HIV Genotyping: Sharing Experiences and Protocols

1. HIV Genotyping: Sharing Experiences and Protocols Justen Manasa PhD Candidate Africa Centre for Health and Population Studies Virology laboratory based at the Nelson R. Mandela School of Medicine University of KwaZulu Natal

2. Introduction • Drug resistance genotyping labs to increase • Surveillance • Clinical management

3. Introduction • Lack of common standards • Limitations in lab infrastructure • Cost and complexity of genotypic resistance testing

4. AIM Ensure access to validated, quality assured genotyping services in SATuRN member countries and collaborating institutions

5. Objectives • Create laboratory mentorship programmes for Labs setting up genotyping services • Create a low cost EQA programme for resource limited settings • Create an open access online resource for sharing genotyping protocols for HIV and TB genotyping

6. WHO Accredited Labs

7. Laboratory Protocols @ Bioafrica.net

8. Laboratory Protocols Capillary Sequencing Next Generation Sequencing Allele Specific PCR

9. Protocols for sequencing the HIV whole genome or specific gene fragment using capillary electrophoresis • The HIV genome or its segments have been and are currently being sequencing for many reasons including; • Viral evolution • Phylogeography • Drug resistance • Epidemiology • The links below provides some of the protocols that have been validated and used for HIV sequencing using capillary electrophoresis. Please click on the segment of your interest to go to the protocol. Complete Genome (9719 bases) tat rev LTR GAG vif vpu nef POL vpr ENV LTR PR RT IN Page last updated by Justen Manasa, 16 November 2013

10. Protocols for sequencing the HIV whole genome or specific gene fragment using capillary electrophoresis Here we provide detailed full protocols. The protocols are linked to the primary papers in which they were 1st published. You can also access a summary of publications that have used the same protocol and referenced the primary paper. For each protocol there is also a comment section which should be used by users to discuss issues related to implementation of the protocol Protocols, Primary Publication (orded by publication date) Ranked by number of citations Antiretroviral drug resistance surveillance among drug-naive HIV-1-infected individuals in Gauteng Province, South Africa in 2002 and 2004. Antiviral Therapy (2008). Citations (9) Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings. Plos One (2010). Citations (6) Affordable in-house antiretroviral drug resistance assay with good performance in non-subtype B HIV-1. J. Virol Methods (2010). Citations (5) A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only. J ClinMicrobiol (2013). Citations (4) In-house human immunodeficiency virus-1 genotype resistance testing to determine highly active antiretroviral therapy resistance mutations in Hong Kong. Hong Kong Med J (2010). Citations (3) An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings. Jove (In Press) Citations (2) SATuRN/Life Technologies: Citations ()Comments An Affordable HIV-1 Drug Resistance Monitoring Method for Resource Limited Settings. MANASA, J., DANAVIAH, S., PILLAY, S., PADAYACHEE, P., MTHIYANE, H., SEEBREGTS, C., RINKE DE WIT, T. F., VILJOEN, J., KATZENSTEIN, D. & DE OLIVEIRA, T. 2013. Jove (in press). Paser Ultra-light: Citations ()Comments A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only. AITKEN, S. C., BRONZE, M., WALLIS, C. L., STUYVER, L., STEEGEN, K., BALINDA, S., KITYO, C., STEVENS, W., RINKE DE WIT, T. F. & SCHUURMAN, R. 2013. J Clin Microbiol, 51, 1757-61. CDC: Citations ()Comments Optimization of a low cost and broadly sensitive genotyping assay for HIV-1 drug resistance surveillance and monitoring in resource-limited settings. ZHOU, Z., WAGAR, N., DEVOS, J. R., ROTTINGHAUS, E., DIALLO, K., NGUYEN, D. B., BASSEY, O., UGBENA, R., WADONDA-KABONDO, N., MCCONNELL, M. S., ZULU, I., CHILIMA, B., NKENGASONG, J. & YANG, C. 2011. PLoS One, 6, e28184. Wallis: Citations ()Comments Affordable in-house antiretroviral drug resistance assay with good performance in non-subtype B HIV-1. WALLIS, C. L., PAPATHANASOPOULOS, M. A., LAKHI, S., KARITA, E., KAMALI, A., KALEEBU, P., SANDERS, E., ANZALA, O., BEKKER, L. G., STEVENS, G., DE WIT, T. F. & STEVENS, W. 2010. J Virol Methods, 163, 505-8. Chen: Citations ()Comments In-house human immunodeficiency virus-1 genotype resistance testing to determine highly active antiretroviral therapy resistance mutations in Hong Kong. CHEN, J. H., WONG, K. H., LI, P. C., CHAN, K. K., LEE, M. P., TO, S. W. & YAM, W. C. 2012. Hong Kong Med J, 18, 20-4. NICD: Citations ()Comments Antiretroviral drug resistance surveillance among drug-naive HIV-1-infected individuals in Gauteng Province, South Africa in 2002 and 2004. PILLAY, V., LEDWABA, J., HUNT, G., RAKGOTHO, M., SINGH, B., MAKUBALO, L., BENNETT, D. E., PUREN, A. & MORRIS, L. 2008. Antivir Ther, 13 Suppl 2, 101-7.

11. Discussion • Registration and comments • Blog • Moderation of user comments • General Trouble shooting guidelines • Structure of protocol • Videos • Any other request?

12. Thank You