Abstract:
Background: Antiretroviral treatment (ART) has grown to be one of the most effective
tool in the fight to control HIV/AIDS morbidity and mortality worldwide. However, due to
the emergence of drug resistant HIV, ART efficacy can be jeopardized. Drug resistant
HIV strain has a potential of becoming a major public threat, as its limit treatment options
on people living with HIV. With several findings worldwide reporting drug resistant HIV to
be currently being transmitted to ART-naïve persons, measures have been taken to
genotype drug resistant HIV prior to treatment initiation. However, in resource limited
countries such measures are not executed especially in public sectors due to the costs
associated with the required assays for genotyping.
Objective: The objectives of the study was to establish a deep sequencing protocol (Next
Generation Sequencing-NGS) using an Illumina MiniSeq Platform and subsequently
apply it to genotype HIV in chronically infected drug naïve persons for resistance
mutations and viral genotypes
Methods: HIV positive Individuals without any exposure to ART (Treatment-naive) were
recruited. Partial pol fragment (complete protease and ~1104bp reverse transcriptase)
were amplified and purified. Libraries were prepared using Nextera XT library preparation
kit, fragmented, tagmented, pooled and denatured then sequenced with Illumina MiniSeq
instrument. Consensus sequences were derived, aligned and phylogenetically analysed.
The Stanford HIV Drug Resistance Algorithm was used to infer the presence of drug
resistant mutants, at the viral minority and majority population levels.
Results and discussion: An NGS protocol to generate nucleotide sequences for drug
resistance inference was established. No major drug resistance mutations were detected
against protease, reverse transcriptase inhibitors in the study subjects investigated.
Nevertheless, V179D change was observed in one patient (8.3%). V179D has been
shown to impact a low-level resistance to NNRTI. On the other hand, several secondary
and unusual mutations at known drug sites were detected even at minority threshold level
of <20%.
Conclusion: No major drug resistance mutations was detected in the drug naïve study
population. This finding suggests that there is no risk of treatment failure to the
investigated subjects, however it is important to assess the potential phenotypic
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significance of the identified secondary resistance mutations in the context of HIV-1
subtype C. The established NGS protocol should be applied in subsequent HIV drug
resistance studies.
