ABSTRACT
OBJECTIVE: The Roche LightCycler 480 (LC480) system was evaluated for quantitative molecular diagnosis of opportunistic viral infections caused by human cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus 6 (HHV-6), and BK virus (BKV), in comparison with "in-house" real-time PCR assays. PATIENTS AND METHODS: A total of 253 whole blood specimens obtained from transplant recipients were tested. RESULTS: Both the "in-house" and the LC480 methods were highly correlated (Spearman correlation coefficient Rho> or =0.85; p<0.0001) with an excellent overall qualitative agreement (90.5%) and no significant quantitative difference between both techniques for the four viruses tested. The accuracy of the LC480 protocols were confirmed further by the results obtained with the 44 samples from the Quality Control for Molecular Diagnosis (QCMD) 2008 proficiency panel. CONCLUSION: The LC480 system constitutes a suitable and versatile real-time PCR platform in a routine laboratory setting for the diagnosis and monitoring of opportunistic viral infections in transplant recipients, by measuring HCMV, EBV, HHV-6, and BKV loads in whole blood samples.
Subject(s)
BK Virus/isolation & purification , Computer Systems , DNA, Viral/blood , Herpesviridae Infections/virology , Herpesviridae/isolation & purification , Polymerase Chain Reaction/methods , Polyomavirus Infections/virology , Reagent Kits, Diagnostic , Tumor Virus Infections/virology , Viral Load , Viremia/virology , BK Virus/genetics , Herpesviridae/genetics , Herpesviridae Infections/blood , Humans , Organ Transplantation , Polyomavirus Infections/blood , Postoperative Complications/blood , Postoperative Complications/virology , Quality Control , Reproducibility of Results , Sensitivity and Specificity , Tumor Virus Infections/bloodABSTRACT
Recently, it has been shown that a new mutational pattern (the E44D/A and/or V118I mutation) confers moderate phenotypic lamivudine resistance in the absence of the M184V mutation. The E44D/A and/or the V118I mutation does not exist in drug-naive patients, and the prevalence increases with the number of treatment regimens and lamivudine experience. The mutations can preexist in nucleoside-experienced but lamivudine-naive patients. They are always associated with zidovudine resistance-associated mutations, even in the absence of M184V. These mutations are more stable than the M184V substitution during antiretroviral treatment interruptions.
Subject(s)
HIV Reverse Transcriptase/genetics , HIV-1/genetics , Alanine/genetics , Amino Acid Substitution , Anti-HIV Agents/pharmacology , Anti-HIV Agents/therapeutic use , Aspartic Acid/genetics , Drug Resistance, Microbial/genetics , Gene Frequency , Glutamic Acid/genetics , HIV Infections/drug therapy , HIV-1/drug effects , HIV-1/enzymology , Humans , Isoleucine/genetics , Mutation , Valine/geneticsABSTRACT
Recently, d4T/ddI combination has been associated with the selection of thymidine analogue mutations (TAMs) in 50% of patients with a detectable viral load after 6 to 12 months of this bi-therapy (ALBI, STADI and BMS A1460 tests). We evaluated the rate of selection of the TAMs in naive patients with a viral load of > 200 copies/mL after: 6 months to 1 year of d4T/3TC bi-therapy (group 1); 1 year or more of a treatment including d4T/3TC (group 2); and more than 6 months of tri-therapy including d4T/ddI (group 3). The reverse transcriptase gene has ben studied in 33 patients in group 1, 17 patients in group 2 and ten patients in group 3. For the latter patients, the tri-therapies were as follows: d4T/ddI/PI (n = 5), d4T/ddI/NNRTI (n = 4), d4T/ddI/NRTI (n = 1). For the group 1 patients, at baseline, two patients already had TAMs. At M6, all the patients acquired the 3TC-associated mutations, M184V. Only one patient selected a MDR mutation profile (F116Y, Q151M). At M12, 26 of 33 plasmas were analysed. Only one patient selected one TAM (T215Y). For the group 2 patients, only three patients selected TAMs after more than 30 months of treatment. For the group 3 patients, at baseline, only one patient already harbored TAMs. None of the other patients had selected TAMs. In patients who received d4T/ddI/3TC, only the M184V, the 3TC-associated mutation, was selected. In conclusion, stavudine in association with 3TC selected a low rate of TAMs; in patients receiving a treatment including d4T/3TC, time of exposure to stavudine seemed to be an important parameter for the selection of TAMs; and in contrast to results obtained on d4T/ddI, tri-therapies including d4T/ddI did not select any TAMs, whatever the combination (NRTI, NNRTI, PI).
