Perinatal Acquisition of Drug-resistant HIV-1 Infection: Mechanisms and Long-term Outcome

Constance Delaugerre; Marie-Laure Chaix; Stephane Blanche; Josiane Warszawski; Dorine Cornet; Catherine Dollfus; Veronique Schneider; Marianne Burgard; Albert Faye; Laurent Mandelbrot; Roland Tubiana; Christine Rouzioux


Retrovirology. 2009;6(2):85 

In This Article

Patients and Methods

Study Population

Since 1985, the ANRS French Perinatal Cohort (CO 01-ANRS-EPF, Agence Nationale de Recherche sur le SIDA-Enquête Périnatale Française) has prospectively collected data on HIV-infected pregnant women and their children in 90 centers throughout France. Informed consent is obtained from the mothers during pregnancy or at the time of delivery. The children receive standard care, including clinical and biological examinations at birth and 1, 3, 6, 12 and 18–24 months, as previously reported.[15] The cohort study was approved by the Cochin Hospital Institutional Review Board and by the French computer database watchdog commission (CNIL). Mother and infant plasma and cells were collected between 1990 and 2005 and stored in Necker hospital virology laboratory.

HIV-1 infection was diagnosed in the newborn when at least two separate samples were positive by HIV-1 RNA/DNA detection or by a viral culture. A positive test at birth or before 7 days of age indicates intrauterine transmission, while a negative test at birth and a positive test more than 7 days later indicate intrapartum transmission. An infant is considered uninfected when two tests performed one month after discontinuation of antiretroviral prophylaxis are negative.

Newborns were included in this analysis if: (1) they were born and enrolled in metropolitan France in centers participating in the EPF cohort between 1997 and 2004; (2) they were HIV-1-infected; and (3) if frozen samples were available for resistance testing.

For each mother-child pair, we analyzed the first available HIV-1-positive sample(s) from the infant's delivery sample and the mother's. If drug resistance was detected in the newborn diagnostic sample, available follow-up samples from the infant were tested for genotypic resistance.

Other data, including the mothers' viral load values and the mothers' and infants' antiretroviral treatment histories, were obtained from the ANRS-EPF database.

HIV-1 RNA Quantification

Plasma HIV-1 RNA was quantified with the Cobas Amplicor HIV-1 Monitor 1.5 assay kit (Roche Diagnostics, Meylan, France; detection limit 400 or 40 copies/mL).

Resistance Genotyping

The ANRS consensus method was used for population-based nucleotide sequence analysis of the whole protease gene (codons 1 to 99) and codons 1 to 305 of the reverse transcriptase gene on HIV-1 RNA in plasma and HIV-1 DNA in PBMC.[16] Drug resistance mutations were identified by following the International AIDS Society-USA 2007 Drug Resistance Group guidelines[17] Specifically, we considered the following mutations (relative to the reference wild-type (WT) strain HXB2): protease inhibitors (PI): D30N, L33F/I, M46I/L, G48V, I50L/V, V82A/F/L/S/T, I84A/C/V, and L90M; nucleoside reverse transcriptase inhibitors (NRTI): M41L, A62V, K65R, D67N, K70R, L74V, V75I, F77L, Y115F, F116Y, Q151M, M184V, L210W, T215Y/F/C/D/E/S/I/V/A/G/H/L/N and K219E/Q/R; and non nucleoside reverse transcriptase inhibitors (NNRTI): L100I, K103N, V106A/M, V108I, Y181C/I, Y188C/H/L, G190A/S, P225H, M230L, and P236L. Mixtures of WT and mutant sequences were considered drug-resistant. Interpretation of genotypic drug susceptibility was done according to the 2007 French ANRS algorithm

Clonal Analysis of Resistance in Three Mother-child Pairs

In order to characterize the plasma and cellular viral quasispecies, clonal analyses were performed on samples from three mother-child pairs. The maternal samples were obtained at delivery and the children's samples were obtained both at birth and subsequently. These three pairs were chosen as being representative of three different situations, and because suitable plasma/cell samples for them were available. The RT or protease gene was amplified. Purified PCR products were cloned into the pCR Topo 2–1 plasmid (TOPO TA Cloning kits, Invitrogen BV, the Netherlands) as recommended by the manufacturer. DNA was purified with the Mini-Prep kit (Qiagen) and clones were analyzed by dye terminator sequencing on an ABI Prism 3100 genetic analyzer.

Phylogenetic Analysis

Mother-child clustering of pol sequences was confirmed by phylogenetic analysis. All sequences of HIV-1 RNA and DNA clones from each mother-child pair were aligned with Clustal W 1.7 software. Pairwise evolutionary distances were estimated with DNADist using Kimura's two-parameter method. The phylogenetic trees were then constructed with a neighbor joining method (Neighbor program implemented in the Phylip package).[18] The reliability of each tree topology was estimated from 100 bootstrap replicates.[18]


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