Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Detection and differentiation between pathogenic and saprophytic Leptospira spp. by multiplex polymerase chain reaction.

A multiplex polymerase chain reaction (PCR) was developed for diagnosing leptospirosis and differentiating pathogenic and saprophytic leptospires. Specific primers were designed to amplify 23S rDNA from pathogenic Leptospira and saprophytic Leptospira spp. PCR products from 27 pathogenic and 5 (including 1 intermediate) saprophytic serovars were 615 and 316 base pairs (bp), respectively. After the restriction enzyme's digestion of PCR products, the fragments by SacI of pathogenic serovars and by PstI of saprophytic serovars were 339 and 276 bp and 202 and 114 bp, respectively. The PCR primers enabled amplification of DNA from L. meyeri serovar Ranarum as a pathogenic Leptospira spp. The PCR assay could detect 1 to 2 cells of leptospires and not amplify DNA from other 18 bacterial species. The sensitivity and specificity of this PCR in rat kidney, using isolation as gold standard, were 98.6% and 100%, respectively. The most appropriate sample preparation of blood for detecting DNA was buffy coat. Among the sample preparations from 7 laboratory-confirmed leptospirosis cases, leptospiral DNA was detected in all 7 buffy coat preparations, whereas leptospiral DNA was detected in only 3 plasma or serum samples. The PCR assay may be useful as a diagnostic tool for 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.

Identification of clinical isolates of Leptospira spp by pulsed field gel-electrophoresis and microscopic agglutination test.

Twenty-five clinical isolates of Leptospira spp were characterized by microscopic agglutination test (MAT) and pulsed field gel-electrophoresis (PFGE) in comparison with 23 reference Leptospira serovars and with the saprophytic L. biflexa serovar Patoc. PFGE DNA profiling was more specific and reliable than MAT.

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.

Comparison of leptospiral serovars identification by serology and cultivation in northeastern region, Thailand.

Blood from patients suspected of leptospirosis 148 specimens were cultured for leptospira. Twenty two specimens were positive (15%). The isolated leptospira were tested against the 24 serovars of standard antisera by Microscopic Agglutination Test (MAT). It was found that all 22 leptospira isolates reacted strongly against L. autumnalis, except 1 isolate that also reacted against serovar djasiman. The patient's sera were collected from only 14 cases. When the sera of the 14 patients were tested with the 24 reference serovars by MAT it was found that sera reacted the most against L. australis and in decreasing order against L. bratislava, L. autumnalis, L. rachmati, L. copenhageni, L. javanica. There had some cross reactions against several serovars in a single patient. The present study showed inconsistency between culture results and serum assays. Since sera showed cross reactivities against several serovars, it was not possible to determine which serovar was etiologic. Therefore, the isolation of leptospira though time consuming is specific in the identification of the serovar.

Chlamydophila pneumoniae specific antibodies in Thai patients with myocardial infarction.

To investigate the correlation between Chlamydia pneumoniae infection and acute myocardial infarction (AMI), a total of 101 serum specimens collected from patients with AMI admitted to the coronary care unit, Bhumibol Adulyadej Hospital, and serum specimens collected from healthy blood donors (control group) were examined by using the micro-immunofluorescence test. C. pneumoniae antibody-positive cases were found in 52 (52%) patients, consisting of 30 males and 22 females, though no significant difference of prevalence rate was observed when compared with the rate in the control group. However, the level of IgG titers in patients was higher than that in the controls, and this finding may support an association between C. pneumoniae infection and AMI. Among patients with AMI, several cases were suspected to have current infections because of a fourfold or higher titer increase in IgG or titers in IgM antibody of 1:32 or 1:64. There is no significant correlation between serologic test results and diabetes mellitus, hypertension, hyper cholesterol, a smoking habit, or the location of myocardial infarction among patients with AMI.

Prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in low and high endemic areas in Thailand.

OBJECTIVE: To investigate the prevalence of antibodies to Leptospira serovars in rodents and shrews trapped in urban and rural areas in low and high endemic areas in Thailand. MATERIAL AND METHOD: A total of 1,664 serum samples were collected from rodents and shrews in areas of low and high endemicity for leptospirosis. Four areas classified by case rates (CR) per 100,000 population of leptospirosis were urban Area I Bangkok (CR = 0.07), rural Area II (CR = 0.24), rural Area III (CR = 1.97) and rural Area IV (CR = 48.20). All serum samples were investigated for antibodies to leptospires by microscopic agglutination test (MAT) using antigens from each of the 22 pathogenic serovars of Leptospira interrrogans: australis, autumnalis, ballum, bangkok, bataviae, bratislava, canicola, celledoni, copenhageni, djasiman, grippotyphosa, hardjo, hebdomadis, icterohaemorrhagiae, javanica, pomona, pyrogenes, rachmati, saigon, sejroe, tarassovi and wolffi and one non-pathogenic strain of L. biflexa serovar patoc. RESULTS: Ninety-four (5.6%) serum samples were positive for Leptospira antibodies. The most commonly detected antibodies were to serovars pyrogenes (39.1%), sejroe (19.1%), bataviae (10.0%), pomona (6.4%), autumnalis (5.5%), copenhageni (3.6%) and javanica (3.6%). The positive rates in Area I, II, III and IV were 7.6 per cent, 2.9 per cent, 4.6 per cent and 7.1 per cent, respectively. The seroprevalence in rural areas tended to increase significantly with high endemicity for leptospirosis (Chi-square for trend, p = 0.04). The seropositive rates by animal species were 39/496 (7.9%), 22/322 (6.8%), 23/492 (4.7%), 6/170 (3.5%), 4/175 (2.3%), 0/4 (0%) and 0/5 (0%) in Rattus norvegicus, Rattus exulans, Rattus rattus, Bandicota indica, Bandicota savilei, Mus musculus and Suncus murinus, respectively. There was a statistical trend between seropositive rates in R. exulans and endemicity for leptospirosis (Chi-square for trend, p = 0.04). CONCLUSION: The 5.6 per cent of rodents and shrews trapped in urban and rural areas in Thailand were reservoirs of leptospires. The results of high seroprevalence in rats also indicate the high endemicity for leptospirosis.

Use of protein-specific monoclonal antibody-based latex agglutination for rapid diagnosis of Burkholderia pseudomallei infection in patients with community-acquired septicemia.

A latex agglutination test employing monoclonal antibody specific to a 30-kDa protein of Burkholderia pseudomallei was used to detect the organisms in blood culture specimens from 1,139 patients with community-acquired septicemia. The sensitivity, specificity, and positive and negative predictive values of the test were 96.75%, 99.61%, 96.75%, and 99.61%, respectively.

Diagnosis of human leptospirosis by monoclonal antibody-based antigen detection in urine.

Hybridomas secreting specific monoclonal antibodies (MAb) to all members of the genus Leptospira (clone LF9) and those that are specific only to the pathogenic species (clones LD5 and LE1) were produced. MAb LF9, which was immunoglobulin G1 (IgG1), reacted to a 38-kDa component of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell lysates of all Leptospira spp., while MAb LD5 and MAb LE1, which were IgG1 and IgG2a, respectively, reacted to the 35- to 36-kDa components of all serogroups of the pathogenic species of LEPTOSPIRA: The MAb LD5 was used in a dot blot-enzyme-linked immunosorbent assay (dot-ELISA) for detecting Leptospira antigen in urine samples serially collected from two groups of patients diagnosed with leptospirosis, i.e., 36 clinically diagnosed patients and 25 Leptospira culture confirmed patients. Their serum samples were tested serologically by IgM Dipstick assay, indirect immunofluorescence assay (IFA), and/or microscopic agglutination test (MAT). Urine samples of 26 patients diagnosed with other illnesses and 120 healthy individuals served as controls. For the first group of patients, who had been ill for an average of 3.4 days before hospitalization, the IgM Dipstick test, IFA, and MAT were positive for 69.4, 70.0, and 85.7% of patients, while the Leptospira antigenuria tested by the MAb-based dot-ELISA was positive for 75.0, 88.9, 97.2, 97.2, and 100% of patients on days 1, 2, 3, 7, and 14 of hospitalization, respectively. All but 1 of 11 patients whose serum samples collected on the first day of hospitalization were IgM seronegative, were positive by urine antigen test on day 1. This is strong evidence that detection of antigen in urine can provide diagnostic information that could be useful in directing early therapeutic intervention. The MAT was positive in 10 of 12 patients (83.3%) of the 25 culture-positive Leptospira patients who had been ill for an average of 5.04 days before hospitalization, and the Leptospira antigen was found in 64.0, 84.0, 96.0, 100, 100, 100, and 100% of the patients' urine samples collected on days 1, 2, 3, 4, 5, 6, and 7 of hospitalization, respectively. Leptospira antigenuria was found in 3 of the 26 patients diagnosed with other illnesses and 1 of the 120 healthy controls. The reasons for this positivity are discussed. The detection of antigen in urine by the monoclonal antibody-based dot-ELISA has high potential for rapid, sensitive, and specific diagnosis of leptospirosis at a low cost.