UNIVERSITY OF THE WITWATERSRAND, JOHANNESBURG

Genotyping Laboratory

Mission

The genotyping laboratory was set-up in 2004 with an aim to

1) Provide a research and training sequencing facility for both the department of Molecular Medicine and Hematology
2) Provide routine HIV drug resistance testing and
3) Provide a training centre and laboratory support for other genotyping laboratories in Africa.

Service

The genotyping unit has been accredited for the last three years by SANAS and more recently by PPD and Qualogy. The laboratory is divided up into several different dedicated areas to ensure quality sequences are obtained. The areas are: nucleic isolation, cDNA synthesis, DNA amplification and a sequencing room, where the ABI 3100 is housed. Currently, the unit?s primary focus is sequencing the pol region of HIV. This provides clinicians with valuable knowledge on HIV-1 drug resistance for both the reverse transcriptase and protease inhibitors. The unit is also the genotyping reference laboratory for the AIDS Clinical Trials Group, the central laboratory for the PASER project and provides clinicians in Namibia and South Africa with routine real-time HIV-1 drug resistance testing.


  HIV-1 Drug resistance monitoring in the public sector

Currently there is no provision for individual patient monitoring on the antiretroviral (ARV) national roll-out program. This Pepfar funded project allows for HIV-1 drug resistance monitoring of all patients, with virological failure, attending the ARV roll-out clinic at Helen Joseph Hospital in Johannesburg. Furthermore, complex cases of patients failing the ARV roll-out program at the Johannesburg Hospital are sent for resistance testing.

  Monitoring of resistance in Acute Infections

The SPARTAC project is funded by the Wellcome Trust and is evaluating the use of ARV therapy in HIV-1 recent infections. The genotyping unit is involved in this study as it is providing HIV-1 drug resistance testing for all the patients entering this study. This will provide valuable data on HIV-1 drug transmission rates in South Africa.

  Adult AIDS Clinical Trial Group

The genotyping unit is the reference laboratory for Africa for the Adult AIDS Clinical Trial Group (ACTG) and is involved in several retrospective trials looking at HIV-1 drug resistance testing on patients being treated with varying drug regimens.
Research and Development

  CIPRA 'Safeguard the Household' 

The CIPRA 'Safeguard the Household' study is funded by the National Institute of Health (NIH). The genotyping unit is involved in both the adult and pediatric arms of the study. The unit is involved in two projects on the adult arm as well as a sub-study. The one project will retrospectively monitor for HIV-1 drug resistance in patients who encountered virological failure on this study. The second project is to develop more affordable HIV-1 drug resistance testing. This mainly focuses on investigating and/or developing an in-house drug resistance assay and several point mutation assays, which monitor certain mutations linked to a particular drug. The monitoring of point mutations is well suited for South Africa as the National Roll-out has a define drug regimens.

  PASER

The PASER project is funded by the Netherlands Government (AIDS FUND) and is focused on ?bringing resistance testing to Africa?.The project will continue for at least five years.The first step of the project is research and development and this unit has been instrumental in setting-up a more affordable population based sequencing assay from dried plasma spots.This in-house assay has been set-up and is currently being validated against the FDA approved ViroSeq Assay.Once the assay has been validated, it will be used to monitor for HIV-1 drug resistance in patients either failing ARV therapy or having been recent diagnosed as HIV positive.This project will be multi-centered and serve 15 countries in Africa.The project is made up of two arms: PASER-M for monitoring resistance profiles of patients accessing therapy and PASER-S for surveillance of transmitted resistance in recently infected individuals.Initially our laboratory will service all of these countries running 1500 tests per year increasing to a total of 4000 test in year 3, onwards.In approximately two years two additional laboratories will be set up in East and West Africa with the technology transfer and personnel training occurring at the genotyping unit at the University of the Witwatersrand.

  NACCAP II

The NACCAP II project is a European Funded grant and focuses on research in HIV-1 drug resistance. This project will occur over three years and focuses on the following: training and set-up of new HIV-1 drug resistance laboratories; looking at novel transport devices for plasma to save on expensive shipping costs of plasma; developing point mutation assays and establishing a HIV subtype C phenotyping facility.

  Host and Viral Factors which influence Treatment Outcome

This project will investigate both Host and Viral factors which influence treatment outcome in HIV-1 infected patients. Initially enzymes which metabolize and absorb the ARV?s will be investigated, namely cytochromes 450 and P-glycoprotein.The genes which code for these enzymes contain genetic polymorphisms, which result in varying expression levels of these enzymes and varying absorption and metabolism of ARV. This study will determine frequency of these polymorphisms in the South African population, as they have previously been linked to race and gender.The HIV-1 drug resistance mutations will also be further investigated in HIV-1 subtype C samples.

  SATuRN project

This network has been set-up in South Africa to perform surveillance testing on HIV-1 drug resistance in the public sector. Our laboratory will be performing resistance testing for this network as well as be involved in data analysis of the resistance data. Furthermore, this network will attempt to establish resistance surveillance guidelines for South Africa.

Publications

1. Wallis C, Mahomed I, Chidarikire T, Stevens G, Rekhviashvili N and Stevens W . A Pilot Evaluation of an Oligonucleotide Ligation Assay (OLA) for detection of NNRTI mutations in HIV-1 subtype C. Presented at the 2nd EU HIV Drug Resistance Workshop (Rome, March 2004)and University of the Witwatersrand, Faculty of health Science Research day, 4th August, Johannesburg.
2. Mohomed I, Wallis C, Rekhviashvilli, Stevens G, Stevens W,. Evaluation of the Oligonucleotide Ligation Assay (OLA) in HIV-1 subtype C. University of the Witwatersrand, Faculty of health Science Research day, 4th August, Johannesburg
3. Wallis CL , I Mahomed, L. Morris, T. Chidarikire, G. Stevens, N. Rekhviashvili and W Stevens . Evaluation of an Oligonucleotide Ligation Assay (OLA) for detection of mutations in HIV-1 subtype C individuals who have high level resistance to nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors. J of Virological Methods, 2005;125(2):99-109.
4. Wallis CL , Rekhviashvili N., Stevens G., Stevens W. An assay for monitoring HIV-1 drug resistance to all known non-nucleoside reverse transcriptase inhibitors in resource poor settings. 2nd SA-AIDS, Durban 2005
5. Fiscus S., Glencross D., Rehkviashvili N., Sherman G,. Wallis C., Marshall T., Stevens W. Symposium on Affrodable HIV Diagnosis and Monitoring for Scaling up ARV Treatment Programmes. 2nd SA-AIDS, Durban 2005.

6. Stevens W., Fiscus S., Glencross D., Rehkviashvili N., Sherman G., Wallis C., Marshall T. 2005. Affordable HIV Diagnosis and monitoring for scaling up ARV treatment programs. South African Journal of HIV Medicine 2005; 20:38-41.

7. Wallis CL, Rekhviashvili N, Stevens G, Papathanasopoulos MA, Stevens W. A Novel Point Mutation Assay for Monitoring HIV-1 Drug Resistance in South Africa. 4th European HIV-1 Drug Resistance Workshop, Monaco 2006.

Awards

Carole Wallis received an award at the Faculty of Health Sciences research day for ?the most promising young researcher in the field of HIV and infectious diseases?. Cindy Gonsalves was promoted to a medical scientist in the genotyping laboratory.