Genotyping of human platelet antigens in ethnic Northeastern Thais by the polymerase chain reaction-sequence specific primer technique.

Human platelet antigens (HPA) are important in neonatal alloimmune thrombocytopenia (NAITP), post-transfusion purpura (PTP), refractoriness to platelet transfusion therapy and population genetics. The distribution of HPA in a Northeast Thai population was studied. 300 healthy, unrelated, and ethnic Northeastern Thais were randomly selected. Using the polymerase chain reaction-sequence specific primer technique (PCR-SSP), the frequency of HPA-1, -2, -3, -4, -5 and -6 were determined. The phenotype frequencies were 100 per cent for HPA-1a, 4a, 5a, and 6a. For HPA-1b, 2a, 2b, 3a, 3b, 5b and 6b, the frequencies were 5.7, 99.7, 12.3, 78.0, 71.3, 7.3 and 3.0 per cent, respectively. The HPA-4b was not found. The HPA frequencies in our subjects were quite similar to other Asian populations but were different from Caucasians. The distribution of HPA genotypes encountered in our study indicate that HPA-1a, -4a, -4b, -5a and -6a will not be involved in NAITP, PTP and refractoriness to platelet transfusion therapy in Northeastern Thais. Moreover, HPA-1b, -2a, -2b, -3a, -3b, -5b and -6b may induce alloantibodies in these patients.

Anti-P1: the most common unexpected antibodies in northeastern-Thais.

The prevalence of unexpected antibodies in the Northeastern-Thai population was studied. Sera were collected from 25,673 blood donors including 18,209 males and 7,464 females. The sera were screened for unexpected antibodies by saline and enzyme techniques. The sera which gave a positive antibody screening test were identified for specificity of antibody. The result demonstrated that 3,928 from 25,673 samples (15.30%) were positive for the antibody screening test and only 3,883 samples could be identified for specificity of antibody. The most common unexpected antibodies were anti-P1, anti-lewis and anti-P1 + anti-lewis with the frequency of 70.8, 18.6 and 10.1 per cent, respectively. The prevalence of anti-P1 in this study was higher than that reported in Central Thailand and Southeast Asia which may due to the high prevalence of liver fluke infection in the Northeastern-Thai population.

Anti-HIV antibody titer: an alternative supplementary test for diagnosis of HIV-1 infection.

The diagnosis of HIV infection is based on screening of HIV antibodies and confirmed by a more specific supplementary test. The most common confirmation test is Western blot, which is expensive, time consuming and subject to technical skill. The present study was carried out to evaluate whether the anti-HIV-1 antibody titer is valid as a supplementary test for diagnosis of HIV-1 infection. Anti-HIV-1 antibody titers of 2,414 anti-HIV-1 positive sera determined by the particle agglutination (PA) method were analysed in comparison with the Western blot analysis. The Western blot negative result was found in 11 of 2,414 (0.46%) anti-HIV-1 positive sera, these sera also gave negative anti-HIV by ELISA. The PA titers of these sera were found in the range of 16 to 64. Seventeen samples (0.70%) with anti-HIV-1 in the titer range of 16 to 256 showed indeterminate Western blot analysis. The rest, 2,386 of these 2,414 sera (98.84%), were shown to be positive by Western blot. However, all of the 2,356 sera with antibody titers > or = 512 (97.6%) demonstrated positive Western blot results. Five cases among the 17 (29.4%) indeterminate sera were examples of early seroconversion of HIV infection, which were confirmed in follow up specimens. The results suggest that only the samples with antibody titers < 512 are required to be confirmed for HIV infection by Western blot. It is possible that early seroconversion may be inferred from anti-HIV titers. Therefore, in order to reduce time and cost, the PA anti-HIV titer can be used as an alternative supplementary test for diagnosis of HIV-1 infection in most positive screened anti-HIV samples. Western blot is needed for testing in only a few cases.

Production and evaluation of Taq DNA polymerase.

Taq DNA polymerase is an enzyme essential in performing Polymerase Chain Reaction (PCR) which has recently become a basic technology in research and diagnostic laboratories. In order to reduce the cost of research work in Thailand, recombinant Taq DNA polymerase was locally produced from pTaq cloned in E. coli. The enzyme was characterized and evaluated in comparison with the commercial Taq DNA polymerase produced by Perkin Elmer Cetus, U.S.A. The yield of enzyme was 6.72 mg/ml and the activity of 9,524 units/mg protein with the total of 448,000 units/litre of the bacterial culture. The preparation was free of DNase based upon its ability to degrade Lambda DNA evaluated by gel electrophoresis. Although the enzyme produced gave a high DNA polymerase activity, the preparation was not as pure as the enzyme produced by Perkin Elmer Cetus. Immunoblot analysis indicated that the enzyme preparation contained the products of enzyme degradation obtained during preparation and bacterial protein contaminations. In spite of the existence of bacterial proteins in the preparation, the Taq enzyme produced was proved to be applicable in performing PCR such as the PCR-SSP (Sequence Specific Primers) typing for HLA-DR. The cost of enzyme preparation was about 256 times less than that of the commercial enzyme. Economically, the locally produced Taq DNA polymerase can be used efficiently in the research laboratories performing PCR based typing of the HLA genes.

DNA typing of the HLA-A, -B and -C genes: possible MHC class I haplotypes in the northeastern-Thais.

The phenotype and gene frequencies of HLA class I were studied in the Northeastern Thai population. Blood samples were collected from 100 unrelated healthy northeastern-Thais. HLA-A, -B and -Cw alleles were determined using the polymerase chain reaction- amplification refractory mutation system (PCR-ARMS). 12 HLA-A, 20 HLA-B and 14 HLA-Cw alleles were found. Linkage disequilibrium analysis indicated the existence of 7 HLA-A-B and 19 HLA-B-Cw haplotypes. A*0207-Cw*01-B*4601 was the most common possible haplotype in this population. These results provide regional basic information for further studies in anthropology, organ transplantation and MHC disease associations in the northeastern-Thais.

Seroepidemiology of HTLV-I infection in northeast Thailand: a four year surveillance.

The human T-lymphotropic virus type I (HTLV-I) can be transmitted through blood transfusion, sexual contact, perinataly and by breast feeding. We carried out a four years seroepidemiology surveillance study of HTLV-I infection among northeast Thai population by screening for antibodies to HTLV-I (anti-HTLV-I) in 1992, 1993, 1995 and 1997. A total of 8,323 blood samples were collected from 6,228 blood donors, 832 pregnancies, 219 multitransfused patients, 53 HIV positive intravenous drug users and 1,000 northeast-Thai workers at different periods of time. The serum samples were tested for anti-HTLV-I by particle agglutination (PA) technique and confirmed by Western blot. One sample from a multitransfused patient collected in 1992 and one sample from a blood donor collected in 1995 demonstrated positive anti-HTLV-I screening by PA but negative by Western blot. This finding indicates that at present HTLV-I is not a public health problem in the northeast of Thailand but surveillance should be continually conducted.

Association of HCV and Treponema pallidum infection in HIV infected northeastern Thai male blood donors.

The study was performed to determine the association of seroprevalence of hepatitis C virus (HCV) and Treponema pallidum (T. pallidum) infection among HIV infected first time male blood donors (HIV group) in comparison with the HIV seronegative blood donors (control group) in the Northeast of Thailand (NET). Serum samples were collected from 10,321 first blood donation voluntary male donors. All samples were screened for anti-HIV and anti-HCV by particle agglutination test, and syphilis antibody by RPR. The anti-HIV positive sera were repeated by EIA and confirmed by western blot. The reactive anti-HCV samples were confirmed by EIA whereas reactive syphilis antibody samples were confirmed by TPPA. Fisher's exact test was used for statistical analysis. The prevalence of anti-HIV in first time male donors was 0.70 per cent (72/10,321). The age of HIV group and 10,018 male control group ranged from 17-50 years old. The prevalence of HIV among 21-40 years old age group was significantly higher than the 17-20 years old (p = 0.00003). The 17-20 years old HIV group showed significantly higher sero-prevalence of TPPA (p = 0.003). The 21-30 years old HIV group gave significantly higher sero-prevalence of anti-HCV (p = 0.0008) and TPPA (p = 0.045), but the seroprevalence of anti-HCV and TPPA among the 31-50 year old group were nonsignificantly different (p > 0.05). The concurrence of anti-HCV and TPPA in HIV groups was not found. This result indicated that HIV infection among NET voluntary male blood donors was significantly associated with T. pallidum infection in young adults and the HCV infection in mature adults.

Distribution of HLA-A and B antigens in northeastern-Thais.

The phenotype and gene frequencies of HLA class I were studied in the Northeastern Thai population. Blood samples were collected from 100 unrelated healthy Northeastern Thais. HLA-A and B antigens were typed by using the standard microlymphocytotoxicity test. Twelve HLA-A and twenty-five HLA-B antigens were found in this population. HLA-A2, A24, A11 and the HLA-B46, B15, B22 antigens are commonly found in this group. Linkage disequilibrium analysis indicated the existence of fifteen haplotypes. The HLA-A2, B46 haplotype was the most common. These results will be useful for further studies in anthropology, organ transplantation and MHC associated disease in Northeastern Thais.

Reducing the risk of HIV transmission through blood transfusion by donor self-deferral.

A cross sectional study was conducted to evaluate the validity of implementing a blood donor self-deferral form for reducing the risk of HIV transmission through blood transfusion. The self-deferral form which was given to all blood donors, included questions about HIV risk factors in the three month period prior to blood donation. Donors were asked to declare confidentially whether their blood was safe for transfusion or not. Blood was collected and examined for HIV antigen, anti-HIV antibodies, HBsAg and syphilis antibodies. All of the serological markers detected among high risk donors and general donors were compared and analysed by Yates corrected X2 test and one-tailed Fisher's exact test with a significance level of 0.05. There were 401 self-deferred high risk donors and 15,523 general donors. The HIV antigen was found as a single marker in only one male high risk individual. The prevalence of anti-HIV antibodies, HBsAg and syphilis antibodies among the general donors was 0.61%, 5.29% and 1.17%, respectively. The anti-HIV, HBsAg and syphilis antibodies in the high risk donors were 1.99%, 7.98% and 1.25%, respectively. In comparison with the general donors, the high risk donors demonstrated statistically significant higher prevalence rates of HIV antigen (p < 0.05), anti-HIV (p < 0.005) and HBsAg (p < 0.05). In conclusion, donor self-deferral is valid for reducing the risk of HIV transmission through blood transfusion and its implementation should be encouraged when recruiting blood donors.

Preliminary survey of anti-HTLV-I in northeastern-Thais.

The anti-HTLV-I prevalence was studied by particle agglutination technique (PA) in 2,609 samples including: 403 antenatal care women from Srinagarind Hospital, Faculty of Medicine, 342 high-risk sexually transmitted disease blood donors and 1,864 randomly selected blood donors from the Blood Transfusion Centre, Faculty of Medicine, Khon Kaen University. All of the 2,609 samples gave negative anti-HTLV-I. The result revealed that HTLV-I infection is not common in northeast-Thailand. However, as the sample size from this study may be too small, further study should be carried out.

Immunoblot analysis to demonstrate antigenic variability of clinical isolated. Pseudomonas pseudomallei.

Pseudomonas pseudomallei (Ps.ps.) is the causative organism of melioidosis, and is widely distributed in Southeast Asia and Northern Australia. Clinical manifestations range from subclinical infection to fulminant septicemia. To demonstrate the antigenic variability of Ps.ps., 62 clinical isolates from 31 blood, 13 sputum, 9 pus, 3 urine and 6 body fluid culture specimens were studied by SDS-PAGE and immunoblotting. In SDS-PAGE, there were approximately 20 antigenic components with molecular weights ranging from 14 to 66 kilodaltons (KD) which suggested that there was antigenic variability among these 62 clinical isolates of Ps.ps. Attempts to correlate immunoblot profiles with clinical illness or sources of specimens were not successful but 6 common antigens were identified with molecular weight of 17.5, 21, 33, 34, 40 and 45 KD, respectively. Among these antigens, the 45 KD component was recognised by all patients' sera. Thus, the 45 KD protein antigen may be useful for the future approach in immunodiagnosis of melioidosis.