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Intracellular DNA Levels of Drug Resistant and Drug Sensitive Human Immunodeficiency Virus Infections and its Correlation with Disease Progression Markers in a Population of Africans

Affram, Yvonne

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Abstract

There has been significant reduction in the scale of the global HIV pandemic over the last decade due to increased awareness and the effectiveness of Highly Active antiretroviral therapy (HAART). However, new challenges have emerged which when not effectively addressed may hinder the progress made so far. One important area of difficulty in the management of HIV infection is the emergence and transmission of drug resistant mutations (DRM), which threatens the long-term use of current HAART. Also, there is a growing need for more effective and relatively cheaper markers for monitoring HIV especially in resource-limited areas. One of such potential markers is HIV-l intracellular (IC) DNA load. We analyzed plasma and buffy coat samples as well as clinical data of 86 HIV-1 infected drug naïve patients and eight follow-up patients with persistently high viral loads after 24 weeks of HAART from Nouna in Burkina Faso. The participants were predominantly females. Among drug naïve patients, paired RNA and DNA templates were polymerase chain reaction (PCR) amplified and Sanger sequenced for the detection of DRMs. Templates encompassed 1461 base pairs sequenced from the protease and reverse transcriptase region of HIV-1 pol. A total of eight patients harboured transmitted drug resistance mutations (TDRMs). Six had TDRMs to non-nucleoside reverse transcriptase inhibitors (NNRTIs), one to nucleoside reverse transcriptase inhibitors (NRTIs), and one to protease inhibitors (PIs). Given that interest is growing in the use of viral DNA sequencing to complement or replace RNA for DRM monitoring, we compared sequences from RNA and DNA templates, for similarities and differences in regions with DRMs and for nucleotide differences. A high level of concordance 78 (94%) was observed in regions with DRMs. Also, nucleotide sequences of paired templates were highly similar (84%). Furthermore, observed nucleotide differences greater than 10 within viral sequence pairs were resolved with deep sequencing using the so-called “Nextera tagmentation” approach. Deep sequencing of thirteen RNA and DNA template pairs revealed predominantly major reverse transcriptase (RT) mutations M230I and M184I within the minority viral population. It also confirmed differences in DRMs observed in Sanger sequencing and showed that patterns of synonymous and non-synonymous nucleotide changes were similar to those seen in Sanger sequencing. Predominant HIV-1 subtypes found in patients were CRF02_AG and CRF06_cpx. With the eight follow-up patients, DRMs and HIV-1 subtyping were determined from RNA templates only. Drug resistance mutations contributing to virological failure after HAART were assessed. Major DRMs to RTIs mainly K103N, E138Q, G190A and M230L were detected. Finally we assessed the utility of HIV-1 IC DNA load as a tool for disease monitoring and found that HIV-1 IC DNA levels did not correlate with traditional HIV-1 disease monitoring markers such as CD4+ T-cell counts (r2=0.017; p=0.23) and plasma viral loads (r2=0.003; p=0.60), as well as other markers of disease progression. Also, there was no association (p=0.26) found between HIV-1 IC DNA levels of drug resistant (median 2.96 log10 copies/106 cells, IQR 2.30-3.52) and drug susceptible (median 2.62 log10 copies/106 cells, IQR 2.23-3.06) strains of HIV-1, signifying that the presence of TDRMs does not affect HIV-1 IC DNA levels. However, a significantly higher baseline HIV-1 IC DNA level (p=0.045) was found in patients failing HAART after 24 weeks of therapy (median 3.16 log10 copies/106 cells, IQR 2.75-3.62) as opposed to those who did not (median 2.63 log10 copies/106 cells, IQR, 2.12-3.04). Our findings show that the prevalence of TDRMs is high and new DRMs develop over time making it necessary to institute resistance testing for more effective clinical management. Also the high concordance in DRMs between viral RNA and DNA templates suggests that DNA could be used for resistance monitoring, as it is cheaper and relatively easy to handle. HIV-1 IC DNA load is an independent marker that could be used alone or together with plasma viral load, CD4+ T-cell counts and other markers to monitor disease progression. A larger follow-up study is recommended to confirm these findings.

Item Type: Dissertation
Supervisor: Kräusslich, Prof. Dr. Hans-Georg
Date of thesis defense: 2 March 2016
Date Deposited: 16 Mar 2016 12:53
Date: 2016
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
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