Simultaneous extraction of hepatitis C virus (HCV), hepatitis B virus, and HIV-1 from plasma and detection of HCV RNA by a reverse transcriptase-polymerase chain reaction assay designed for screening pooled units of donated blood.

BACKGROUND: Testing for viral nucleic acids should reduce the residual risk of transmitting viral infections by transfusion of blood components. The AmpliScreen Hepatitis C (HCV) Test, Version 2.0, was designed for screening pools composed of samples from individual units of blood or plasma. STUDY DESIGN AND METHODS: An ultracentrifugation step during sample processing simultaneously extracts and concentrates HCV, HIV type 1, and hepatitis B virus particles from plasma. An HCV internal control RNA serves as an extraction and amplification control for each processed sample. Processed samples are amplified by reverse transcriptase polymerase chain reaction and detected by hybridization of the amplified products to HCV- and internal-control-specific oligonucleotide probes. Plasma samples containing known quantities of HCV were used to evaluate analytical sensitivity and precision. RESULTS: The analytical sensitivity of the test was 25 IU of HCV per mL of pooled plasma; all HCV genotypes were detected with similar efficiency. The test did not react with other blood-borne viruses. The within-run and total coefficients of variation were 1.3-13.0 percent and 1.7-22.0 percent, respectively, with low copy samples producing the more variable results. The test performed well using plasma collected in either EDTA, ACD, or PPT Vacutainer tubes. Plasma samples containing elevated levels of hemoglobin, albumin, triglycerides, or bilirubin did not interfere with the test. The test detected HCV RNA 23 to 32 days prior to seroconversion for four of the five seroconversion panels tested. CONCLUSION: The AmpliScreen HCV Test, Version 2.0 provides a reproducible and specific method for screening pooled blood units for HCV. Theoretically, this test has sufficient sensitivity to detect a single infected unit containing 2.4 x 10(3) IU of HCV per mL in a pool with 95 uninfected units and should reduce the window period by at least 20 to 30 days.

Assay of plasma samples representing different HIV-1 genetic subtypes: an evaluation of new versions of the amplicor HIV-1 monitor assay.

Quantification of plasma HIV-1 RNA levels has rapidly become the main tool for monitoring disease progression and treatment. However, some first-generation assays do not accurately quantify all HIV-1 subtypes. This study compares the first-generation and two newer prototype Amplicor HIV-1 Monitor assays (Roche Diagnostic Systems) in terms of their performance in quantifying HIV-1 RNA in stored plasma samples from 101 individuals infected with various genetic subtypes of HIV-1 (28 subtype A, 18 B, 26 C, 20 D, 2 E, 3 G, 2 H, and 2 J). The HIV-1 subtype had previously been determined by direct sequencing of the V3 domain of the env gene. The results from the three assays agreed for subtypes B and C, as well as for most subtype D samples. In contrast, the first-generation assay reported significantly lower plasma HIV-1 RNA levels than did the two newer versions of the assay for most subtype A, E, G, and H samples. There were no differences in mean plasma HIV-1 RNA levels between individuals infected with subtypes A, B, C, and D if the results from the two newer test versions were used, and if an adjustment was made between subtypes for differences in CD4 count. Thus, this study confirms that the first-generation assay does not accurately quantify HIV-1 RNA levels in many individuals infected with subtype A, E, G, and H. These problems appeared to have been greatly reduced in the two new prototype versions.

Development of calibrated viral load standards for group M subtypes of human immunodeficiency virus type 1 and performance of an improved AMPLICOR HIV-1 MONITOR test with isolates of diverse subtypes.

Accurate determination of plasma human immunodeficiency virus type 1 (HIV-1) RNA levels is critical for the effective management of HIV-1 disease. The AMPLICOR HIV-1 MONITOR Test, a reverse transcription-PCR-based test for quantification of HIV-1 RNA in plasma, was developed when little sequence information on HIV-1 isolates from outside North America was available. It has since become apparent that many non-subtype B isolates, particularly subtypes A and E, are detected inefficiently by the test. We describe here the AMPLICOR HIV-1 MONITOR Test, version 1.5, an upgraded test developed to minimize subtype-related variation. We also developed a panel of HIV-1 standards containing 30 HIV-1 isolates of subtypes A through G. The virus particle concentration of each cultured viral stock was standardized by electron microscopic virus particle counting. We used this panel to determine the performance of the original AMPLICOR HIV-1 MONITOR Test and version 1.5 of the test with HIV-1 subtypes A through G. The original test underestimated the concentration of HIV-1 subtype A, E, F, and G RNA by 10-fold or more, whereas version of the 1.5 test yielded equivalent quantification of HIV-1 RNA regardless of the subtype. In light of the increasing intermixing of HIV-1 subtypes worldwide, standardization of PCR-based tests against well-characterized viral isolates representing the full range of HIV-1 diversity will be essential for the continued utility of these important clinical management tools.

Efficiencies of four versions of the AMPLICOR HIV-1 MONITOR test for quantification of different subtypes of human immunodeficiency virus type 1.

Three versions of a commercial human immunodeficiency virus (HIV) type 1 (HIV-1) load test (the AMPLICOR HIV-1 MONITOR Test versions 1. 0, 1.0+, and 1.5; Roche Diagnostics, Branchburg, N.J.) were evaluated for their ability to detect and quantify HIV-1 RNA of different genetic subtypes. Plasma samples from 96 patients infected with various subtypes of HIV-1 (55 patients infected with subtype A, 9 with subtype B, 21 with subtype C, 2 with subtype D, 7 with subtype E, and 2 with subtype G) and cultured virus from 29 HIV-1 reference strains (3 of subtype A, 6 of subtype B, 5 of subtype C, 3 of subtype D, 8 of subtype E, 3 of subtype F, and 1 of subtype G) were tested. Detection of subtypes A and E was significantly improved with versions 1.0+ and 1.5 compared to that with version 1. 0, whereas detection of subtypes B, C, D, and G was equivalent with the three versions. Versions 1.0, 1.0+, and 1.5 detected 65, 98, and 100% of the subtype A-infected samples from patients, respectively, and 71, 100, and 100% of the subtype E-infected samples from patients, respectively. Version 1.5 yielded a significant increase in viral load for samples infected with subtypes A and E (greater than 1 log10 HIV RNA copies/ml). For samples infected with subtype B, C, and D and tested with version 1.5, only a slight increase in viral load was observed (<0.5 log10). We also evaluated a prototype automated version of the test that uses the same PCR primers as version 1.5. The results with the prototype automated test were highly correlated with those of the version 1.5 test for all subtypes, but were lower overall. The AMPLICOR HIV-1 MONITOR Test, version 1.5, yielded accurate measurement of the HIV load for all HIV-1 subtypes tested, which should allow the test to be used to assess disease prognosis and response to antiretroviral treatment in patients infected with a group M HIV-1 subtype.

The AMPLICOR® HCV Tests for the Detection and Quantitation of Serum or Plasma HCV RNA.

Clinical diagnosis of HCV infection is generally accomplished by using immunoserological assays to detect the presence of anti-HCV antibodies. Such immunoserological assays have been approved for blood donor screening, thereby reducing the incidence of post-transfusion hepatitis in the United States. Although useful, immunoserological assays have several limitations. Recent evaluations have shown that interpretation of these immunological tests often is difficult, since 25-90% (depending on the risk group under evaluation) of samples repeatedly reactive in the screening assay are negative on supplemental evaluation with a recombinant immunoblot assay (RIBA) (1,2). Also, the presence of anti-HCV antibodies indicates prior exposure to HCV infection, but cannot be considered a marker for current infection. Nor can anti-HCV antibody levels be used to monitor response to therapeutic agents. Finally, in cases of acute HCV infection resulting from accidental needlestick exposure, many patients fail to produce antibody to HCV (3), which makes diagnosis of HCV infection impossible using immunoserological techniques. At the present time, an immunological assay for direct detection of HCV antigen is unavailable.

Quality control of the polymerase chain reaction.

The polymerase chain reaction (PCR) has revolutionized both the basic and the applied aspects of the biomedical field with more than 40,000 papers having been published employing this technique. PCR has become an indispensable tool for basic research applications, such as cloning (1), sequencing (2), mRNA analysis (3), DNA and RNA quantitation (4-6), mutagenesis (7), and gene detection (8). Because of its ability to amplify minute quantities of nucleic acid specifically, PCR has also been applied with great success in clinical diagnostics (9-11), genetic testing (12-15), forensics (16,17), and environmental testing (18,19).

Ultrasensitive reverse transcription-PCR assay for quantitation of human immunodeficiency virus type 1 RNA in plasma.

With the recent introduction of combination therapy, human immunodeficiency virus type 1 (HIV-1) RNA levels in plasma have been dramatically reduced, frequently to below the limit of quantitation (400 copies/ml of plasma) of the AMPLICOR HIV-1 MONITOR Test (Roche Diagnostic Systems). To achieve enhanced sensitivity of the AMPLICOR HIV-1 MONITOR Test, a modified specimen preparation procedure that allows input of RNA from 10-fold more plasma per amplification reaction was developed. This "ultrasensitive" method allows the accurate quantitation of plasma HIV-1 RNA levels as low as 50 copies/ml. A precision study yielded average within-run and between-run coefficients of variation (CV) of 24.8 and 9.6%, respectively. A multicenter reproducibility study demonstrated that the laboratory-to-laboratory reproducibility of this assay is good, with an average CV of 32%. The linear range of this test is between 50 and 50,000 copies/ml of plasma. RNA concentrations measured by the ultrasensitive and standard HIV-1 MONITOR tests exhibited good agreement within the shared linear range of the two methods. The two measurements were within a factor of 2 for 91% of the specimens tested, with the concentration measured by the ultrasensitive method being only slightly lower (median, 22% lower). Preliminary studies suggest that this assay will prove to be useful for predicting the stability of viral suppression in patients whose RNA levels drop below 400 copies/ml in response to highly active antiretroviral therapy.

An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance.

We constructed internal controls (ICs) to provide assurance that clinical specimens are successfully amplified and detected. The IC nucleic acids contain primer binding regions identical to those of the target sequence and contain a unique probe binding region that differentiates the IC from amplified target nucleic acid. Because only 20 copies of the IC are introduced into each test sample, a positive IC signal indicates that amplification was sufficient to generate a positive signal from targets present at the limit of test sensitivity. The COBAS AMPLICOR Chlamydia trachomatis, Neisseria gonorrhoeae, Mycobacterium tuberculosis, and human hepatitis C virus tests exhibited inhibition rates ranging from 5 to 9%. Approximately 64% of these inhibitory specimens were not inhibitory when a second aliquot was tested. Because repeatedly inhibitory specimens were not reported as false negative and because additional infected specimens were detected during retesting, test sensitivities were 1 to 6% greater than they would have been if the IC had not been used.

Blood collection on filter paper: a practical approach to sample collection for studies of perinatal HIV transmission.

The use of dried blood spots lends itself to widespread application in large field studies, especially in remote areas. We present experience gained during a perinatal HIV transmission study in southern Africa in which dried blood spot samples were used for polymerase chain reaction (PCR) tests. In this study, 15,810 filter paper cards with dried blood spots were collected. Infants were seen at age 6 and 12 weeks, and PCR was routinely done in duplicate on each sample. Of 186 negative controls (infants born to HIV-negative women), two (1.1%) had a single strongly reactive PCR result; the repeated duplicates were both negative. In contrast, all 24 known positive samples were strongly positive in both tests. Results were available from 1,976 duplicate tests on 1,235 infants born to HIV-infected women. Based on the PCR result on a later sample, the positive predictive value was 97.6% if both replicates were strongly positive (absorbance: 0.8 OD450 U), 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive (but none strongly positive). When both replicates were negative, the negative predictive value was > or = 96.2%. Thus, when a single HIV PCR test has a strongly positive result, the infant is very likely to be infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months. Suggestions for sample collection, storage, and PCR testing are provided.

PIP: 15,810 filter paper cards with dried blood spots were collected during a perinatal HIV transmission study in southern Africa to be subjected to polymerase chain reaction (PCR) testing. Infants were seen at ages 6 and 12 weeks, with PCR routinely done in duplicate on each sample. Of 186 infants born to HIV-negative mothers, two had a single strongly reactive PCR result, while the repeated duplicates were both negative. All 24 known positive samples were strongly positive in both tests. Results were available from 1976 duplicate tests on 1235 infants born to HIV-infected women. Based upon the PCR result of a later sample, the positive predictive value was 97.6% if both replicates were strongly positive, 100% when one of the replicates was strongly positive, and 27% when one or both replicates were weakly positive. When both replicates were negative, the negative predictive value was greater than or equal to 96.2%. Therefore when a single HIV PCR test has a strongly positive result, the infant is very likely infected. A positive PCR result after age 1 month was 98.9% accurate in predicting antibody positivity after 15 months.

Screening potential corneal donors for HIV-1 by polymerase chain reaction and a colorimetric microwell hybridization assay.

PURPOSE: Current screening of potential corneal donors for human immunodeficiency virus type 1 (HIV-1) involves serologic detection of antibodies to the virus. However, this approach cannot detect infection during the seronegative window period of the disease. We therefore evaluated the polymerase chain reaction (PCR) assay for viral nucleic acid as a possible alternative to screening cadaveric blood for HIV-1. METHODS: Blood specimens from cadavers diagnosed at autopsy with acquired immunodeficiency syndrome (AIDS) (n = 21), at high risk for HIV-1 infection (n = 47), and at no known risk (n = 350) were screened by PCR for HIV-1 proviral DNA and human leukocyte antigen (HLA)-DQ alpha sequences, and for HIV antibodies. RESULTS: All AIDS group samples were seropositive; of these, 18 (86%) and 20 (95%) of 21 were positive for HIV by PCR of proteinase K- and Chelex-extracted pellets, respectively. The seropositive samples negative by PCR testing were shown to inhibit PCR amplification. Nine (19%) of 47 high-risk specimens were HIV-positive. The no-known-risk group yielded negative results. The overall sensitivities for PCR in the proteinase K- and Chelex-treated groups were 90% and 97%, respectively, compared with Western blot reactivity. If PCR-inhibitory samples and HLA-DQ alpha-negative samples had been eliminated, sensitivity would have been 100%. Specificity was 100% for each group. CONCLUSIONS: Screening cadaveric blood by PCR may be feasible, but further refinement of the assay and blood specimen collection practices will be necessary for it to become routine. Future studies should focus on optimizing specimen procurement and preparation to reduce or eliminate specimens that inhibit PCR.

Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe.

Nucleic acid amplification techniques such as the PCR are very useful in the rapid diagnosis of infections by Mycobacterium tuberculosis. However, recent studies have shown that the accuracy of results can vary widely when tests are performed with nonstandardized reagents. We have developed a PCR assay for the detection of M. tuberculosis that is both rapid and accurate. The assay reagents are standardized and quality controlled. False-positive results due to carryover contamination are prevented by the incorporation of dUTP coupled with uracil-N-glycosylase restriction. This assay also employs pan-Mycobacterium amplification primers, allowing for flexibility in the mycobacterial species that can be identified from a single amplification reaction. The amplification is very sensitive; amplification products generated from as few as three bacteria can be detected by agarose gel electrophoresis. DNAs isolated from 33 of 34 mycobacterial species tested were amplified efficiently. Only DNA from Mycobacterium simiae did not amplify. The amplification is also very specific. Amplification products were generated only from the DNAs of bacteria in closely related genera such as Corynebacterium. The nonmycobacterial amplicons do not pose a problem, as they do not hybridize to mycobacterium-specific probes. Hybridization of amplicons to an M. tuberculosis-specific probe allows for the unambiguous identification of M. tuberculosis complex organisms. The clinical performance of this PCR assay was evaluated against that of culture in 662 respiratory specimens. Sensitivities of 100 and 73.1% were obtained from smear-positive and -negative respiratory specimens, respectively. The corresponding specificities were 100 and 99.8%. The high sensitivity and specificity, coupled with the potential for detecting a wide range of mycobacteria, make this assay a useful tool in the clinical management of mycobacterial infections.

Rapid screening for early detection of mother-to-child transmission of human immunodeficiency virus type 1.

The testing of dried blood spots (DBSs) for the presence of human immunodeficiency type 1 (HIV-1) proviral DNA by PCR was first described in 1991. The technology has proven to be particularly valuable for resolving the infection status in HIV-1-indeterminate infants born to HIV-1-seropositive mothers. To broaden the applicability of DBS PCR, we adapted it to a standardized, commercially available microwell plate amplification and detection kit, Amplicor HIV-1, produced by Roche Diagnostic Systems. The microwell assay is rapid and easy to perform and uses equipment that is readily available in routine diagnostic laboratories. The high level of performance of the assay was demonstrated in 1,168 duplicate tests performed on 584 DBSs from 178 uninfected and 100 HIV-1-infected individuals, including 56 children with perinatally acquired HIV-1. Of 12 infants who were followed prospectively from birth, 3 (25%) were infected in utero (PCR positive at birth) and 9 (75%) were infected intrapartum (PCR negative, culture negative at birth). Overall, HIV-1 DNA was identified in 3 of 11 (27.3%) DBSs collected from infected infants during the first 4 days of life, 8 of 9 (88.9%) DBSs collected between 10 and 15 days postpartum, and 166 of 167 (99.4%) DBSs collected after 15 days of age. All 320 DBSs from uninfected children were PCR DNA negative. These findings indicate that use of the Roche microwell DBS PCR assay provides a powerful new approach for large-scale perinatal screening programs and population-based studies of vertical transmission.

Diagnosis of enteroviral meningitis by using PCR with a colorimetric microwell detection assay.

A 5-h, user-friendly PCR assay for the diagnosis of enteroviral meningitis was developed. Reverse transcription and amplification were performed in a one-step reaction using rTth polymerase. Carryover contamination was prevented with dUTP and uracil N-glycosylate. Detection was performed colorimetrically on a microwell titer plate. Sensitivity, specificity, positive predictive value, and negative predictive value were 94.7, 97.4, 94.7, and 97.4%, respectively.

Single copies of HIV proviral DNA detected by fluorescent in situ hybridization.

A fluorescent in situ DNA hybridization assay employing a biotinylated DNA probe was used to visualize single copies of human immunodeficiency virus (HIV) proviral DNA in the nuclei and metaphase chromosomes of infected cells. In clonal cell lines that contain either one or two copies of proviral DNA, the efficiency of detection of individual loci of proviral DNA was 57% to 78%. Only 1% of uninfected control cells exhibited a false-positive signal. HIV proviral DNA could be accurately identified in mixed populations comprised of only 5% infected cells. Thus, this assay could be used to identify cells that harbor HIV proviral DNA and to monitor the status of proviral DNA throughout the course of HIV infection.

Differential expression of histone sequences in Drosophila following heat shock.

The expression of the sequences encoding the four nucleosomal histone proteins was examined following heat shock of a variety of Drosophila cells and was found to be highly differential. In Drosophila melanogaster KC-O cells grown in suspension culture, there is a continuation of the synthesis of all four of the nucleosomal histone proteins following heat shock. Analysis of RNA from these cells confirms that histone messengers are transcribed and located on polysomes. This exact same pattern of histone protein synthesis occurs in KC-O cells grown to low density on plates. In contrast, KC-O cells grown to high density on plates exhibit a dramatic elevation of H2b protein synthesis relative to the synthesis of the other core histones. Organs from D melanogaster third instar larvae were examined to ascertain whether histone protein synthesis continues following heat shock in the organism. Different tissue types exhibited differential histone synthesis. Imaginal disks excised from heat-shocked larvae continue to synthesize nucleosomal histones in a variable fashion. In contrast, neither fat bodies, brains, nor salivary glands continues to synthesize core histone proteins at a significant level. D hydei plated cell cultures and larval tissues fail to synthesize histones at any detectable level following a heat shock. Based on these observations, we propose that there is a differential synthesis of nucleosomal proteins in Drosophila that is highly dependent on the state of the cells prior to the heat shock.

Cell position regulates endodermal differentiation in embryonal carcinoma cell aggregates.

It has been suggested that cell position regulates endodermal differentiation in mouse embryo inner cell masses and in aggregates of embryonal carcinoma (EC) cells. This hypothesis states that cells at the interface between the cell mass and blastocoel fluid or culture medium differentiate into endoderm, whereas internally located cells follow alternative developmental pathways. To test the cell position hypothesis, pluripotent PSA-1 cells were aggregated with hypoxanthine phosphoribosyltransferase-deficient, parietal-like, endodermal cells. The resulting aggregates consisted of cores of PSA-1 cells surrounded by endodermal cells. Autoradiography was used to distinguish between endodermal cells that were the products of EC cell differentiation and the exogenous endoderm. Alkaline phosphatase staining was used to distinguish EC cells from endodermal cells. As predicted by the cell position hypothesis, the PSA-1 EC cells, all of which were internally located, did not differentiate into endodermal cells. Nonspecific inhibition of differentiation did not account for the lack of PSA-1-derived endoderm since the PSA-1 cells in such aggregates did differentiate into columnar ectodermal-like cells. Similar experiments were also conducted with F9 cells. In this case, aggregation cultures contained retinoic acid to induce F9 cells to differentiate into visceral endoderm. In cultures containing F9 cells surrounded by parietal-like endodermal cells, no F9-derived endoderm was detected either autoradiographically or by assaying for alpha-fetoprotein production, a visceral endoderm marker. Thus, retinoic acid-induced endodermal differentiation was also regulated by cell position. Collectively, the above results provide strong evidence for the hypothesis that cell position regulates endodermal differentiation in aggregates of EC cells.