Poster (Scientific congresses and symposiums)
Performance of three HIV-1 DNA real-time PCRs for early diagnosis in infants and adults
Iserentant, G.; Masquelier, C.; Lambert, C. et al.
20119th European Workshop on HIV & Hepatitis
 

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Abstract :
[en] Background Early pediatric HIV-1 diagnosis is a challenge in resource limited countries to identify HIV-infected newborns to provide early ART treatment. HIV DNA PCR testing is an accurate method for diagnosis in newborns and could be valuable for monitoring HIV infection in adults. We have evaluated the analytical, clinical performances and genotype inclusivity of 3 real-time Taqman PCRs targeting the ltr, int and env genes. Material and methods Total DNA was extracted from whole blood and Dried Blood Spots (DBS) using the NucleoSpin Blood kit (Macherey Nagel) and the Chelex resin. DNA amplification was performed using the Qiagen Multiplex PCR kit and detected on a 7300 Real time PCR system (Applied Biosystems). Method validation was realized as required by ISO 15189 standard. The accuracy profile methodology was applied. HIV-1 DNA of ACH2 cells (0, 25, 50, 100, 1000 and 5000 copies/PCR) mixed in whole blood were measured in 4 different series (2 PCR kits and 2 operators) of 5 independent DNA extraction of each standard. Linear and quadratic weighted and non weighted regression models were tested. Trueness, precision, limit of quantifications of the method and accuracy of the results were obtained for each model and compared. 93 whole blood samples of ART treated-patients with undetectable or low viraemia of 16 B and 77 non-B subtypes were assessed. Clinical evaluation of the assay was performed on 26 whole blood samples and 238 DBS from infants of Guinea Conakry determined HIV+ using the rapid Bioline HIV test Results The calibration model providing the most accurate results was the linear regression with an r² equal to 0.92 for ltr, 0.88 for int and 0.95 for env. Trueness, precision and accuracy were highly acceptable for the 3 sets of primers/probe not exceeding -0.23 log(copies/PCR) for bias and 0.5 log(copies) for intermediate precision standard deviation, respectively. The estimated lower limit of quantification was 41, 475 and 25 copies/PCR for ltr, int and env respectively. The accuracy profile of each gene showed that each future measurement using this assay has 80% probability to be within a limit of 0.5 log (copies/PCR) around the reference or true value of copies of each gene. 52/93 whole blood samples of ART-treated patients had 3 positive genes (Ct<40 cycles), 38/93 had two positive genes and 4/93 had only one positive gene (ltr). Among these patients, 3 were assigned as long term progressors and one was a subtype C strain. Clinical evaluation of the assay identified 1 HIV-1 infected newborn and 3 adults (at least 2 positive genes) for which HIV-1 infection was confirmed either by serology or viral load techniques as well as 96 HIV-1 infected infants using DBS. Conclusions This assay is suitable for detection of proviral HIV-1 DNA in infants and adults on whole blood and DBS samples. Despite an estimated lower limit of quantification higher than ltr and env, the int PCR was required to achieve a good genotype inclusivity. The method was fully validated as required by ISO 15189 and showed to provide sufficiently reliable results.
Disciplines :
Pharmacy, pharmacology & toxicology
Author, co-author :
Iserentant, G.
Masquelier, C.
Lambert, C.
Baurith, T.
Arendt, V.
Zachariah, R.
Chaillet, P.
Schmit, J. C.
Rozet, Eric ;  Université de Liège - ULiège > Département de pharmacie > Chimie analytique
Devaux, C.
Language :
English
Title :
Performance of three HIV-1 DNA real-time PCRs for early diagnosis in infants and adults
Publication date :
March 2011
Event name :
9th European Workshop on HIV & Hepatitis
Event place :
Paphos, Cyprus
Event date :
23-25 march 2011
Audience :
International
Available on ORBi :
since 25 March 2011

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