Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. ACTG PCR Working Group, and the ACTG PCR Virology Laboratories

Jackson, J. Brooks; Drew, James; Lin, Hsiang Ju; Otto, Patricia; Bremer, J; Hollinger, F. Blaine; Wolinsky, Steven M.

doi:10.1128/jcm.31.12.3123-3128.1993

Cited by 60 publications

(17 citation statements)

References 20 publications

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“…In many cases those studies were limited because they involved the use of nonstandardized assays, tested a limited number of samples, used different and sometimes poorly defined endpoints, and did not involve rigorous statistical analysis (1,3,16). While PCR-based assays are generally more sensitive than hybridization assays for nucleic acid quantification, in-house PCR assays tend to be very poorly standardized (9,17,21).…”

Section: Discussionmentioning

confidence: 99%

Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

et al. 1998

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Quantification of hepatitis B virus (HBV)DNA in serum is used to establish eligibility for treatment and to monitor therapeutic response. With the trend toward centralized testing, defining the conditions that preserve sample integrity is of paramount importance. We therefore evaluated the stability of HBV DNA in 26 previously frozen (PF) and 5 fresh, never previously frozen serum specimens. PF specimens, covering a 3-log 10 HBV DNA dynamic range, were thawed and stored at ؊70, 4, 23, 37, and 45°C (؎1.5°C) for 0, 24, 72, and 120 h (؎2 h) and were refrozen at ؊70°C prior to testing. Five fresh specimens were split into two groups. Both group FG1 and group FG2 specimens were handled as described above; however, group FG1 specimens were subsequently maintained at 4°C and were never frozen prior to testing. Linear regression analysis of PF specimens demonstrated no significant HBV DNA degradation at <4°C over 5 days; however, HBV DNA levels decreased by 1.8, 3.4, and 20% per day at 23, 37, and 45°C, respectively. Three independent statistical methods confirmed that the probability of specimen failure, defined as a loss of 20% or more of HBV DNA and/or coagulation of serum, was lowest at <4°C and increased with temperature. Because only 10 to 20% of individual patient specimens demonstrated losses of HBV DNA of >20% at 23 or 37°C, sufficient numbers of serum specimens must be evaluated to determine overall statistical trends. In conclusion, HBV DNA integrity in separated serum specimens is preserved for at least 5 days when the specimens are stored at ؊70 or 4°C.

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Section: Discussionmentioning

confidence: 99%

Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

et al. 1998

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“…This test is based on single-step reverse transcription and amplification of enterovirus RNA, and it incorporates enzymatic elimination of amplicon contamination, thus harmonizing and simplifying laboratory procedures and reducing the risk of false positivity due to carryover of previously amplified nucleic acid. However, quality assurance procedures will be required to ensure test sensitivity, specificity, and reliability; experience with PCR assays for other viruses has revealed considerable interlaboratory variation in sensitivity, specificity, and reliability, which is partially but not completely alleviated by the use of standardized methods or commercial assays (46,93,185,351,442,485,486).…”

Section: Enterovirus Detection By Pcrmentioning

confidence: 99%

Molecular Typing of Enteroviruses: Current Status and Future Requirements

et al. 1998

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Human enteroviruses have traditionally been typed according to neutralization serotype. This procedure is limited by the difficulty in culturing some enteroviruses, the availability of antisera for serotyping, and the cost and technical complexity of serotyping procedures. Furthermore, the impact of information derived from enterovirus serotyping is generally perceived to be low. Enteroviruses are now increasingly being detected by PCR rather than by culture. Classical typing methods will therefore no longer be possible in most instances. An alternative means of enterovirus typing, employing PCR in conjunction with molecular genetic techniques such as nucleotide sequencing or nucleic acid hybridization, would complement molecular diagnosis, may overcome some of the problems associated with serotyping, and would provide additional information regarding the epidemiology and biological properties of enteroviruses. We argue the case for developing a molecular typing system, discuss the genetic basis of such a system, review the literature describing attempts to identify or classify enteroviruses by molecular methods, and suggest ways in which the goal of molecular typing may be realized.

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“…However, even when such a reference panel is available, the comparison of in-house PCR assays between centers can prove to be disappointing, as was the case with hepatitis C virus (19), for which erroneous results were reported by 52% of the laboratories. Moreover, even when centers used the same commercially available kit for the PCR detection of HIV, the results were not always in agreement (10), emphasizing the fact that staff expertise and laboratory facilities can also affect the outcome.…”

Section: Discussionmentioning

confidence: 97%

“…When the virus and/or viral nucleic acid is associated with the plasma fraction, reference panels can be set up by ''spiking'' plasma with different amounts of viral nucleic acid and/or virus (19). For HIV, reference panels have been set up by spiking blood with lymphoid cell lines infected with HIV in vitro (10), as well as by using whole blood specimens from well-characterized patients known to be consistently PCR positive (10). Neither of these latter approaches is easily adopted for CMV, since we do not have an in vitro model which reflects the physiologic state of CMV in the blood and CMV viremia is usually of shorter duration and of lower magnitude than viremia with viruses such as hepatitis C virus.…”

Section: Discussionmentioning

confidence: 99%

A three-center European external quality control study of PCR for detection of cytomegalovirus DNA in blood

et al. 1996

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The presence of cytomegalovirus (CMV) in the blood has important consequences for patient management, and an external quality control study of its detection by the PCR was conducted by the Infectious Disease Working Party of the European Group for Blood and Marrow Transplantation. Forty-eight coded peripheral blood samples from bone marrow transplant recipients were processed in parallel in three European centers by using the routine in-house PCR assay. Protocols varied in choice of primers, specificity and amplificability controls, and sample processing. Results for 38 of 47 samples agreed, 35 being negative and 3 positive. Of the 12 samples reported as positive by at least one center, only 3 were found to be positive by all three centers, 1 was found to be positive by two centers, and the remaining 8 were found to be positive by one center only. The nine discrepant samples appeared to contain around 1,000-fold less viral DNA than the three concordant positive samples. CMV detection was affected both by the number of leukocytes from which DNA was extracted and by the number of cell equivalents added per PCR. External quality control schemes for CMV PCR are clearly necessary in order to compare data from different centers, and recommendations for standardizing the PCR detection of CMV in blood leukocytes are made. Patients and samples. Forty-eight peripheral venous blood samples were taken in triplicate from 34 patients, 13 from Tübingen, 12 from Huddinge/ Stockholm, and 9 from London. Six had received autologous marrow, and 28 had received allogeneic marrow. Samples were taken between Ϫ1 and 17 weeks

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Establishment of a quality assurance program for human immunodeficiency virus type 1 DNA polymerase chain reaction assays by the AIDS Clinical Trials Group. ACTG PCR Working Group, and the ACTG PCR Virology Laboratories

Cited by 60 publications

References 20 publications

Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

Assessment of Hepatitis B Virus DNA Stability in Serum by the Chiron Quantiplex Branched-DNA Assay

Molecular Typing of Enteroviruses: Current Status and Future Requirements

A three-center European external quality control study of PCR for detection of cytomegalovirus DNA in blood

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