Dawit Assefa Ethiopia Health and Nutrition Research Institute Dawit Assefa Ethiopia Health and Nutrition Research Institute Evaluation of an in-house HIV drug resistance testing assay

In Ethiopia free antiretroviral therapy (ART) program was launched in 2005 Rapid scale up of ART, as of June health facilities 247,805 patients were on ART Back ground With the expansion of ART, a proportion of treated cases can be expected to develop drug resistance, risking transmission of resistant HIV strains Year

Monitoring the prevalence of drug resistance would be an effective strategy to monitor program effectiveness asses the public health impact of the roll-out of ART Commercial genotyping methods Commercial genotyping methods are very expensive validated for HIV subtype B viruses sub-optimal amplification rates of subtype C - Engelbrecht et al., SA AIDS 2007, Eshleman et al., J Clin Microbiol 2004 Back ground

To evaluate a less-expensive in-house HIV-1 drug resistance testing assay for use to monitor HIV-1 drug resistance in Ethiopia Objective

Methods Specimens 51 samples were used for evaluation of the in-house assay 41 blood samples (plasma and serum) collected from treatment naïve patients for the base line HIV drug resistance and threshold survey in Addis Ababa in 2005 Median log 10 HIV-1 RNA copies/ml was 5.18 [IQR: 4.73 – 5.65] Ranges (4.26 to 6.4) log 10

Methods Quality assurance: 10 VQA samples obtained from Rush University, Virology Quality Assurance Laboratory, USA – Viral loads ranging from 3,557 to 57,005 copies/ml – Each sample was re-run 3 times Lower limit of detection was assessed by using 7 plasma samples collected from patients on ART – Viral loads ranging from 500 to 2,000 copies/ml

ViroSeq TM HIV-1 genotyping system VirSeq TM version 2.0 HIV genotyping system was considered as reference method for the evaluation of this in- house assay Extraction, amplification and sequencing: each sample test was performed following the manufacturers instruction Sequences obtained were assembled and analyzed on the VirSeq TM HIV genotyping system software version 2.6 Methods

HIVDR report Sequence data editing Automated Sequencing Purification Cycle sequencing Purification Confirmation of AMP. Amplification RNA extraction QIAamp Viral RNA mini kit Six primer (3 forward, 3, reverse ) QIAquick purification kit 1% agarose gel electrophorosis RT-PCR & Nested PCR Stanford HIVDR database ChromasPro ABI 3100 genetic analyzer Isopropanol

13-99 PR RT RT-PCR Pro Out 3Fv (F1) RTgeg4R (R1) Nested PCR PAFIV (F2) 215/219/3R (R2) 3 reverse primers AV44,HIV90V, 215/219 3R 3 forward primers A35V, AV36V,PAFIV Forwarded primer Position HXB2 1. Pro Out 3Fv PAFIV A35V AV36V Reverse primer Position HXB2 1. RT-geg 4R /219 3R AV HIV90V CYCLE SEQUENCING RT-PCR & NESTED PCR PR1-254 RT

HIVDR mutation and subtype HIVDR mutation and subtype determination were done using Stanford Genotypic Resistance Interpretation Algorithm The in-house primer sets specificity to the HIV pol gene was assessed by using blast algorithm of NCBI Phylogenetic analysis Phylogenetic analysis was done using MEGA 4 & BioEdit (Version ) Nucleotide sequence similarity Nucleotide sequence similarity was analyzed using Vector NTI Data analysis

Sequences obtained by both assays were compared based on the analysis of amino acid positions, of PR and of RT Concordant :- Concordant :- if both assays gave the same result Partially concordant:- Partially concordant:- if mixture of amino acid detected by one assay but not by the other Discordant Discordant:- if the two assays detected different amino acids Data analysis

Sequencing performance of the in-house assay All the samples processed by the in-house assay were successfully amplified and sequenced, including Samples with low plasma viral load > 500 copies/ml Subtype and CRFs (B, C, F, G, CRF01_AE & CRF02_AG) The in-house primer sets were 100% specific to the HIV pol gene among the sequence submitted to the NCBI blast algorithm. Result

Sequence similarity Mean nucleotide similarity of paired pol gene sequences covered by both assays was 99.35±0.5 % (mean, SD) and ranged between 98.0 % and 100% Phylogenetic analysis also confirmed that sequences generated by both assays clustered together monophyletically Result

pol geneCodon analyzed ConcordantPartial concordant Discordant PR RT Total Comparison of HIVDR mutation report by the two assays Total amino acid position 13,981= (87 PR+ 254 RT)x41 pair of samples Percent of discordance between the two assay, 0.19% (26/13,981) PR region, 0.17% (6/3,567) RT region, 0.25% (26/10,414) Result

Reproducibility of the assay Using VQA samples Using VQA samples Identical (100%) major HIVDR mutation profile in the PR and RT region were detected in the VQA samples when repeated High concordance (sequence homology) to the reference data provided by participating laboratories that uses different genotyping systems, including ViroSeq TM, Trugene® and in- house systems Result

This in-house assay has comparable sensitivity, specificity and efficiency with commercial HIV-1 drug resistance genotyping assay (ViroSeq TM ) in detecting drug resistance mutations Our results also demonstrate an excellent performance of this in- house assay for genotyping major HIV-1 subtypes,CRFs Suitable for areas with high genetic diversity. Conclusion

Compared to FDA approved commercially available ViroSeq TM,this in-house assay reduce the cost by 33 to 50 % per each test making it suitable for HIVDR monitoring in resource limited countries It can be used to monitor the emergence and transmission of HIV-1 drug resistance isolates within the population where different subtypes circulate at a less expensive cost Conclusion

Ethiopian Health and Nutrition Research Institute, (EHNRI) School of Pharmacy, Addis Ababa University, Ethiopia Center for Disease Control and Prevention, (CDC) Acknowledgements

Dr. Almaz Abebe Dr. Teferi Gedif Dr. Belete Tegbaru Mesfin Kebede Tesfaye Tilahun Hiwot Birhanu Woldaregay E.Abegaz Collaborators