Overcoming the errors of in-house PCR used in the clinical laboratory for the diagnosis of extrapulmonary tuberculosis.

Our experiences from 1993 to 1997 in the development and use of IS6110 base PCR for the diagnosis of extrapulmonary tuberculosis in a routine clinical setting revealed that error-correcting processes can improve existing diagnostic methodology. The reamplification method initially used had a sensitivity of 90.91% and a specificity of 93.75%. The concern was focused on the false positive results of this method caused by product-carryover contamination. This method was changed to single round PCR with carryover prevention by uracil DNA glycosylase (UDG), resulting in a 100% specificity but only 63% sensitivity. Dot blot hybridization was added after the single round PCR, increasing the sensitivity to 87.50%. However, false positivity resulted from the nonspecific dot blot hybridization signal, reducing the specificity to 89.47%. The hybridization of PCR was changed to a Southern blot with a new oligonucleotide probe giving the sensitivity of 85.71% and raising the specificity to 99.52%. We conclude that the PCR protocol for routine clinical use should include UDG for carryover prevention and hybridization with specific probes to optimize diagnostic sensitivity and specificity in extrapulmonary tuberculosis testing.

Detection of HIV-1 window period infection in blood donors using borderline anti-HIV results, HIV-1 proviral DNA PCR, and HIV-1 antigen test.

Prevention of transmission of HIV-1 via blood transfusion has been carried out by the National Blood Center by screening donated blood with anti-HIV and HIV antigen tests. To increase the safety measure, detection of proviral DNA by PCR has been proposed; however, it was impractical to test all samples by PCR. From August 1994 to September 1995, there were 296,169 blood donors with 0.32 per cent prevalence of anti-HIV positive. From these donors, 153 samples of which the anti-HIV enzyme immunoassay optical density (OD) between cutoff and 80 per cent of cutoff value (borderline results) were selected for PCR testing. One out of 153 borderline cases showed positive by PCR test for HIV-1 proviral DNA. However, this case was also positive by HIV antigen test. Therefore, most of the samples with borderline anti-HIV results were true negative for HIV infection. On the other hand, there were 8 HIV antigen positive samples which had anti-HIV OD below the borderline value determined in this study. This finding confirmed the necessity of using both the anti-HIV and HIV antigen tests for screening of donated blood.

Detection of human immunodeficiency virus type 1 (HIV-1) proviral DNA in breast milk and colostrum of seropositive mothers.

There is increasing evidence of vertical transmission of HIV-1 to infants through breast feeding of milk from HIV-1 infected mothers. It has been postulated that transmission occurs mainly via ingestion of infected cells in breast milk and colostrum. In this study, detection of HIV-1 proviral DNA was used to prove that cells from colostrum and milk do contain HIV. DNA were extracted from these cells of colostrum and milk of 18 seropositive mothers and amplified by nested PCR for HIV-1 gag and pol and 44 per cent were positive mostly by two primers. All ten negative control samples from seronegative mothers were negative. This study demonstrated the infectivity of breast milk and colostrum. Nevertheless, recommendation against breast-feeding should be weighed against poor alternatives in low socioeconomic families.

Enzyme-linked immunosorbent assay for leptospirosis immunoglobulin M specific antibody using surface antigen from a pathogenic Leptospira: a comparison with indirect hemagglutination and microagglutination tests.

A search for a sensitive and specific test for human leptospirosis was made by enzyme-linked immunosorbent assay for immunoglobulin M specific antibody (IgM ELISA) using a surface antigen from L.interrogans serovar bataviae, L. interrogans serovar pyrogenes and L.interogans serovar icterohaemorrhagiae. The IgM ELISA tests using each of the three antigens were evaluated in 103 sera primarily positive by microagglutination test (MA). Optical density of these IgM ELISA tests showed good correlation. The IgM ELISA using antigen from serovar bataviae was compared with MA and indirect hemagglutination (IHA) in 20 sera primarily positive by IHA, and 103 sera primarily positive by MA. IgM ELISA and IHA using antigen prepared from serovar bataviae in 103 sera positive for MA had a sensitivity of 98.06 and 92.23 per cent respectively. In 20 sera primarily positive by IHA, IgM ELISA and MA showed sensitivity of 80 and 45 per cent respectively. The surface antigen used in IgM ELISA is broadly specific making IgM ELISA a sensitive and specific test for human leptospirosis. IHA agreed more with IgM ELISA in comparison to MA. As MA is not sensitive for early infection, IHA and IgM ELISA should be in routine use in general laboratories.

Antibodies to hepatitis C virus among patients with hepatocellular carcinoma and blood donors in Thailand.

HCC is the most cancer among Thai men. It is not known if HCV plays an oncogenic role in HCC in this country where HBV is endemic. Anti-HCV and HBsAg were assayed in 154 sera from HCC and 3,387 voluntary blood donors. The prevalence of anti-HCV in HCC (8.4%) was significantly higher than blood donors (1.38%). The prevalence of HBsAg in HCC (61%) was also significantly higher than blood donors (5.28%). The prevalence of anti-HCV in HCC was lower than that of Spain, Italy, Africa and Taiwan. Anti-HCV was found associated with a small portion of patients with HCC while HBV was found closely associated with the larger proportion of HCC. HCV in normal Thais was as common as those in southern Europe and HCV was found associated with HCC. However, HBV remains the major etiological factor of HCC in Thailand.

Laboratory diagnosis of congenital and maternal rubella infection: a review.

Physicians are aware of the congenital rubella syndrome. Serodiagnosis is usually used to detect rubella infection in pregnant women and their fetuses. Although being considered the cornerstone of serodiagnosis, the hemagglutination inhibition test is gradually being replaced by new more convenient methods. Tests to detect IgM eliminate the need for paired sera to diagnose acute rubella infection. However, because of the possibilities of false positive, IgM results should be interpreted with caution. Detection of IgM in cord blood and new genetic technology made the diagnosis of infection in utero possible. The evidence of reinfection in people considered to be immune is abundant; however, discovering new antigenic determinants correlating with immunity may solve the problem and a new vaccine and antibody test that is truly associated with immunity will be available in the future.