الفهرس 
TitleOnly 14 pages are availabe for public view
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Abstract
Human immunodeficiency virus (HIV) belongs to the genus Lentivirus of the family Retroviridae with its genome consisting of two identical positive-sense single stranded RNA. Globally 38 million people are infected with HIV, out of them; 81% knew their status, 67% were on treatment and 59% were virally suppressed.
Aiming at better sustained viral replication suppression with minimal side effects, recovery of the immune system functions and improved tolerability, a combination of at least three inhibitors drugs (two NRTIs and either a PI or a NNRTI or an INSTI) have been developed which specifically target key regions of the HIV genome known as, highly active antiretroviral therapy (HAART).
The reverse transcriptase enzyme, responsible for reverse transcription of viral RNA into a complementary DNA (cDNA), the protease which cleaves gag and gag-Pol precursor proteins to form functional proteins and viral enzymes and the integrase which incorporates the viral DNA into the host cell genome are the most important targets during viral replication.
However, the high genetic diversity and mutation rate lead to development of viral quasispecies with varying degrees of resistance to antiretroviral agents which increase the potential of antiretroviral therapy failure.
The aim of this study was to determine the HIV-1 subtypes and the major resistant mutations to NRTIs, NNRTIs and PIs among HIV patients in Alexandria, Egypt.
Fifty-seven HIV-1 positive patients were selected from outpatient AIDS clinic of Hepatology, Gastroenterology and Fever Hospital in Alexandria, from August 2017 to July 2019. Demographic data were collected (age, sex) as well as data regarding treatment. Blood samples were collected from each patient and separated sera were divided into aliquots and stored at – 80°C for the following investigations:
HIV RNA extraction using Qiagen QIAamp viral RNA mini spin kit.
cDNA synthesis using High-Capacity cDNA Reverse Transcription Kit.
Amplification of partial gag-pol domain by nested PCR on Veriti Thermal Cycler (Applied Biosystem) using pairs of specifically designed primers.
The PCR product were analyzed on 1.4% agarose gel stained with ethidium bromide.
Cycle Sequencing of the amplified regions using BigDye Terminator V 3.1 Cycle Sequencing Kit.
Trimming and assembly of sequences using BioEdit version 7.0.5.3.
Summary, Conclusion and Recommendations
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Identification of HIV-1 sequences subtypes by phylogenetic analysis and seven different online tools (Stanford HIV database, NCBI, COMET, Rega, SCUEAL, RIP, jpHMM-HIV).
Detection of NRTIs, NNRTIs and PIs resistance mutations using Stanford HIV database, ANRS and Rega algorithms.
The current study included fifty-seven HIV-1 positive patients. Out of them, twenty-seven patients were excluded because of their low viral loads (less than 1000 copies/ml). Among the thirty cases selected for the study, partial gag-pol domain was amplified only in twenty-one (70%) cases.
Among the twenty-one HIV-1 strains available for subtyping, twelve (57.1%) strains were CRF02_AG, five (23.8%) strains were subtype B, two (9.5%) strains were CRF35_AD, one (4.8%) strain was subtype A and one (4.8%) strain was CRF06_cpx according to phylogenetic analysis.
Stanford HIV database showed the highest accuracy (100%) among the used HIV subtyping tools, followed by NCBI (85.7%) and COMET (76.2%). On the other hand, Rega and SCUEAL showed intermediate accuracy [(71.4%) and (42.9%) respectively]. RIP (33.3%) and jpHMM-HIV (28.6%) showed the least accuracy among the used tools.
Among the three (14.3%) NRTIs resistant cases, M41L, D67N, K70R, M184V, L210W and T215F/Y were reported as a major NRTIs resistance mutations, while E44D, V75M, K219E were reported as minor NRTIs resistance mutations according to Stanford HIV Database. Also, Rega drug resistance interpretation algorithm, reported the same mutations causing resistance to NRTIs among our cases. For ANRS algorithm, V75M was not reported as a mutation causing resistance.
Among the five (23.8 %) NNRTIs resistant cases, K101P/E, K103N/S, Y188L, G190A and P225H were reported as a major NNRTIs resistance mutations, while V106I, E138A, V179E and H221Y were reported as minor NNRTIs resistance mutations according to Stanford HIV Database. Rega and ANRS also reported, V90I as mutation causing resistance, which was considered as “other mutation” in Stanford HIV Database.
No major PIs resistance mutations were reported. Only one accessory mutation (L89V) was detected according to Stanford HIV database.
In this study, discrepancies were observed among the three used online genotypic drug resistance interpretation algorithms, Stanford HIV database, ANRS and Rega, regarding susceptibilities to ARV drugs. For NRTIs, all the three reported resistant patients were discordant for didanosine, two patients (6 and 10) for abacavir and also two (patients 6 and 13) for tenofovir disoprovil fumarate, while only patient 6 for zidovudine and patient 10 for emtricitabine and lamivudine.
As for NNRTIs, discrepancies between the three algorithms were observed in all the five reported resistance patients. Four patients (1,6,7 and 10) were discordant for etravirine, three patients (7,10 and 13) for doravirine, while only one patient (6) for rilpivirine.