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

Disclosures

Retrovirology. 2009;6(2):85 

In This Article

Results

Study Population

From January 1997 to December 2004, 6170 mother-child pairs were enrolled in the ANRS-EPF cohort, representing approximately 70% of births to HIV-1-infected mothers in France. 92 newborns were infected during this period despite prophylaxis. It is important to note that the newborn samples were used to diagnose HIV infection and that the remaining stored samples were usually very limited.

HIV-1-positive plasma and/or PBMC samples from 60 children (33 boys and 27 girls) were available for drug resistance studies. Samples were also available from 32 of these children's mothers. The children's samples were obtained at a median age of 29 days (1 to 313 days), and 72% of plasma samples were collected less than 60 days after birth. The children's median HIV-1 RNA viral load at diagnosis was 4.5 log10 copies/ml (2.1 to 7.3 log10).

Drug Resistance at HIV-1 Diagnosis in the Infant

Twelve (20%) of the 60 newborns had resistant variants at diagnosis of HIV-1 infection, according to the 2007 IAS (International AIDS Society) list (Table 1). Six of these children were infected in utero and four intrapartum; the timing of infection could not be determined in the remaining two children as no birth sample was available. The mutations were associated with resistance to NRTI in 10 cases [thymidine analog mutations (TAMs) in six cases, T69N in one case, M184V in one case, and both mutations in two cases], NNRTI in two cases, and PI in one case.

According to the 2007 ANRS algorithm, 6 of the 12 children had variants with resistance to at least one antiretroviral drug [overall frequency 10% (6/60)]. Resistance to NRTI, NNRTI and PI was observed in four children, two children and one child, respectively. One child had variants resistant to both NRTI and NNRTI (child #10, Table 1).

In all but one case, the neonates' drug resistance profiles were related to the antiretroviral drugs received by the mother and/or by the child (Table 1). Infant #10 harbored viruses with mutations associated with NNRTI resistance, without being exposed perinatally to this drug class. His mother had never received NNRTI, but she had probably been infected with NNRTI-resistant virus transmitted by her husband, who was treated with a regimen containing nevirapine, stavudine and lamivudine.

The viral subtypes were determined in 53 children, and were subtype CRF02_AG in 23 cases (43%), B in 19 cases (36%), A in 5 cases (9%) and another subtype in 6 cases (11%). Among the 10 subtyped resistant viruses, 5 (50%) belonged to subtype B, three (30%) to CRF02_AG, one to A and one to F.

DNA-based resistance results were available for 8 of the 12 children with resistant viruses in plasma. In 6 cases HIV-1 RNA and DNA harbored the same resistance mutations (Table 1), while no mutation was detected in HIV-1 DNA in the other two cases.

Comparison of Resistance Mutations in the Children and Their Mothers

Samples from 32 mother-child pairs were available, including 10 of the 12 children with resistant virus in the plasma (Table 1). The resistance pattern was the same in six mother-child pairs. In the remaining four cases the mothers harbored different mutations or no mutation. Interestingly, child #9, whose mother harbored PI resistance mutations L10I, L63P and L90M and RT resistance mutations Y181C, L210W and T215D, only harbored the PI resistance mutations. The mother was receiving didanosine, saquinavir and lopinavir/ritonavir, probably leading to the selection of a dominant PI-resistant quasispecies. Among the 22 remaining mother-child pairs, 20 mothers had wild-type viruses (in plasma), while the other two mothers harbored resistant viruses that were not transmitted to the child.

Longitudinal Resistance Analysis in Infected Children

Longitudinal resistance studies were performed in 8 of the 12 cases in which serial samples were available (median 4 samples per child), over a median period of 52 months (Table 2). The same resistance mutations persisted in the plasma and PBMC for 6 months to 5 years, regardless of the antiretrovirals used in six children. Additional mutations had accumulated in the RNA and the DNA during failing regimens. In two children (#6, #12), no zidovudine resistance mutations were detected when zidovudine prophylaxis was discontinued. Interestingly, no resistance mutations were detected in mother samples and in birth children cells (Table 1 and 2).

Clonal and Phylogenetic Analysis of HIV-1 in Three Mother-newborn Pairs

To better understand how drug-resistant HIV-1 strains detected in newborns are acquired, we conducted clonal analyses of plasma and PBMC viral populations in three mother-child pairs. The maternal samples were taken at delivery, and the children's samples were taken both at birth and at a later time.

In mother-child pair #9, 110 protease gene clones were sequenced (Figure 1). In the mother, all 21 plasma clones harbored the L90M major mutation and other minor mutations. Her PBMC harbored heterogeneous variants (12/21 wild-type, 8/21 L90M and 1/21 I84V), according to the temporal archiving of resistant variants in lymphocytes during therapeutic regimens that contrasted with the homogeneity reported in the plasma under selective therapeutic pressure. In her child, who was infected in utero, all plasma and cellular variants harbored the L90M mutation (40/40 at birth and 28/28 at month 30), even during the period without PI selective pressure. Phylogenetic analysis confirmed the homogeneity of the child's specimens at birth with a genetic intravariability of protease gene that increased over time (from 0.003% to 0.01%). This case suggests the perinatal transmission of L90M variants with early archiving in the child's lymphocytes and persistence over time.

Figure 1.

Resistance analysis of HIV-1 RNA and DNA from the mother-child pair #9. Time course of HIV-1 RNA and DNA levels in children with resistance mutations as detected by population-based sequencing and clonal analysis (box). Antiretroviral treatment is indicated above. Maternal antiretroviral treatment at delivery, viral RNA load, and the number of wild-type (WT) or resistant clones are indicated. In the phylogenetic tree, maternal viral clones are represented by circles and newborn viral clones by squares. M indicates the time to genotype testing in month. Wild-type quasispecies are represented by open circles and squares, and resistant quasispecies by full circles and squares. HIV-1 RNA results are in blue, and HIV-1 DNA results are in pink. The arrow indicates the maternal viral clone closest to the infant's quasispecies.

In mother-child pair #11, 70 RT gene clones were sequenced (Figure 2). The mother acquired HIV-1 infection during pregnancy and was rapidly treated with zidovudine, lamivudine and indinavir/ritonavir. The child was infected in utero, despite elective Cesarean section and the intensification of postnatal zidovudine prophylaxis by the addition of lamivudine. All plasma and cellular quasispecies detected in the newborn (35/35 at month 3 and 26/26 at month 7) harbored the M184V lamivudine resistance mutation. However, this mutation was not detected in the mother's delivery plasma sample (9/9 wild-type). Phylogenetic analysis confirmed low genetic intravariability (mean 0.006%) of the RT gene in the mother and her child, in keeping with the high homogeneity due to the primary infection in the child and his mother. M184V variants may have arisen during lamivudine treatment of the mother and prophylaxis of the infant, leading to the massive early lymphocyte infection and persistence of lamivudine resistance. However, we cannot exclude an abacavir-selective pressure on the M184V resistance-associated mutation or a minor maternal M184V variant transmission.

Figure 2.

Resistance analysis of HIV-1 RNA and DNA from the mother-child pair #11. Time course of HIV-1 RNA and DNA levels in children with resistance mutations as detected by population-based sequencing and clonal analysis (box). Antiretroviral treatment is indicated above. Maternal antiretroviral treatment at delivery, viral RNA load, and the number of wild-type (WT) or resistant clones are indicated. In the phylogenetic tree, maternal viral clones are represented by circles and newborn viral clones by squares. M indicates the time to genotype testing in month. Wild-type quasispecies are represented by open circles and squares, and resistant quasispecies by full circles and squares. HIV-1 RNA results are in blue, and HIV-1 DNA results are in pink. The arrow indicates the maternal viral clone closest to the infant's quasispecies.

In mother-child pair #12, 61 RT gene clones were sequenced (Figure 3). The mother had advanced HIV-1 disease and poor adherence to treatment as reflected by high viral load (4.4 log10 copies/mL). Resistance was undetectable even by clonal analysis (28/28 wild-type). Zidovudine prophylaxis was initiated at birth and continued for 6 weeks despite the diagnosis of HIV-1 in utero infection in the newborn. In the child, the K70R mutation was detected in 42% of clones (10/24) at month 1 and in 0% at month 12. Genetic intravariability was low (0.005%) in the child, as expected, during primary infection. In this case, wild-type viruses were detected concomitantly in the RNA from the mother and in the DNA from the child (only 1/10 resistant clones), suggesting that most archived viruses in the child were WT viruses transmitted by the mother. Zidovudine resistance, present at the time of diagnosis, occurred during suboptimal zidovudine pressure. Zidovudine discontinuation led to the re-emergence of wild-type variants in the plasma at month 12, confirming that the reservoir consisted mainly of wild-type viruses.

Figure 3.

Resistance analysis of HIV-1 RNA and DNA from the mother-child pair #12. Time course of HIV-1 RNA and DNA levels in children with resistance mutations as detected by population-based sequencing and clonal analysis (box). Antiretroviral treatment is indicated above. Maternal antiretroviral treatment at delivery, viral RNA load, and the number of wild-type (WT) or resistant clones are indicated. In the phylogenetic tree, maternal viral clones are represented by circles and newborn viral clones by squares. M indicates the time to genotype testing in month. Wild-type quasispecies are represented by open circles and squares, and resistant quasispecies by full circles and squares. HIV-1 RNA results are in blue, and HIV-1 DNA results are in pink. The arrow indicates the maternal viral clone closest to the infant's quasispecies.

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