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Burkholderia pseudomallei is the causative agent of melioidosis, a severe and potentially fatal infectious disease in humans known to be endemic in Southeast Asia and northern Australia. The infection is also increasingly recognized in various animal species with a potential to spread to humans. With the potential as a biological warfare agent, specific serodiagnosis of melioidosis for surveillance in large populations at risk, humans or animals, would be highly valuable. In this study, a competitive enzyme-linked immunosorbent assay (ELISA) using a lipopolysaccharide-specific monoclonal antibody was developed. The assay provides high specificity, based on a previously described monoclonal antibody to a specific epitope on the lipopolysaccharide (LPS) of B. pseudomallei. The assay sensitivity of 96.0% and specificity of 100% were achieved at a cutoff value of 50% inhibition in human culture-proven melioidosis cases. An optimal cutoff value of 65% inhibition for sera from a melioidosis endemic area was obtained by ROC analysis and resulted in an assay specificity of 86.2%, while maintaining assay sensitivity of 92.0%. A potential application of the assay in the serodiagnosis of melioidosis in animal species was also evaluated usina dolphin sera with satisfactory results.

Nonstructural 3 (NS3) protein of hepatitis C virus (HCV) is one of the antigens commonly used in diagnostic assays for antibody to hepatitis C virus. However, immune response to the NS3 protein from one genotype may not cross-react with that from other genotypes. In the development of an anti-HCV assay, the NS3 genes from genotypes 1 and 3 commonly found in Thailand were amplified and cloned into a bacterial expression system. These recombinant NS3 proteins were immunogenic and reacted with plasma samples of Thai patients infected with various HCV genotypes. Interestingly, the NS3 proteins from the Thai genotypes could react with 3 plasma samples from HCV infected Thai blood donors, which could not bind to the NS3.1 protein in the commercial HCV immunoblot kit using antigen from HCV genotype 1. This finding supports our prior observation that the appropriate HCV antigens used in a diagnostic assay should be derived from the virus genotypes commonly found in that geographical region.

We measured the cell-mediated immune response to GAD and bovine beta-casein in 38 type 1 and 37 type 2 diabetic patients who visited diabetic clinics or who were hospitalized in Bangkok, Thailand, and in 43 normal controls, by using a lymphoproliferation assay. Positive results against GAD were found in 29/38 (76.3%) type 1, 6/37 (16.2%) type 2 diabetic patients and 1/43 (2.3%) normal controls. Positive results against bovine beta-casein were found in 18/38 (47.4%), 5/37 (13.5%) and 1/43 (2.3%) of these subjects, respectively. The frequencies of the positive results and the magnitude of the responses to both antigens in type 1 diabetic patients were significantly higher than those in the other two groups (P<0.001). In addition, the prevalence of a positive lymphoproliferative response to these antigens in type 1 diabetic patients was higher than that of anti-GAD antibody positivity in the same group of subjects (26.3%). Thus, the prevalence of positive lymphoproliferative response to GAD in type 1 diabetic patients studied was higher than the prevalence of other autoimmune markers previously reported in type 1 diabetic patients in Thailand.

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal tropical infectious disease endemic in Southeast Asia. Environmental isolates of B. pseudomallei have two distinctive biotypes. Some soil isolates are arabinose-assimilators (Ara+ biotype) and are non-virulent in experimental animals. The others cannot assimilate arabinose (Ara- biotype) and are virulent in experimental animals. The Ara- biotype is found in almost all B. pseudomallei clinical isolates. In the present study, a panel of eight monoclonal antibodies that agglutinate the bacteria were produced and tested. The first group, Bps-D2, -D3, -D5, -L1, and -L2 agglutinated 100% of Ara+ clinical and soil isolates of B. pseudomallei. Another group Bps-A1, -A2, and -D1 agglutinated 92.9% and 90.9% of Ara- clinical and soil isolates, respectively. This panel of monoclonal antibodies may be useful for rapid differentiation between non-virulent Ara+ and virulent Ara- B. pseudomallei.

Melioidosis is an important public health problem in Southeast Asia and Northern Australia. This disease is caused by the gram-negative bacilli, Burkholderia pseudomallei. Wide spectra of clinical manifestations are observed in melioidosis ranging from asymptomatic to septicemic infection. Although serodiagnostic methods of melioidosis have been improved significantly in recent years, a highly specific diagnostic test that can differentiate asymptomatic seropositive individuals and melioidosis patients remains to be the subject of current investigations. In this study, a B. pseudomallei-specific gene, pBps-1, expressing a novel 18.7 kDa recombinant protein was selected from genomic libraries of two B. pseudomallei virulent isolates by using pooled sera from septicemic melioidosis patients. Nucleotide sequence analysis demonstrated that this gene is unique and does not show substantial similarity with any known genes in the Genbank database. The Bps-1 recombinant protein was evaluated for its potential in serodiagnosis of melioidosis by Western blot analysis. A high degree of specificity was demonstrated using sera from healthy individuals in the endemic (98.5%) and non-endemic areas (100%), with moderate sensitivity (69.7%) in melioidosis patients. The study demonstrated that this approach can be used to obtain highly specific recombinant antigens such as that described in the present report. A combination of such antigens should provide materials for successful serodiagnosis of melioidosis in the endemic areas.

Hepatitis is one of the most important infectious diseases in Thailand. The knowledge of host factors that influence the course of the disease is still limited. In this study, the HLA class I and class II phenotypes were analyzed in the 2 groups of HCV-infected Thai populations. The first group included 43 individuals with transient HCV infection (HCV antibody positive, HCV RNA PCR negative), and the second included 57 individuals with persistent chronic HCV infection (HCV antibody positive, PCR positive). HLA class I typing was performed by 2-stage microlymphocytotoxicity test, and HLA class II typing, by PCR-SSO. No significant difference in the frequencies of HLA-A and -B antigens was observed between the 2 groups of HCV-infected individuals. The frequency of DRB1*0301 and DQB1*0201 was significantly higher in the persistent-infection group than in the transient-infection group (Pc = 0.03, Pc = 0.04, respectively). In addition, DRB1*0701 and DQA1*0201 were significantly decreased in all the HCV-infected patients compared with levels in the normal controls (Pc = 0.003, Pc = 0.001, respectively). This study demonstrated that DRB1*0301 and DQB1*0201 are associated with persistent HCV infection, whereas DRB1*0701 and DQA*0201 are associated with protection against HCV infection.

Melioidosis is an important infectious disease endemic in Southeast Asia and the Northern territories of Australia. Septicemic melioidosis, is the leading cause of fatality from community acquired septicemia in northeastern part of Thailand where death often occurs within a few days after hospitalization. The present study was carried out to investigate the polymorphisms of the position -308 promoter region of the TNF-alpha gene, as well as of the intron 1 of the TNF-beta gene in patients with melioidosis compared with normal uninfected controls in the same endemic area. The gene frequency of TNF2 allele was significantly higher in melioidosis patients compared with control subjects (p = 0.0097, relative risk 2.32). The increase in TNF2 allele in melioidosis patients was found in both heterozygous and homozygous forms. In addition, the increase in TNF2 allele was most apparent in patients who had fatal outcome from septicemic melioidosis (p = 0.017), but was also observed with lesser degree in other groups of melioidosis patients. However, no difference in the frequency of TNF-beta polymorphism the melioidosis patients was observed.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in Southeast Asia. The septicemic form of melioidosis is the leading cause of death due to community-acquired bacteremia in the northeastern part of Thailand. The delay in isolation and identification of the causative organism is a major contributing factor to the high mortality. The present study describes the evaluation of a latex agglutination test for rapid identification of the bacteria directly from blood cultures. The Bps-L1 monoclonal antibody recognized the lipopolysaccharide antigen of 96.8% of B. pseudomallei clinical isolates and was highly specific for B. pseudomallei. The diagnostic value of the latex agglutination test based on Bps-L1 monoclonal antibody was prospectively evaluated in an area endemic for melioidosis. The agglutination test kit was evaluated in 88 blood cultures with gram-negative bacteria identified with Gram staining. The sensitivity and specificity of the test kit were both 100%. These results indicated that the detection of B. pseudomallei lipopolysaccharide by specific monoclonal antibody in a latex agglutination format is clinically useful for the rapid identification of the bacteria in blood cultures in areas endemic for melioidosis.

The genotypic distribution of hepatitis C virus (HCV) isolated from blood donors from four major regions of Thailand was studied by reverse hybridization assays. PCR-amplified products from the 5' noncoding and core regions of the viral genome were hybridized to genotype- and subtype-specific probes which were immobilized on the nitrocellulose membrane. Of 332 anti-HCV-positive plasma samples studied, 71% contained HCV RNA. HCV genotype 3a was the most prevalent genotype (39%), followed by genotype 1b (20%) and genotype 6 group variants (18%). HCV genotype 1a was identified among 9% of all isolates. Other genotypes (genotype 1 which was neither 1a nor 1b, genotype 3b, and an unclassified genotype) were uncommon. There was no difference in the mean age of the donors infected with different HCV genotypes. The genotypic distribution pattern of HCV was similar among HCV isolates from different regions of Thailand.

Previous studies on the prevalence of hepatitis C virus (HCV) infection in Asian countries reported an average prevalence of less than 1.5%. In this study a combination of second- and third-generation enzyme immunoassays (EIAs), immunoblot analysis, and polymerase chain reaction was used to evaluate the prevalence of HCV infection in 3,255 volunteer blood donors in northeastern Thailand. Antibodies to HCV were detected in 6.5% of male blood donors and 0.9% of female blood donors, giving an overall prevalence of 5.6% in this population (gender-adjusted prevalence of 3.7%). The prevalence was higher in males than in females (P < 0.0001) and increased with age, reaching a peak at 31-40 years of age. More than 90% of the EIA-positive samples tested positive by immunoblot analysis, giving an estimated minimal prevalence of antibodies to HCV in the blood donors of 5.2%. Approximately 80% of the EIA-positive blood donors were viremic as determined by the presence of HCV RNA detected by the polymerase chain reaction, indicating that at least 4.5% of volunteer blood donors had detectable HCV RNA and were considered potentially infectious. The prevalence of HCV infection in this population was higher than that in previous reports for central and northern Thailand, while the prevalence of HBV infection was similar to that in other regions of the country. This study clearly demonstrated a very high prevalence of HCV infection in northeastern Thailand, especially in the male population.

Melioidosis, an infection caused by Burkholderia pseudomallei, is endemic in southeast Asia. The septicemic form of melioidosis is the leading cause of death from nonhospital-acquired septicemia in the northeastern part of Thailand. A major factor that contributes to the high mortality is the delay in isolation and identification of the causative organism. The present study was undertaken to evaluate the use of enzyme-linked immunosorbent assays based on an immunoaffinity-purified antigen for detecting specific IgG and IgM antibodies to this organism as a rapid serodiagnostic method for melioidosis. The diagnostic value of these tests was evaluated in an actual clinical situation in an area endemic for melioidosis. The specificity of specific IgG test (82.5%) and the specific IgM test (81.8%) were significantly better than that of the indirect hemagglutination (IHA) test (74.7%). The sensitivity of the specific IgG assay (85.7%) was higher than that of the IHA test (71.0%) and the specific IgM test (63.5%). Specific IgG antibody was detected in a majority of septicemic melioidosis (87.8%), as well as in localized forms (82.6%). The specific IgG test was also better than the specific IgM test and the IHA test in identifying acute melioidosis cases in the first five days after admission. In addition, the IgG antibody level to this antigen remained high over a period of more than five years in those who had recovered from melioidosis and remained clinically free of the disease. These results indicate that the detection of specific IgG antibody is clinically useful for the diagnosis of acute melioidosis in an endemic area.

An improved system for amplification of hepatitis C virus genome (HCV) was developed based on a multiplex nested polymerase chain reaction format. Two sets of oligonucleotide primers were used simultaneously. One was derived from the conserved sequences in the 5' non-coding region of the viral genome which can bind to the viral genome of all genotypes. The other set of primers was designed from a sequence in the nonstructural-5 region of HCV. HCV genotypes 1 and 3 can be differentiated by the banding patterns of amplified DNA products. All of 39 samples containing the HCV genotype 1 could be amplified with primers in the 5' non-coding region only, whereas 92% of those with genotype 3 could be amplified by both primer sets. In addition, HCV RNA can be detected in 81% of 84 anti-HCV-positive blood donors and in 0% of 34 anti-HCV-negative cases. Of the HCV RNA-positive specimens, 69% showed genotype 1-like patterns while 31% showed genotype 3-like patterns. The detection rate of HCV RNA in this study was much higher than that in our previous report due to the improvement of new primers which can detect all genotypes of the virus. In conclusion, this improved amplification system is a sensitive method for rapid identification of HCV RNA in clinical specimens that can simultaneously differentiate the two most common genotypes of HCV found in Thailand.

The gene encoding nucleocapsid (core) protein of hepatitis C virus (HCV) was isolated from a Thai blood donor infected with HCV genotype 1b. The nucleotide sequence of this clone showed a high degree of homology to that of 4 HCV strains isolated from other Thai blood donors as well as that of the HCV prototypes of genotypes 1a, 1b, 2a and 3a. The entire region of the core gene was cloned into an expression plasmid pGEX-3X to be expressed as a fusion protein with glutathione-S-transferase (GST). E. coli transformants containing this plasmid did not express the fusion protein. However, GST-HCV core fusion protein could be produced when the core gene was truncated at the 3' end resulting in a gene encoding only the first 123 amino acid residues of the core protein. This fusion protein was insoluble in standard buffers, but could be solubilized by sarkosyl and thus subsequently purified using glutathione-Sepharose 4B. The purified fusion protein was immunogenic and could react with antibodies from blood donors infected with all genotypes of HCV found in Thailand. In addition, two murine hybridoma clones secreting monoclonal antibodies specific to the recombinant HCV core protein were produced. The purified HCV core protein and the monoclonal antibodies to the recombinant protein will be useful for developing assay systems for detecting anti-HCV antibodies and HCV antigen, respectively.