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

Kim Steegen, Els Demecheleer, Nancy De Cabooter, Dieudonné Nges, Marleen Temmerman, Peter Ndumbe, Kishor Mandaliya, Jean Plum, Chris Verhofstede

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44 Citations (Scopus)


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. A total of 377 clinical samples, collected from 374 HIV-infected patients of diverse geographic origin, were tested. 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. 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.

Original languageEnglish
Pages (from-to)137-145
Number of pages9
JournalJournal of Virological Methods
Issue number2
Publication statusPublished - May 2006
Externally publishedYes


  • Genotyping
  • HIV-1
  • Qualitative detection
  • RT-PCR


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