title: A sensitive in-house RT-PCR genotyping system for combined detection of plasma HIV-1 and assessment of drug resistance.
abstract: 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.
authors.affiliation: International Centre for Reproductive Health, University Hospital, Ghent, Belgium.
authors.forename: Kim
authors.initials: K
authors.lastname: Steegen
authors.forename: Els
authors.initials: E
authors.lastname: Demecheleer
authors.forename: Nancy
authors.initials: N
authors.lastname: De Cabooter
authors.forename: Dieudonné
authors.initials: D
authors.lastname: Nges
authors.forename: Marleen
authors.initials: M
authors.lastname: Temmerman
authors.forename: Peter
authors.initials: P
authors.lastname: Ndumbe
authors.forename: Kishor
authors.initials: K
authors.lastname: Mandaliya
authors.forename: Jean
authors.initials: J
authors.lastname: Plum
authors.forename: Chris
authors.initials: C
authors.lastname: Verhofstede
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology; D001483:Base Sequence; D015331:Cohort Studies; D003365:Costs and Cost Analysis; D024882:Drug Resistance, Viral / Q000235:genetics*; D005260:Female; D005814:Genes, Viral; D005838:Genotype; D015658:HIV Infections / Q000097:blood; D016333:HIV Protease / Q000235:genetics*; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000187:drug effects / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D010802:Phylogeny; D012367:RNA, Viral / Q000097:blood; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load
chemical_list: D019380:Anti-HIV Agents; D012367:RNA, Viral; D054303:HIV Reverse Transcriptase; D016333:HIV Protease