title: Development and evaluation of an affordable real-time qualitative assay for determining HIV-1 virological failure in plasma and dried blood spots.
abstract: Virological failure (VF) has been identified as the earliest, most predictive determinant of HIV-1 antiretroviral treatment (ART) failure. Due to the high cost and complexity of virological monitoring, VF assays are rarely performed in resource-limited settings (RLS). Rather, ART failure is determined by clinical monitoring and to a large extent immunological monitoring. This paper describes the development and evaluation of a low-cost, dried blood spot (DBS)-compatible qualitative assay to determine VF, in accordance with current WHO guideline recommendations for therapy switching in RLS. The assay described here is an internally controlled qualitative real-time PCR targeting the conserved long terminal repeat domain of HIV-1. This assay was applied to HIV-1 subtypes A to H and further evaluated on HIV-1 clinical plasma samples from South Africa (n = 191) and Tanzania (n = 42). Field evaluation was performed in Uganda using local clinical plasma samples (n = 176). Furthermore, assay performance was evaluated for DBS. This assay is able to identify VF for all major HIV-1 group M subtypes with equal specificity and has a lower detection limit of 1.00E+03 copies/ml for plasma samples and 5.00E+03 copies/ml for DBS. Comparative testing yielded accurate VF determination for therapy switching in 89% to 96% of samples compared to gold standards. The assay is robust and flexible, allowing for "open platform" applications and producing results comparable to those of commercial assays. Assay design enables application in laboratories that can accommodate real-time PCR equipment, allowing decentralization of testing to some extent. Compatibility with DBS extends access of sampling and thus access to this test to remote settings.
authors.affiliation: University Medical Centre Utrecht, Utrecht, the Netherlands. s.c.aitken@umcutrecht.nl
authors.forename: Susan C
authors.initials: SC
authors.lastname: Aitken
authors.forename: Aletta
authors.initials: A
authors.lastname: Kliphuis
authors.forename: Michelle
authors.initials: M
authors.lastname: Bronze
authors.forename: Carole L
authors.initials: CL
authors.lastname: Wallis
authors.forename: Cissy
authors.initials: C
authors.lastname: Kityo
authors.forename: Sheila
authors.initials: S
authors.lastname: Balinda
authors.forename: Wendy
authors.initials: W
authors.lastname: Stevens
authors.forename: Nicole
authors.initials: N
authors.lastname: Spieker
authors.forename: Tulio
authors.initials: T
authors.lastname: de Oliveira
authors.forename: Tobias F
authors.initials: TF
authors.lastname: Rinke de Wit
authors.forename: Rob
authors.initials: R
authors.lastname: Schuurman
mesh_terms: D001769:Blood / Q000821:virology*; D003890:Desiccation; D016903:Drug Monitoring / Q000379:methods; D015658:HIV Infections / Q000188:drug therapy / Q000821:virology*; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013019:South Africa; D013048:Specimen Handling / Q000379:methods*; D013636:Tanzania; D014454:Uganda; D019562:Viral Load / Q000379:methods*