Multi-Site PCR-based CMV viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology.

Viralload (VL) assessment of cytomegalovirus (CMV) by real-time PCR is an important tool for diagnosing and monitoring CMV viremia in patients with compromised immune systems. We report results from a sample exchange organized by members of the Association for Molecular Pathology that compared PCR results from 23 laboratories; 22 such laboratories used a laboratory-developed real-time PCR assay and one laboratory used a competitive PCR assay. The samples sent to each laboratory were comprised of a dilution panel of CMV virion-derived reference materials that ranged from 0 to 500,000 copies/ml. Accuracy, linearity, and intralaboratory precision were established for the different laboratory-developed assays. Overall, PCR results were linear for each laboratory (R(2) > 0.97 in all but two). While 13 laboratories showed no significant quantitative assay bias, 10 laboratories reported VLs that were significantly different compared with expected values (bias range, -0.82 to 1.4 logs). The intralaboratory precision [mean coefficient of variance of 2% to 5% (log-scale)] suggested that changes in VLs of less than 3- to fivefold may not be significantly different. There was no significant association between laboratory-specific technical variables (PCR platform, calibrator, extraction method) and assay linearity or accuracy. These data suggested that, within each laboratory, relative VL values were linear, but additional method standardization and a CMV DNA reference standard are needed to allow laboratories to achieve comparable numeric results.

Results of the 2001 AcroMetrix HIV-1 resistance proficiency program.

BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) drug resistance mutation testing has become a useful tool in assessing antiretroviral treatment and managing patient care. As these complex molecular genotyping procedures move into routine use in clinical laboratories, the need arises for quality control procedures that will ensure that drug resistance associated mutations are accurately identified. OBJECTIVES: The AcroMetrix HIV-1 Resistance Proficiency Program was designed to assess proficiency in the identification of HIV-1 drug resistance mutations using molecular genotyping methods. This study describes results obtained from a variety of laboratories participating in the 2001 proficiency program. STUDY DESIGN: The 2001 program included three challenges, each comprised of five blind-labeled panel members containing mutations in the reverse transcriptase (RT) and/or protease (PR) genes. Challenge HIV-R01 was distributed to 19 laboratories in May 2001, challenge HIV-R02 was shipped to 25 participants in August 2001, and challenge HIV-R03 was distributed to 31 laboratories in November 2001. RESULTS AND CONCLUSION: Participants correctly identified 74% of the total number of mutations and 82% of the panel members (i.e. all appropriate mutations detected in each panel member) in HIV-R01, 88% of the mutations and 91% of the panel members in HIV-R02, and 75% of the mutations and 52% of the panel members in HIV-R03. The AcroMetrix HIV-1 Resistance Proficiency Program, along with AcroMetrix's Internet-based reporting system (http://www.labqc.org), provides a useful tool for assessing operator proficiency within individual laboratories, and for comparing HIV-1 resistance testing proficiency among laboratories and technology platforms worldwide.