الفهرس 
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Abstract
AIDS is one of the most overwhelming infectious diseases in history that caused by HIV virus. HIV is classified into two major types: HIV-1 and HIV-2. HIV-1 is grouped into 4 phylogenetic groups: M, N, O and P group. HIV-1 group M is the main cause of AIDS pandemic worldwide. It is further classified into 9 “pure” subtypes in addition to numerous CRFs and URFs.
The main purposes of this study were to determine the current status of HIV-1 subtypes circulating in Alexandria, Egypt. and to detect the genetic related-mutations to NNRTIs, NRTIs and PIs. This study presented a unique attempt for detection of DRMs to either RTIs and/or PIs among treatment-naïve or treatment-experienced HIV-1 patients.
The goals of using ART for HIV infection are to achieve and maintain virologic suppression, thereby preventing disease progression and transmission. The HIV-1 drug resistance has become a growing public health problem worldwide. An alarming surge in HIV-1 drug resistance is recently uncovered by WHO surveys in 2019.
This study was conducted in Medical Research Institute on 60 randomly collected non-relative HIV-1 patients except a mother and her 8-year old daughter that were enrolled from HIV department of Alexandria Hepatology, Gastroenterology and Fever Hospital, during the period of October 2017 to July 2019. Of them 15 were excluded because their viral loads were less than 1000 copies/mL during treatment. The remaining 45 HIV-1 participants included had CD4 count ranged from 45-810 mm3 and viral load from 1000 to 1.3 * 107copies/mL.
The viral genomic RNA was extracted using QIAamp Viral RNA Mini™ Kit and reverse transcribed using High-Capacity cDNA Reverse Transcription™ Kit. cDNA was amplified by nested PCR reaction. Out of 45 HIV-1 patients, the viral pol genes encoding PR and RT regions of 21 HIV-1 isolates were successfully amplified then sequenced using BigDye terminator cyclesequencing kit.
All 21 amplified HIV-1 isolates were genotyped using five subtyping tools: NCBI viral genotyping tool, Stanford subtyping program, REGA, COMET and RIP. Both NCBI and Stanford were in agreement in almost all HIV-1 patients while REGA agree with RIP in 80% of HIV-1 patients. COMET was the oddest tool with high percent of uncertainty of HIV-1 subtype.
The final subtype assignment was achieved using practical subtyping algorithm and performing molecular phylogenetic analysis. Viral subtyping revealed that the most common HIV-1 subtype was CRF02_AG (57.1%), followed by subtype B (23.8%), CRF35_AD (9.5%), subtype A1 and CRF06_cpx (4.8% each).
To investigate the relationships between the 21 HIV-1 isolates. The PR and RT regions of the pol gene covering ~ 1000 bp of the 21 HIV-1 were analyzed using MEGA X program for generation of a phylogenetic tree using NJ methodwith 1000 replicates bootstrapping. Three clusters were presented. One cluster encompassing 9 HIV-1 CRF02_AG isolates and the secondcluster covering 2 HIV-1 CRF02_AG isolates. The third cluster was including the rest of 21 HIV-1 isolates.Another molecular phylogenetic analysis was conducted using a set of representative polreference sequences (716 pol sequences) called GSPS. There was only one dissimilarity compared with final subtype assignment by used practical algorithm. where isolate 10 was subtyped as CRF54_01B with molecular phylogeny and as subtype B according to the practical algorithm.
The analysis of HIV-1 sequences was pursued in order to elucidate a mutation profile for each patient and to determine HIV-1 drug resistance levels toward NNRTIs, NRTIs and PIs. All major, accessory and other mutations or polymorphisms were searched for according to both Stanford HIVDB and the IAS-USA list, 2019 edition.
NNRTI DRMs and NRTI DRMs were described with penalty scores according to Stanford HIVDB. On the other hand, IAS-USA list, 2019 edition is more comprehensive than Stanford HIVDB in describing PI related-mutations.
Both major NNRTIs and NRTIs DRMs were recognized in the current study nevertheless, only minor PI mutations were observed among all study participants. A total of 15 NNRTI related-mutations were documented. The most frequent NNRTI DRM was K103N (14.3%). Notably, a mutation conversion was observed in one HIV-1 isolate in this study. A total of 13 NRTI related-mutations were acknowledged. The most frequent NRTI DRMs were M41L and V75M. A total of 18 minor PI mutations were identified according to IAS- USA, 2019 list.
Several mutation patterns related to NNRTIs and NRTIs were recognized based on Stanford HIVDB however, only one universal PI pattern was observed. Six different NNRTI DRM patterns were detected that were previously reported by Stanford HIVDB. Four NRTI DRM patterns were detected of which 2 were previously reported by Stanford HIVDB.
CPR tool provided by Stanford HIVDB was utilized to search for SDRMs among 21 HIV-1 isolates. Several suggestive NNRTI and NRTI SDRMs were detected. No PI SDRMs were found.No TDR was detected among treatment-naïve patients in this study.
Genetic variability in HIV-1 PR and RT regions of pol gene were assessed. Highly polymorphic PR codons such as: S37, L63, I72 and L89 were observed. Highly polymorphic RT codons: V35, E122, D123, I135, S162, K173, Q174, D177, T200, Q207 were observed. Finally, each HIV-1 isolate had a unique distinct mutation profile.
Genotypic drug resistance interpretation was performed through submitting HIV-1 sequences into HIValg program on Stanford HIVDB. The drug resistance level toward 5 NNRTIs: DOR, EFV, ETR, NVP, and RPV and 7 NRTIs: ABC, AZT, d4t, DDI, FTC, 3TC and TDF and 7 ritonavir boosted PIs: ATV/r, DRV/r, FPV/r, IDV/r, LPV/r, SQV/r and TPV/r were assessed. HIValg provided comparative tables with 3 different rule-based drug resistance interpretation algorithms: HIVDB, ANRS and REGA. French ANRS algorithm suggested different results from Stanford and REGA regarding PIs drug resistance levels in this study.
HIV drug resistance to each class was estimated. A high PI drug resistance (85.8%)toward TPV/r according to ANRS could not be neglected followed by NNRTI drug resistance (28.6%) according to Stanford HIVDB algorithm. The NRTI drug resistance (14.3%) was the least detected among the 21 HIV-1 patients.
Total drug resistance in relation to subtype was calculated. DRMs conferring resistance for NNRTI and NRTI were estimated as 100%, 66.67%, &16.7% and 50%, 20%, &8.3% in CRF35_AD, subtype B, CRF02_AG, respectively. Other subtypes, subtype A1 and CRF06_cpx did not show any RTI DRMs. On the other hand, resistance to PI due to minor mutations were estimated as 100% in all subtypes except subtype B (40%).
To the best of our knowledge, this is the first study in Egypt to tackle HIV-1 subtyping among heterogeneous group of patients. The results of the current study serve as an initial crucial step in determining the overall prevalence of HIV-1 drug resistance in Egypt.