Abstract:
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ABSTRACT
Human Immunodeficiency Virus (HIV) infection remains a major global public health concern with 36.9 million people infected. The HIV health burden is most felt in Sub-Saharan Africa, where about 70% of the infection occurs. The unprecedented scale-up of access to antiretroviral therapy (ART) has improved the management of HIV and reduced HIV-associated morbidity and mortality. However, long term sustainability of this success requires treatment monitoring and surveillance of emerging HIV drug resistance in patients during combination Antiretroviral Therapy (cART). This study aimed at characterizing HIV-1 drug resistance mutations (HIVDRM) in children and adolescents failing treatment; investigating the relatedness of the circulating viral isolates, and modifying and assessing performance characteristics of the Thermofisher HIV drug resistance genotyping assay. Fifty plasma samples collected from children and adolescents experiencing virologic failure were used to characterize drug resistance mutations and an additional set of 26 plasma samples used to assess the performance of the modified assay. RNA was extracted from 500μl of plasma and subjected to reverse transcriptase (RT) PCR before PCR amplification. The amplicons were purified and sequenced using the ABI 3730 genetic analyzer platform. The modified assay was assessed by testing its accuracy, precision, reproducibility, and amplification sensitivity. Out of the 50 participants tested in this study, 42 harbored at least one major drug resistance mutation. Mutations to nucleotide reverse transcriptase inhibitors (NRTIs), non-nucleotide reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) were present in 34/50, 38/50 and 2/50 participants respectively. HIV-1 subtype A was the most prevalent (73%). The accuracy, precision, and reproducibility of the modified assay were 98.5% (CI, 97.9 – 99.1%); 98.67% (CI, 98.1 – 99.23), and 98.7% (CI, 98.1 – 99.3) respectively. Test for concordance between the two assays showed no difference in mutations detected by both assays (χ2 = 2.358, df=1, p<0.05). The modified assay had an amplification sensitivity of 62.5% for viremia between 200 and 999 copies/ml and 100% for viremia above 1000 copies/ml. Assay modification resulted in a 38.5% reduction in reagent cost per test. The study showed that HIV-1 drug resistance remains to be a major barrier to disease management in children and adolescents. To implement routine HIVDR testing, there is a need to adopt validated cost-effective methods for HIV drug resistance surveillance.
