Detection of M41L resistance mutation in patients infected with HIV by Real-Time Polymerase Chain ??Reaction

HIV / AIDS, has been reported as most frequent cause of death in sub-Saharan Africa, and ranks fourth on the the world wide list. One of the most important problems in the treatment of AIDS is the resistant mutants for antiretroviral drugs. Currently, routine or epidemiological HIV antiviral resistance tests are commonly performed by conventional sequence assays. However in recent years, more sensitive methods have been developed, to detect drug resistant mutant variants in the minority of the total viral population which adversely affect the success of the treatment, compared to the conventional sequence assays. In this study, we investigate M41L mutations, for zidovudine, the first and still an important drug in HIV treatment and for Stavudine, which is for one of the common mutations, and has not been previously investigated by real-time PCR method. Point-mutation-specific Real-Time Polymerase Chain Reaction (PMS-RT-PCR) method which is more sensitive than conventional sequence analysis method is performed in this study. Sixty HIV?1 genotype B positive samples which were examined by conventional sequence analysis (ABI Prism 310, Foster City, USA) in the Laboratory of Molecular Microbiology, Cerrahpaşa Faculty of Medicine. Point mutation spesific Real- time PCR method (PMS-RT-PCR; which was reported as a more sensivite and easy to interpret test compared to conventional sequence assays, developed and validated by Heneine et al. to detect HIV-1 genotype B, consisting nine major drug mutations; was performed on LightCycler 2.0 (Roche Applied Science, Germany) system. Recommended primers and TaqMan probe for detection of M41L mutation for resistance were used. Three samples were excluded due to poor quality of the RNA quality. To distinguish between wild type and mutant variants, in order to determine the criteria for positivity, difference between Ct values (?threshold cycle?) of the mutant variant and the Ct values of viral copies of total reaction were evaluated.According to the results of the PMS-RT-PCR; for 57 samples of HIV-1genotype B, when Ct value of the amplification of the total viral copies less than 10 (reexamination with the need for dilution due to the high number of copies), and higher than 26 (low copy number due to uncertain results ) were applied, a total of 25 samples which would be used as measure of positivity were exculeded from the study to determine the value of ?Ct threshold. 32 samples, used to calculate the threshold. The threshold was determined according to the distribution of central tendency and median ?Ct value (5.81). When ?Ct value was at threshold level, samples were considered negative for the mutation M41L. M41L resistance mutation were detected in 16 of 32 samples which were evaluated by this threshold according to the PMS-RT-PZR. Six of these samples, were M41L resistance mutations which were positive according to the results of conventional sequence analysis. Results of the seven of 13 samples which were reported to be positive by conventional sequence analysis, were accepted uncertain as the total viral copy Ct's were above 26 according to the results of the PMS-RT-PCR assay. In these examples, because of low viral loads, after pre-amplification stage, to minimize the risk of false results, the test was performed. However, as the storage conditions were not proper (too much freeze- thaw) and since the quality of RNA were unacceptable, these samples were not examined again. Since both conventional sequence analysis and PMS-RT-PCR method were not at the status of a reference method, the reliability of comparison between the two methods, was measured by Kappa Statistics. According to Kappa statistics, between the two methods, a fair agreement (k = 0,375, P <0.007) was found. To our knowledge, this is the first study in Turkey which investigated the minority resistance mutations to antiviral drugs used to treat HIV infection with a more sensitive and easier method than conventional sequence assay. PMS-RT-PCR method, presently has been developed for testing only one group of mutations for HIV. PMS- RT-PCR method provides near to ultra-deep sequencing sensitivity for exploring an important group of minor resistance mutations. Therefore, testing with this method is expected to contribute to better understanding of epidemiologic dynamics related to antiviral resistance in HIV infections and increase the success of treatment.