced by amyloid. Progressive obsolescence of the glomeruli, tubular atrophy, interstitial inflammation and fibrosis associated with massive proteinuria and deterioration of renal function appeared in week 10 after infection toward the end of the experiment in week 38 after infection.
    Journal of the Medical Association of Thailand = Chotmaihet thangphaet 02/1992; 75 Suppl 1:7-19.
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    ABSTRACT: The protein compositions of homogenized metacercaria and adult Opisthorchis viverrini were characterized by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The two different stages of O. viverrini parasite contained several protein components with very similar mobilities on SDS-PAGE gel. The reaction in immunoblots of sera from patients with opisthorchiasis demonstrated antibodies against heterogeneous protein components of metacercariae and adult O. viverrini parasites. Only the components of metacercariae with molecular weights of 190-200 kD, 132 kD and 107 kD reacted specifically with opisthorchiasis sera. This characteristic reaction pattern was not observed in any control sera of normal subjects or those infected with other parasites. Identical reactions were however also recognized in the sera of experimental infected hamsters with metacercariae, but did not occur with adult worm as the antigen for the immunoblotting reaction. This may indicate that these metacercariae-associated protein components have potential diagnostic value for opisthorchiasis.
    Clinical & Experimental Immunology 01/1989; 74(3):355-8. · 3.41 Impact Factor
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    ABSTRACT: In this work, we consider a deterministic model for the transmission of leptospirosis which is currently spreading in the Thai population. The SIR model which incorporates the features of this disease is applied to the epidemiological data in Thailand. It is seen that the numerical solutions of the SIR equations are in good agreement with real empirical data. Further improvements are discussed.

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