HIV Genotyping: Sharing Experiences and

Protocols

Justen Manasa PhD CandidateAfrica Centre for Health and Population Studies

Virology laboratory based at the

Nelson R. Mandela School of Medicine

University of KwaZulu Natal

Introduction

• Drug resistance genotyping labs to increase

– Surveillance

– Clinical management

Introduction

• Lack of common standards

• Limitations in lab infrastructure

• Cost and complexity of genotypic

resistance testing

AIM

Ensure access to validated, quality assured

genotyping services in SATuRN member

countries and collaborating institutions

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

WHO Accredited Labs

Laboratory Protocols @ Bioafrica.net

Laboratory Protocols

Capillary Sequencing

Next Generation Sequencing

Allele Specific PCR

POL ENV

nefGAG

vpr

vifLTR

LTR

vpu

Complete Genome (9719 bases)

RTPR IN

tat

rev

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 •EpidemiologyThe 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.

Page last updated by Justen Manasa, 16 November 2013

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.

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.

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.

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).

Chen: Citations () CommentsIn-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 () CommentsAntiretroviral 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.

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

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 Clin Microbiol (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)

Protocols, Primary Publication (orded by publication date) Ranked by number of citations

Discussion

• Registration and comments• Blog• Moderation of user comments• General Trouble shooting

guidelines• Structure of protocol• Videos• Any other request?

Thank You