A one-step reverse transcription quantitative PCR (RT-qPCR) assay in combination with rapid RNA extraction was evaluated for routine testing of hepatitis C virus (HCV) RNA. Specific primers and probes were designed for the detection of a 150 bp sequence located in the 5'untranslated region (5'UTR) of HCV RNA. The target sequence was selected as the most conserved region between the six known HCV subtype sequences following an alignment. The assay was able to quantify a dynamic linear range of 10⁸ to 10¹ plasmid copies/reaction (r²  = 0.98) containing the target sequence. Two copies of this HCV plasmid corresponds to one international unit (IU) measured using a standard obtained by serial dilutions of the World Health Organization (WHO) standard. The detection limit of the assay was about 10 IU/mL of HCV RNA (20 copies/mL) in plasma samples. The assay was comparable to Cobas AmpliPrep/Cobas TaqMan® HCV Test, v2.0 Quantitative assay (Roche Molecular Systems, Inc., Branchburg, NJ) with correlation coefficient r²  = 0.98. The present assay could be completed within 3 hours from RNA extraction to data analysis of at least 30 plasma samples. Our test provides sufficient sensitivity, specificity, and reproducibility and proved to be fast, labor-saving, and cost-effective. Indeed, our system will definitely allow low-income countries to monitor accurately this viral infection and to efficiently treat their infected patients.

The use of isotopically labeled endonuclease digestion products allows for the relative quantification of ribonucleic acids (RNAs). This approach utilizes ribonucleases such as RNase T1 to mediate the incorporation of 18O onto the 3'-terminus of the endonuclease digestion product from a solution containing heavy water (H2 18O). The accuracy and precision of relative quantification are dependent on the efficiency of isotope incorporation and minimizing any possible 18O to 16O back-exchange before or during mass spectral analysis. Here, we have investigated the stability of 18O-labeled endonuclease digestion products to back-exchange. In particular, the effects of pH, temperature and presence of RNase on the back-exchange process were examined using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have found that back-exchange depends on the presence of the RNase--back-exchange was not observed once the enzyme was removed from the sample. With RNase present, at all pH values examined (from acidic to basic pH), back-exchange was detected at incubation above room temperature. The rates and extent of back-exchange were similar at all pH values. In contrast, back-exchange in the presence of RNase was found to be especially sensitive to incubation temperature--at temperatures below room temperature, minimal back-exchange was detected. However, back-exchange increased as the incubation temperature increased. Based on these findings, appropriate sample-handling and sample storage conditions for isotopically labeled endonuclease digestion products have been identified, and these conditions should improve the accuracy and precision of results from the relative quantification of RNAs obtained by this approach.

Hepatitis B virus (HBV) infections are still a major health issue, with approximately 350 million people chronically infected with HBV worldwide. Information about the minimum copy number of HBV genomes required for infection would be useful as a reference for drug and vaccine development; for monitoring HBV patients during treatment; for screening of blood, organ, and tissue donors; and for regulating nucleic acid amplification assays for HBV.

Serum samples from chronic carriers (hepatitis B surface antigen-positive and antibody to HBV core antigen-positive) of the three most common subtypes of HBV were studied; their infectivity titers had been evaluated previously in chimpanzees. The genotypes of the HBV samples were determined by DNA sequences and type-specific amino acids of the S gene of HBV. Copy numbers of HBV DNA were quantified by real-time TaqMan polymerase chain reaction (PCR) and by nested PCR applied to limiting dilutions. The copy number determined for each inoculum was compared with previously defined chimpanzee infectivity titers.

The genotypes of the HBV adw, ayw, and adr inocula were A, D, and C, respectively. The concentration of HBV DNA was determined to be 5.4 x 10(9), 2.5 x 10(9), and 3.1 x 10(8) genome equivalents (geq) per mL for serum samples containing the adw, ayw, and adr, respectively. The chimpanzee infectivity titers per milliliter of these initial HBV-containing serum samples were previously determined to be 10(7.5) for adw, 10(7.5) for ayw (MS-2 strain), and 10(8) for adr.

The minimal copy number of HBV DNA in chronic carriers of HBV that can infect the chimpanzee model was estimated to be from 3 to 169 geq based upon the three well-characterized inocula.

We herein focused on identifying biological factors possibly involved in non-parenteral transmission of hepatitis C virus (HCV), such as HCV excretion patterns and antibody-based immunity to the virus in saliva and/or cervicovaginal secretions (CVS).

Paired blood, saliva and cervicovaginal lavage samples were obtained from HCV-RNA plasma-positive hemoglobin (Hb) antigen and HIV-seronegative, HCV-seropositive males (n=13) and females (n=21). HCV-specific antibodies were detected by ELISA in paired samples, and HCV-RNA was detected in cell-free and cell-associated body fluids.

Antibodies to E1 HCV surface glycoprotein of the IgG and IgA isotypes showed similar, but less pronounced, profiles as IgG and IgA to E2. HCV-specific IgG and IgA in mucosal fluids likely originated predominantly from the systemic compartment, because HCV-specific mucosal immunoglobulins involved primarily monomeric antibodies, including monomeric IgA, and because their specific activities for HCV antigens in corporeal fluids were similar to those in paired serum (Se). Viral shedding in saliva or CVS was restricted to cell-associated, non-replicating strand((+)) HCV-RNA in 42% (12 out of 28) of saliva and in 19% (four out of 21) of cervicovaginal fluids.

The association in body fluids of HCV-specific IgG, and to a lesser extent IgA, directed to E1/E2 surface glycoproteins (which may block critical steps of virus-cell interactions), of undetectable free viral RNA, and of occasional non-replicating cell-associated HCV, suggests a resulting poor infectivity of saliva or cervicovaginal fluid in chronically HCV-infected individuals. Taken together, these observations provide the basis for the low risk of non-parenteral transmission of HCV infection.

A reverse-transcription competitive PCR (RT-cPCR) combined with chemiluminescence hybridization was designed for the detection and quantitative determination of serum hepatitis C virus (HCV) RNA. The concentration of HCV RNA was calculated based on an external standard curve that was generated by coamplification of internal competitor and target sequences in serial dilutions. The detection limit of the chemiluminescence RT-cPCR was 100 copies/ml (94 IU/ml). Meanwhile, the linear range for quantitation extended from 850 copies/ml (795 IU/ml) to 4.95x10(7) copies/ml. The performance of the current assay for measuring circulating HCV levels from 26 anti-HCV-antibody positive patients was compared with that of branched-chain DNA (bDNA) and nested RT-PCR assays. Eighteen patients had HCV RNA levels that exceeded the quantitation limit by the chemiluminescence RT-cPCR, but only 11 patients were quantitation-positive by the bDNA. A significant correlation of the quantitation values was found between the chemiluminescence RT-cPCR and the bDNA (R2=0.8391). Among the eight patients with HCV RNA titers below the quantitation limit, four remained positive by the chemiluminescence cRT-PCR, demonstrating the results in agreement with those using the nested RT-PCR. Furthermore, good linearity was revealed for the HCV genotypes 1b, 2a, 2b in 3-order magnitude diluted serum samples. In conclusion, the proposed chemiluminescence RT-cPCR method can detect quantitatively HCV RNA as accurately as the bDNA method and has sensitivity as high as nested RT-PCR.

Because of the difficulties of the chimpanzee model and the genetic differences using the duck model, we developed a cell culture method to measure human hepatitis B virus (HBV) inactivation in vitro. Pooled HBV-infected human plasma that had been exposed to a disinfectant was left in contact for three days with a cell culture of the human hepatoma cell line, HepG2, with 4% polyethyleneglycol and 3 mM sodium butyrate. The mean log10 of the viral titre of unexposed plasma was 4.87 infectious units per mL. Our results showed that 1% glutaraldehyde, sodium hypochlorite at 4700 ppm free chlorine and an iodophor-detergent disinfectant containing 3.6% povidone-iodine reduced viral titres by factors exceeding 10(3)-10(4). However, sodium hypochlorite at 1000 ppm free chlorine had minimal activity and povidone-iodine at 9, 5 and 3.6% had no measurable activity (less than 10-fold reduction). This is the first study using a cell culture model to assess disinfectant activity against HBV. It demonstrates more rapidly than the chimpanzee model that glutaraldehyde and sodium hypochlorite, using standard concentrations and exposure times compatible with clinical practice, were highly active against HBV. However, unexpectedly for an enveloped virus, we found no antiviral activity for iodine in the absence of detergent.

The laboratory diagnosis of hepatitis B virus (HBV) infection is based mainly on serological assays. Yet the detection and quantitation of viral DNA is necessary when addressing directly the question of infectivity or when monitoring the viral load during therapy. Standard hybridization assays allow for exact quantitation, but their sensitivity is limited to 10(5)-10(6) viral genomes per ml of serum. The most sensitive tests for HBV DNA are nested PCR systems, which recognize virtually one molecule of the target DNA per reaction. However, these assays only provide very coarse quantitative statements. To take advantage of both methods, a new assay for HBV DNA is described based on the commercial TaqMan system. This assay is capable of quantifying HBV DNA from the theoretical lower limit up to 10(10) genome equivalents per ml of serum and, thus, covers the complete range of naturally occurring states of infections. The method was calibrated on the basis of serial plasmid dilutions and compared with a well-established nested PCR system. More than 100 HBV positive sera and serial dilutions of the Eurohep standard for both ad and ay subtypes were analyzed. The assay reliably detected all HBV positive samples. It shows minimal run-to-run deviations, allows for quantitation that covers eight orders of magnitude, and finally, completely avoids the risk of cross-contamination by PCR products. Thus, this technique combines the sensitivity of PCR amplification and the quantitation potential of hybridization tests and it is time efficient and safer.

Microchimerism after liver transplantation is a readily observed phenomenon. The immunological implications, however, remain unclear. Moreover, methodological approaches and their detection limits in the study of allogeneic microchimerism have not been studied in detail.

Therefore, the aim of this study was to evaluate the single-step and nested formats of the polymerase chain reaction/sequence-specific priming (PCR-SSP) approach under standardized conditions. For that purpose, a panel of recombinant plasmid clones was generated by PCR cloning. The panel contained the allelic sequences of the second exon of DRB1 covering all DR specificities on a low-resolution level. Using this panel, limiting dilution assays for various DR sequences in the presence and absence of competitor DNA were carried out to determine the minimal number of copies required for detection by single-step and nested PCR-SSP. Subsequently, 22 liver transplant recipients were analyzed in a retrospective study for the presence of allogeneic microchimerism by nested PCR-SSP.

Although at least 10 copies of template DNA could be detected by nested PCR-SSP overall, single-step PCR-SSP was on average 10(2) to 10(3) times less sensitive. Upon the addition of human competitor DNA, the detection limits decreased on average by a factor of 10. In addition, sequence-specific differences in amplification efficiency could be appreciated. Using nested PCR-SSP, peripheral blood allogeneic microchimerism could be observed in 17 of 22 HLA-DR-mismatched liver recipients. Recombinants representing recipient DRB1 specificities were used to exclude false-positive results by lack of cross-reactivities of the donor-specific primers and to evaluate negative results due to sample-related reduced amplification efficiencies in microchimerism-negative recipients. In donor/recipient combinations that differed by at least one DR specificity, allogeneic microchimerism was seen in 87.5% of the cases. In five chimerism-negative cases, sample-related problems were detected in two cases.

The optimization and standardization of the detection of genomic HLA sequences at low copy number may be greatly facilitated using a clonal reference system. Furthermore, a clonal reference system may be used to conduct cross-priming experiments to exclude false-positive results and may allow the determination of sample-specific detection limits for donor-derived HLA-DR specificities in chimerism-negative patients. Our evaluation of the PCR-SSP approach for the study of allogeneic microchimerism indicated that nested PCR-SSP provides the most sensitive format when HLA sequences are targeted. Yet, the detection sensitivity may vary between individual alleles and specificities. Allogeneic microchimerism in liver recipients can be observed in the majority of patients. However, the detection may be subject to the degree of mismatching, the HLA-DR alleles involved, and sample-related impaired PCR amplification efficiency.