|Year : 2015 | Volume
| Issue : 4 | Page : 565-567
HIV reverse transcriptase gene mutations in anti-retroviral treatment naïve rural people living with HIV/AIDS
K Mohanakrishnan1, A Kasthuri2, SK Amsavathani3, G Sumathi1
1 Department of Microbiology, Sri Muthukumaran Medical College and Research Institute, Mangadu, Chennai, Tamil Nadu, India
2 Department of Community Medicine, Sri Muthukumaran Medical College and Research Institute, Mangadu, Chennai, Tamil Nadu, India
3 Department of Microbiology, Meenakshi Medical College and Research Institute, Enathur, Kanchipuram, Tamil Nadu, India
|Date of Submission||30-May-2014|
|Date of Acceptance||08-Jun-2015|
|Date of Web Publication||16-Oct-2015|
Department of Microbiology, Sri Muthukumaran Medical College and Research Institute, Mangadu, Chennai, Tamil Nadu
Source of Support: None, Conflict of Interest: None
This study is designed to find out the mutational variations of reverse transcriptase (RT) gene of HIV, after the traditional drug usage among anti-retroviral therapy naïve rural people living with HIV/AIDS. HIV Reactive patients, who were exposed for indigenous medicines such as Siddha, Ayurveda etc., for a minimum period of 6 months were taken for this study. Among 40 patients, two samples (5.55%) demonstrated high-level mutational resistance variations for nucleoside RT inhibitor (NRTI) and non-NRTI. The predominant polymorphisms detected were K122E (91.7%), V60I (91.7%), V35T (89%), Q207E (89%), D177E (89%), T200A (86.1%), S48T (83.33%), K173A (80.6%).
Keywords: Anti-retroviral therapy, HIV, people living with HIV/AIDS, reverse transcriptase gene
|How to cite this article:|
Mohanakrishnan K, Kasthuri A, Amsavathani S K, Sumathi G. HIV reverse transcriptase gene mutations in anti-retroviral treatment naïve rural people living with HIV/AIDS. Indian J Med Microbiol 2015;33:565-7
|How to cite this URL:|
Mohanakrishnan K, Kasthuri A, Amsavathani S K, Sumathi G. HIV reverse transcriptase gene mutations in anti-retroviral treatment naïve rural people living with HIV/AIDS. Indian J Med Microbiol [serial online] 2015 [cited 2020 Jul 4];33:565-7. Available from: http://www.ijmm.org/text.asp?2015/33/4/565/167326
| ~ Introduction|| |
After knowing the HIV reactive status, most of the people in rural setup choose the native drugs before starting the anti-retroviral therapy (ART), because of the discrimination, false claims by the alternative medical practitioners and the myths about the disease which may have an impact on mutations of the reverse transcriptase (RT) gene. So, it was decided to assess the prevalence of genotypic variations and resistance mutations of the RT gene of HIV, after the native drug pressure among ART naïve rural people living with HIV/AIDS (PLHA).
| ~ Materials and Methods|| |
Study design and study population
A total of 40 PLHA who were exposed for indigenous medicines such as Siddha, Ayurveda, etc., for a minimum period of 6 months were taken from the group of 207 HIV reactive persons. This study was conducted at the Meenakshi Medical College, and the samples were processed for gene sequencing at YRG CARE, Chennai. Drug resistance assay, the HIV-1 genotyping assay was performed using the homebrew methodology. RT regions of the polymerase (pol) gene of the HIV genome were amplified by complementary DNA (cDNA) conversion and nested polymerase chain reaction (PCR) as follows. Viral RNA was extracted from plasma Siam viral RNA extraction kit (Qiagen Inc., Valencia, CA, USA) and reverse transcribed to cDNA with a single temperature cycle (i.e.,) 25°C for 10 min, 42°C for 1 h, and 70°C for 15 min. RT from the pol gene was amplified from cDNA using relevant primers. All PCR amplifications were performed on the Gene Amp PCR System 9700 (Perkin Elmer, Waltham, MA, USA). Amplified products from the first round of PCR were initially analysed on an ethidium bromide-stained agarose (1%) gel. If the amplicon was visible on the gel, it was column purified using a PCR purification kit (iNtRON Biotechnology, Inc., Gyeonggi-Do, Korea). Those samples that were not detected after the first-round PCR were subject to a second-round nested PCR. The amplicon was again checked on an agarose gel and subsequently column purified. The purified PCR products were subjected to bidirectional sequencing on an automated ABI 3100-Avant Genetic Analyser (Applied Biosystems, Foster City, CA, USA). Both forward and reverse sequences were assembled and manually edited with the SeqScape version 2.5 multiple sequence alignment software and exported as a FASTA (Seqscape-Life Technologies FASTA-European Bioinformatics Institute) format consensus RT sequences. The consensus sequences of each specimen were assessed for drug resistance using the Stanford HIV drug resistance database available at http://hivdb.stanford.edu/.
| ~ Results|| |
Among all (n = 40) isolates, only 36 (20 males, 16 females) were successfully amplified at RT region of pol gene. There were major mutations found that were directly associated with high-level resistance to ARV drugs and the overall prevalence of this was 5.55% (n = 2) against nucleoside RT inhibitor (NRTI) and Non-NRTI (NNRTI). One of the isolates was found to harbour mutations (M184V, T215Y, K103S and G190A) associated with reduced susceptibility to NRTI drugs. The other isolate was found to harbour 2 mutations (K103KN and G190AG) associated with reduced susceptibility to NNRTI drugs. In the RT region, the predominant polymorphisms detected were K122E (91.7%), V60I (91.7%), V35T (89%), Q207E (89%), D177E (89%), T200A (86.1%), S48T (83.33%), K173A (80.6%).
| ~ Discussion|| |
Testing for the presence of drug resistance strains prior to initiation of therapy may be economical and beneficial to the individual. In countries where the standard initial and alternate antiretroviral regimens are restricted, it is important to evaluate if transmitted resistance has reached a population level that could affect the effectiveness of ART. Studies from India have not revealed any high prevalence of drug resistance conferring mutation.,, As per WHO protocol, the drug resistance prevalence in a geographical area can be categorised into three categories <5%, 5–15% and 15%. In the current study, RT regions from the pol gene of HIV-1 viral strains from PLHA were characterised. All pol sequences of the study patients clustered with subtype C reference sequences with no evidence of recombination.
A study by Deshpande et al. from Mumbai indicated that two isolates of 128 (1.6%) had the M184V mutation, indicating primary drug resistance to 3TC. A phenotypic study by Hira et al. conducted in Mumbai has shown a higher prevalence (6.7%) of primary drug resistance to RT inhibitors. Another genotypic study by Balakrishnan et al. from Chennai involving drug naïve patients attending HIV clinic has also shown the absence of primary drug resistance. Our study showed 5.55% prevalence for Primary NNRTI drug resistance and 2.77% to Primary NRTI drug resistance. There was not much variation found in drug resistance compared to the alert cut-off level because of the native drugs used by the ART naïve reactive patients. This study, although limited to 40 drug-naive individuals, offers some reassurance that the contemporary subtype C viruses in Southern India are not likely to exhibit high levels of drug resistance. This is consistent with evidence from other non-subtype B studies.
Enormous polymorphic substitutions were observed in the current study. V35T, K122E, S48T, V60I, T200A, Q207E, D177E, K173A were observed in more than 80% of the sequences. In Iqbal et al., at RT region, the predominant polymorphisms detected were V60I (98.2%), S48T (94.5%), K122E (90.9%), T200A (85.5%), and D177E (80%). These observations reveal that HIV-1 polymorphisms differ in different geographical locations and population groups within the same subtype. These polymorphic variations are important in terms of vaccine design.
| ~ Conclusion|| |
In the current study, RT regions from the pol gene of HIV-1 viral strains from PLHA were characterised. HIV-1 drug resistance has been observed among ART-naive PLHA. There was not much variation found in drug resistance compared to the alert cut-off level because of the native drugs used by the ART naïve reactive patients. The presence of drug resistance mutations, though minimal, is important as the drug-resistant strains could spread among PLHA and to their sexual partners. Enormous polymorphic substitutions were observed in the current study. There is a definite need to generate the drug resistance database and the polymorphic pattern of Indian strains for the future clinical management and vaccine design to contain the disease.
| ~ Acknowledgments|| |
The authors would like to thank Dr. Sunithi Solomon, Dr. P Balakrishnan Dr. Saravanan and Dr. M Vidya for their immense help in carrying out the molecular study at YRG CARE.
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