Utility of a Relatively Affordable In-House HIV-1 Genotyping Assay for Drug Resistance Testing among Non B HIV-1 Infected Drug Naive Patients in Nigeria

Background: The introduction of antiretroviral (ARV) in resource-limited settings has increased life expectancy among non-B HIV-1 infected individuals. We used a validated In-house genotyping assay to characterize non-B HIV-1 and to determine drug resistance mutations among treatment-naive patients. Methods: Plasma samples from 105 HIV-1 infected drug-naive adult patients attending a tertiary hospital Jos, Nigeria were subjected to HIV-1 RNA extraction, reverse transcription amplification, and population-based sequencing of the partial pol gene on the ABI 3130xl genetic analyzer. Subtyping and phylogenetic analyses were performed by REGA Subtyping Tool v2.0 and MEGA v5.0 respectively. Drug resistance profiles were evaluated according to IAS-USA 2013 drug resistance mutations list. Result: One hundred samples (95.2%) were successfully genotyped. The distribution of the non-B HIV-1 subtypes were; CRF02_AG-48%, G-41.0%, CRF06_cpx-6.0%, and A-5.0%. Ten percent of the isolates had at least one major drug resistance mutation in the pol gene. The drug-class specific resistance prevalences were 6.0% for NRTIs; M41L-1.0%, K65KR-1.0%, M184IM-1.0%, M184V-2.0%, and T215ADNT-1%, 8.0% for NNRTIs; K103N-2%, 1.0% for K101E, E138A, G190A, P225HP, Y181I, Y188L, Y181C including protease inhibitors’ Q58E How to cite this paper: Anejo-Okopi, J.A., Onywera, H., Abah, I.O., Ebonyi, A.O., Agbaji, O.O., Agaba, A.P., Oguche, S., Olonitola, O.S. and Idoko, J.A. (2018) Utility of a Relatively Affordable In-House HIV-1 Genotyping Assay for Drug Resistance Testing among Non B HIV-1 Infected Drug Naive Patients in Nigeria. Advances in Microbiology, 8, 355-365. https://doi.org/10.4236/aim.2018.85023 Received: April 6, 2018 Accepted: May 27, 2018 Published: May 30, 2018 Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Human Immunodeficiency Virus type 1 (HIV-1) is responsible for chronic infection leading to Acquired Immunodeficiency Syndrome (AIDS) as a result of chronic infection damage [1].The clinical course of HIV-1 infection is due to a high variability of the viral strain.This heterogeneity is a result of the high frequency of viral replication errors; retroviral reverse transcriptase (RT) lacking proofreading functions.As the virus replicates, genetic variations produced has ability for continued viral survival and growth as well as resistance to antiretroviral drugs (ARVs).However, while ARV is reducing mortality rates from AIDS-related causes, the widespread and long-term usage of drugs raises concerns with regards to emergence of ARV associated resistance mutations in evolving HIV strains.As evidence on the impact of large scale treatment programs in reducing incidence and increasing life expectancy in developing countries continues to accumulate, efforts to increase wider use ARVs will be intensified [2] [3].The use of potent ARVs can result in a profound and durable suppression of HIV-1 replication resulting in plasma HIV-1 viral load below levels of detection.The presence of some viral mutations introduced into the HIV-1 genome during replication can compromise efficacy, while others display susceptibility of the virus infectivity in combination with ARVs resistance.With this expected long-term and success of ART in developing countries, the coverage and programmatic effort should be complemented by HIV drug resistance (HIVDR) monitoring programs to ensure good clinical outcomes, and to reduce treatment failure and transmission of drug resistance [4].Thus, there is a need for more and affordable genotyping assays in attempt to effectively monitor and control drug resistance as more individuals are initiated onto ART as recommended by World Health Organization [5].In-house genotypic drug resistance testing has been successfully used in developed countries; for surveillance and monitoring of drug resistance in HIV infected individuals receiving ART, but has not been integrated into the continuum of care in most settings in Africa.
Most international guidelines recommend drug resistance testing for adult or The In-house HIV-1 genotyping assay is sensitive for detection and sequencing of non-B HIV strain variants specifically and it is CDC-supported Genotyping assays that well popular in East African countries [11] which is currently being deplored to CDC supported Laboratories in Nigeria, though not yet widely in use for clinical decisions.There is the need for Nigeria Government to buy into this program and fund it for effective utilization of the available ARVs to reduce emergence and transmission of drug resistance mutations, and associated economic cost.The utility of this assay will not only support researchers, but also impact positively on the success of HIV treatment populations and stop the spread of HIV and ARV-resistant strains.This study aimed to describe frequency of DRMs in HIV-1 genotypes that circulate in drug naive patients, using a cost-effective in-house HIV-1 GRT suitable for non-B HIV-1 variants.

Methods
This

Isolation and Amplification of HIV-1 Partial Pol Gene by RT-PCR and Nested PCR
We use two amplification protocols for HIV pol gene as previously described [9], HIV viral RNA were extracted from plasma using the QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany) followed by reverse-transcription of the

Sequence Analyses, Determination of Drug Resistance Mutation and Subtyping and Phylogenetic Analyses
The generated nucleotide sequences were viewed using the Sequence Analysis Software v3.7, and aligned and edited using Sequencher version 5.0, which assembles the six overlapping sequence segments for the six primers to form a contiguous sequence.Sequences with frame shifts or stop codons were excluded from analysis.The quality of the generated sequences was checked using Sequence Quality Assessment Tool (SQUAT).The sequences in fasta format were then subjected to the Stanford HIVdb algorithm (http://hivdb.stanford.edu/)for subtyping and determination of mutations conferring various antiretroviral drugs resistances.Mutations in the sequences were defined as differences from the consensus B reference sequence and were further characterized as RTI and
The participants whose spouses were on ARV were 14%, and those whose spouses were not on ARV were 86%.

Discussion
With the widespread availability of HAART in RLSs, the demand for sensitive and affordable monitoring of patients on ART is becoming a high priority.However, a better access to ARVs without adequate monitoring might result in a widespread transmission of drug resistant strains which will result to therapeutic failure.The current epidemic of drug-resistant HIV strains in RLSs can only be reduced by deliberate interventional strategies that would guarantee effective patients' regimen options, enhance correct intake of the medication, support uninterrupted drug supply, ensure the drug pharmaco-vigilance, treatment outcomes and provide useful therapeutic guidelines in case of treatment failures.The access to commercial genotypic assays and viral load monitoring in Africa is still limited and beyond the reach of the average patients due to high cost.Although these approved assays has demonstrated good performance on both subtype B non-B HIV-1, the high cost implication hinder their routine application in RLSs [13].
This study used a modified In-house GRT which had improved primer binding quality suitable for testing, the increased sensitivity, and found high degree of HIV-1 subtypes with the detection of diverse and rare mutations.Although recombinant subtype AG is still the most prevalent, it is responsible for only less than half of all the infections in Nigeria [9].Studies have shown the extensive genetic diversity of HIV-1 strains from West Africa including Nigeria and Cameroon with significant representations of the recombinant form CRF02_AG, subtypes G, J, and F [25].Although the performance of the two available genotyping assays can be considered as appropriate, but problems with non-B subtype strains do occur occasionally [21].
The main objective of this report was to present the utility of relatively affordable in-house HIV genotyping system for the monitoring of HIV-infected patients under treatment and surveillance.The method identified diverse major, accessory mutations and polymorphism which could be used as a guide for careful treatment options and further research into drug resistance surveillance, and the test can be performed at a lower cost compared to commercially available genotyping assays.Although, one is not ignorant of the need for expensive instrumentation, well trained personnel, the laboratory equipment requirement, however, the equipped laboratory can also be used for other applications including research.Now that the therapy has become widely available, affordable in-house genotyping for monitoring of patients on ARVs can be adopted and if properly utilized will avoid emergence of drug resistance in the population of HIV-1 strains.

Conclusion
HIV-1 was heterogeneously distributed; CRF02_AG and G predominate and some known major mutations associated with NRTIs, NNRTIs, diverse accessory, and natural polymorphism in the pol gene including one major mutation to PIs(Q58E) were determined.The In-house HIV-1 genotyping assay is suitable for both characterization of non-B HIV-1 subtypes and detection of drug resistance at a significant lower cost than available commercial genotyping assays.
This finding underscores the need to consider use of low-cost In-house genotyping assay as an alternative in resource-limited settings with non-B HIV-1 epidemic, and if well utilized, has potential to guide treatment options thereby keep the emergence of viral resistant strains at an acceptable level.In Nigeria, the burden of costs for treating HIV-infection is high, not only for the average citizens especially with the introduction of some laboratory investigation fees, but also the country's healthcare systems.Therefore the low cost and yet effective genotyping assay for non-B subtypes is a viable and practicable solution to expensive genotyping platforms.
study was carried out at the AIDS Prevention Initiative in Nigeria (APIN) CDC supported HIV clinic at the Jos University Teaching Hospital (JUTH), Jos.DOI: 10.4236/aim.2018.85023358 Advances in Microbiology The entry point for all patients was either through HIV counseling and testing (HCT) or referrals from other services within Jos metropolis and neighboring states.One hundred and five (105) HIV-1 infected treatment-naive patients were recruited sequentially after obtaining informed consent between October 2010 and April 2011.The institutional review board at Jos University Teaching Hospital approved the study protocol.A structured questionnaire was used to collect basic demographic data from each participant with no previous ARV exposure and was aged ≥ 18 years.Blood samples were collected in EDTA containers and plasma was extracted and cryopreserved.The samples were shipped in ice-parked containers to the Kenya Medical Research Institute HIV-Resistance Laboratory, Kisian Kisumu, where genotypic testing using affordable In-house genotyping System was done.Of 105 samples tested, 100 successfully amplified for genotypic drug resistance testing.
RNA into cDNA using the outer primers Prt-F1-forward (2253 -2275 nucleotides, nt) and RT-R1 reverse (nt 3370-3348) for RT-PCR.The cDNA were amplified by nested-PCR with primer Prt-F2 (forward, 2265 -2288 nt) and RT -R2 (reverse, 3326 -3304 nt).The amplified DNA fragments from nested-PCR were verified by visually comparing the intensity of each sample's band to that of the DNA mass ladder's bands of known DNA quantity for expected size by electrophoresis in 1.0% agarose gel stained with 0.5 µg/ml ethidium bromide and photographed under ultraviolet illumination.The fragments were purified using the QIA quick PCR purification kit (Qiagen, Hilden Germany) in spin columns, and direct population-based sequencing performed on both strands using Big Dye Terminator v3.1 Cycle Sequencing kit on ABI 3130X1 Genetic Analyzer.
showed the distribution relationship of different genetic subtypes in the pol region using 21 references from Los Alamos HIV Sequence database.The subtypes AG and G clustered around references from Nigeria and other West and Central African neighboring regions.

Figure 1 .
Figure 1.Phylogenetic Analysis of Isolates among the ARV Treatment naive Patients Jos, Nigeria.
DOI: 10.4236/aim.2018.85023357 Advances in Microbiology pediatric patients failing ART-first-line and second-line [6] [7].However, the affordability due to high-cost of the technology, and infrastructure requirements have limited the implementation of similar approaches to utility of drug resistance monitoring in resource-limited-settings (RLS).
PI associated resistance mutations.HIV-1 subtyping was performed using REGA HIV-1 subtyping tool V2.0 from Stanford HIV drug resistance database (http://hivdb.stanford.edu/),a worldwide subtype references were obtained from Los Alamos database (http://hiv-web.lanl.gov),and sequences were aligned against the known reference strains.Phylogenetic analysis was performed by the neighbor-joining (NJ) method as implemented in MEGA V.0.The boots canning method was used to detect and study recombination, as implemented in the SIMPLOT software v.2.5).The recombination was further confirmed with Recombination Identification Program (RIP) v.3.0 available online (http://www.hiv.lanl.gov/content/sequence/RIP/RIP.html).Subtypes assignments by this method are shown to the right of the tree with isolates clustered around the various references.Analyze the sequence using the HIVDB program at http://hivdb.stanford.edu.

Table 1 .
Prevalence of major ARV Resistance Mutations among the ARV Treatment-naive Patients.
R211K, V179IE, G196EG, V118I, V106I, A98SG, T69N, L228HL, and E44DE, J. A. Anejo-Okopi et al.DOI: 10.4236/aim.2018.85023361 Advances in Microbiology [24]u et al. 2011)thod in the follow-up of patients under treatment will allow the fast detection of emerging drug resistance at a more affordable cost than the commercial tests.The in-house assay is broadly sensitive in the detection of drug resistance genotyping of HIV-1 group M viral strains and has proofed to be more sensitive than the, commercially available assays in detecting mixed viral populations(Zhou et al. 2011).The substantial reagent cost saving and broad sensitivity make this assay more accessible for RLS where HIV drug resistance surveillance is strongly recommended to minimize the development and transmission of HIV drug resistant mutants[21] [22][23][24].