A sensitive in-house RT-PCR genotyping system for combined detection of plasma HIV-1 and assessment of drug resistance | ICRH

Authors and affiliation: 

Kim Steegen (ICRH-Ghent) and Marleen Temmerman (ICRH-Ghent) Els Demecheleer (Aids Reference Laboratory, Ghent University), Nancy De Cabooter (Aids Reference Laboratory, Ghent University) and Jean Plumb, Chris Verhofstede (Aids Reference Laboratory, Ghent University)Dieudonn´e Nges (Faculty of Medicine, University of Yaounde) and Peter Ndumbe (Faculty of Medicine, University of Yaounde)Kishor Mandaliya (CPGH),

Ranking: 

A1

Team members: 

Dr Kim Steegen, Dr Stanley Luchters, Dr Kishor Mandaliya, Mrs. Mercy Mutie, Mr. Bhavin Morjaria, Mr. Henry Jimbi, Mrs. Margaret Magina, Ms. Mary Mwambaru, Mrs. Hannah Mburu, Mrs. Anne Mwangemi, Mr. Lawrence Lewa, Ms. Charity Mwabili, Dr Anisa Baghazar, Ms. Violet Wafula, Ms. Mary Namunyu;

PubMedID: 

PMID: 16375980

Published: 

Journal of Virological Methods 133 (2006) 137–145

Publication Date: 

20/12/2005

Background: Quantification of the viral burden and identification of drug resistant mutations are important laboratory tools in the management of HIV-1 infected patients. However, widespread use of assays for viral load determination and genotyping is still hampered by the high cost. Here, an in-house RT PCR-sequencing assay for HIV-1 drug resistance monitoring with the potential to be used both as a qualitative assay to detect the virus in plasma and as a genotyping system is described. Methods: A total of 377 clinical samples, collected from 374 HIV-infected patients of diverse geographic origin, were tested. Results: The nested RT-PCR for amplification of the protease reverse transcriptase gene was found positive for 350 (92.8%) and 346 (91.8%) of 377 samples, respectively. All amplification-failures were due to viral loads of below 500 copies/ml. However, low viral load does not exclude amplification since 80.2 and 76% of 121 samples with viral loads of less than 500 copies/ml were amplified successfully for protease and reverse transcriptase, respectively. The high sensitivity of the assay was independent of the HIV-subtype, with a broad range of different HIV-1 subtypes tested.  Conclusions: In conclusion the RT-PCR-direct sequencing method is convenient for the sensitive detection and subsequent genotyping of plasma RNA from a broad range of different HIV-1 subtypes. The assay enables the accurate follow-up of patients under treatment at a significantly reduced cost compared to the currently available commercial assays for viral load assessment and genotyping.