Suppression of the inhibitory effect of denatured albumin on the polymerase chain reaction by sodium octanoate: application to routine clinical detection of hepatitis B virus at its infectivity threshold in serum.

Ten to fifteen percent of posttransfusion viral hepatitis cases are still caused by HBV despite mandatory third generation screening procedures for HBsAg. There is thus an urgent need for a simple, time-cost-effective, but very sensitive test for routine HBV DNA detection in serum. Nested-primed PCR has been shown to detect purified HBV DNA at its infectivity threshold in serum. Since this is too labor-intensive for routine testing, we assessed the efficiency of a Fast PCR procedure, of three pairs of primers, and of thirty-five simple serum pretreatments with the aim to achieve the same sensitivity level. Using ten-fold dilution in phosphate buffered saline as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at the 2 x 10(3)/ml level in serum. Using either NaOH denaturation or sodium octanoate thermoprotection as pretreatment and Fast PCR for 99 cycles, we were able to detect HBV DNA at its infectivity threshold in serum, while the classical phenol/chloroform/isoamylic alcohol/isopropanol/ethanol DNA purification procedure enabled us to reach the 10 virus particles/ml level. These results suggest that denatured albumin is responsible for the well known inhibitory effect of serum proteins on Taq polymerase. Because of its simplicity and its lower risk of sample-to-sample cross-contamination, the sodium octanoate thermoprotection method was chosen for routine clinical detection of HBV in serum. The clinical usefulness of this approach is demonstrated by the results obtained with HBsAg-negative acute hepatitis B incubation sera and with anti HBe-positive chronic hepatitis B sera.

Prevalence of high risk genital papillomaviruses in the Belgian female population determined by fast multiplex polymerase chain reaction.

Because in situ/filter hybridisation is not sensitive enough and because classical polymerase chain reaction (PCR) protocols are generally not sufficiently reproducible and specific, there is little accurate information on the prevalence of human papillomaviruses (HPV) 16, 18, and 33 infections in women without dyskaryotic changes of the cervix. In our hands, our Fast Multiplex PCR protocol has always been the most sensitive, specific, and reproducible DNA detection assay in all the microbiological and haematological applications we attempted (Vandenvelde C, Verstraete M, Van Beers D [1990]: Journal of Virological Methods 30:215-228; Vandenvelde C, Scheen R, Corazza F, Van Beers D [1991a]: Journal of Experimental and Clinical Hematology 33:293-297; Vandenvelde C, Scheen R, Van Beers D, Fondu P [1991b]: Journal of Experimental and Clinical Hematology 30:25-29). Using this new technique, cervical scrapes from 336 Belgian women attending the cervical cancer screening clinic were examined for the presence of these three high-risk genital papillomaviruses. Positive results were confirmed using another set of HPV-specific primers. Exactly one sixth of our population was found positive for one or more of these HPVs. Types 33 and 16 were significantly more prevalent than type 18. The nonparametric statistical analysis of the data suggests that some risk factors such as particular sexual habits, that are inversely related to age, must exist.

Fast PCR based therapeutic follow-up of T-cell malignancies.

Available methods for the detection of minimal residual disease in T-cell malignancies are limited by their poor sensitivity and/or by their complexity. With the aim of avoiding these drawbacks, we used the Fast PCR technique in order to amplify V delta 1-(D delta 1)-(D delta 2)-J delta 1 and V gamma I family-J gamma junctional sequences from nucleated cells of boiled bone marrow. We were thus able to detect malignant T-cells down to a dilution of 1 in 665 nucleated marrow cells, in less than 4 hours after sampling. This new quantitative method is promising for monitoring therapy and detecting early disease relapse in T-lymphoproliferative disease, since it is 2 to 35 fold more sensitive than Southern blotting.

Preliminary results of hepatitis B vaccination (HEVAC B) in healthy subjects and haemodialysis patients.

Hepatitis B vaccine (Hevac B, Pasteur) was assessed in 52 healthy and 25 haemodialysis individuals. The percentage of hepatitis B surface antibody seroconversion was 100% in the first group but only 57.7% in the other one. The mean levels of hepatitis B surface antibody, 3 months after the first injection were respectively 222 and 42 milli International Units per ml. Five health-care workers, who experienced an accidental exposure were protected by a combined passive-active immunization.