Chia C. Pao

The detection of mycobacterial DNA sequences in uncultured clinical specimens with cloned Mycobacterium tuberculosis DNA as probes

A plasmid DNA library was constructed from restriction endonuclease digested genomic deoxyribonucleic acid (DNA) of a virulent strain of Mycobacterium tuberculosis isolated from sputum of a patient. The sensitivity and specificity of two of the cloned DNA fragments in detecting M. tuberculosis and its related DNA sequences were analysed by DNA-to-DNA hybridization. The level of detection was determined to be 50 picograms of M. tuberculosis DNA, which is approximately equivalent to 10,000 mycobacterial genomes. These two M. tuberculosis DNA probes did not cross-hybridize to DNA of non-mycobacterial origin, nor with DNA from 9 out of 11 other mycobacterial species. Mycobacterial DNA sequences could be detected in 134 of 441, or 30.4%, of various types of uncultured clinical specimens from 365 patients by the DNA probes, whereas traditional culture method showed only a 19.0% positivity rate for the same specimens (p less than 0.001). The overall sensitivity and specificity of the DNA probes in detecting M. tuberculosis are 90.5% and 83.8% respectively. The DNA hybridization test may become a useful tool for the early and rapid determination of mycobacterial infection in uncultured clinical specimens.

Fetal cells in the maternal circulation during first trimester in pregnancies

To investigate the presence of fetal cells in the maternal circulation during early pregnancy, the polymerase chain reaction was used to test the presence of human Y chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood specimens from 19 women carrying male fetuses and 12 women carrying female fetuses. The presence of fetal cells was suggested as early as 6 weeks gestation in 1 of the 19 women bearing male fetuses. Fetal cells were present in the maternal circulation of 15 of the 19 women by 9 weeks gestation, and in only 1 of the 19 were fetal cells not detected until the 12th week after conception. These results suggest that identification of fetal cells in the maternal circulation is possible with a properly designed and executed polymerase chain reaction. However, there was considerable variation with respect to when these fetal cells first became detectable during pregnancy. These fetal cells are potentially a valuable source of material for biochemical and genetic studies of the fetuses.

Presence of fetal cells in maternal circulation after delivery

Fetal cells can still be detected in maternal blood 8 month postpartum.

Hepatitis Type B Virus DNA in Patients Receiving Hemodialysis: Correlation with Other HBV Serological Markers

Possible presence of hepatitis type B virus (HBV) was assessed in 239 end-stage renal failure patients who were receiving long-term maintenance hemodialysis (average 30.8 months; duration: 1-94 months), and who had not shown any other symptom of HBV infection. Their HBV serological markers, including HBV DNA, were evaluated together with those of normal control individuals. HBV surface antigen (HBsAg) was detected in 42 of the 239 dialysis patients, 15 of whom also positive for HBV DNA (mean +/- SD = 56.2 +/- 23.7 pg/100 microliters of serum). HBV DNA was also found in 22 of the 197 (11.2%) dialysis patients who were negative for HBsAg, with an average of 36.2 +/- 19.0 pg/100 microliters of serum. This rate of detecting HBV DNA in HBV seronegative dialysis individuals was significantly higher than the rate of 1.83% found among healthy HBsAg(-) individuals. Among these 22 dialysis patients who were HBsAg(-) but HBV DNA(+), 15 were found to possess antibodies against HBsAg (anti-HBs) and/or antibody against HBV e antigen (anti-HBe). These data suggested that the absence of serum HBV antigen or the presence of antibodies against HBV markers might not be sufficient to identify possible HBV infection in immunocompromised hosts such as hemodialysis patients.

Presence of Cells of Fetal Origin in Maternal Circulation of Pregnant Women

Fetal cells can be identified by using the polymerase chain reaction to test for the presence of human Y-chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood of women who bear a male fetus. Thirty-one pregnant women were studied in the first trimester to determine when fetal cells become detectable in the maternal circulation. Among the 19 women whose peripheral blood samples were positive for Y-chromosome-specific DNA sequences, the presence of fetal cells was quite case-variable from the 6th to 12th gestational weeks. Twenty-eight women who had given birth to their first male babies were studied postpartum to determine when fetal cells disappear from the maternal circulation. Fetal cells can still be detected in maternal blood 10 months postpartum in some cases. These results suggest that identification of fetal cells in the maternal circulation is possible. Nevertheless, interpretation of fetal cells in maternal circulation should be handled very carefully with respect to when these fetal cells first became detectable and potential interference from previous pregnancies.

Amplification of viral RNA for the detection of dengue types 1 and 2 virus

In vitro DNA amplification by means of the polymerase chain reaction (PCR) was used to amplify dengue types 1 and 2 viral genomes in cultured cells and in the serum of persons infected with dengue virus. Results of the present investigation suggest that the PCR method is type-specific in detecting dengue virus and has a detection sensitivity of less than 100 plaque-forming units (pfu) for both serotypes of the virus. The PCR method may be useful for detecting and typing dengue virus in clinical and epidemiological specimens.

Intra blood-cerebrospinal fluid-barrier detection of hepatitis B virus

Hepatitis B, a major public health concern worldwide, is caused by hepatitis type B virus, a hepdnavirus that infects only human and certain nonhuman primates, and replicates strictly in hepatocytes. By using the techniques of slot and Southern blot DNA hybridization, and electron microscopy, the presence of HBV was identified in the cerebrospinal fluid of three affected individuals.