Protease inhibitor associated mutations compromise the efficacy of therapy in human immunodeficiency virus – 1 (HIV-1) infected pediatric patients: a cross-sectional study
© Malhotra et al; licensee BioMed Central Ltd. 2007
Received: 30 October 2006
Accepted: 09 July 2007
Published: 09 July 2007
Although the introduction of combined therapy with reverse transcriptase and protease inhibitors has resulted in considerable decrease in HIV related mortality; it has also induced the development of multiple drug-resistant HIV-1 variants.
The few studies on HIV-1 mutagenesis in HIV infected children have not evaluated the impact of HIV-1 mutations on the clinical, virological and immunological presentation of HIV disease that is fundamental to optimizing the treatment regimens for these patients.
A cross sectional study was conducted to evaluate the impact of treatment regimens and resistance mutation patterns on the clinical, virological, and immunological presentation of HIV disease in 41 children (25 male and 16 female) at the Robert Wood Johnson Pediatric AIDS Program in New Brunswick, New Jersey. The study participants were symptomatic and had preceding treatment history with combined ARV regimens including protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Fifteen (36.6%) children were treated with NRTI+NNRTI+ PI, 6 (14.6%) with NRTI+NNRTIs, 13 (31.7%) with NRTI+PIs, and the remaining 7 (17.1%) received NRTIs only.
Combined ARV regimens did not significantly influence the incidence of NRTI and NNRTI associated mutations. The duration of ARV therapy and the child's age had no significant impact on the ARV related mutations. The clinico-immunological presentation of the HIV disease was not associated with ARV treatment regimens or number of resistance mutations. However, primary mutations in the protease (PR) gene increased the likelihood of plasma viral load (PVL) ≥ 10,000 copies/mL irrespective of the child's age, duration of ARV therapy, presence of NRTI and NNRTI mutation. Viremia ≥ 10,000 copies/mL was recorded in almost all the children with primary mutations in the PR region (n = 12/13, 92.3%) as compared with only 50.0% (n = 14/28) of HIV infected children without (PR-), P < 0.008. However, CD-4 T cells were not affected by the mutations in the PR gene of the HIV-1 isolates.
Primary PR resistance mutations significantly increase the likelihood for high viral replication in pediatric patients with moderate/severe HIV-1 infection, which may affect the long-term clinical prognosis of the HIV infected children.
Antiretroviral drug resistance produced by drug associated mutations in specific regions of the HIV genome has been recognized as one major problem during the treatment of human immunodeficiency virus type 1 (HIV-1) infected patients [1, 2]. It has been shown that antiretroviral therapy used in the presence of drug-resistant viruses may increase the risk of HIV-1 mutagenesis [2, 3] and the expansion of resistant HIV mutants compromises the efficacy of the antiretroviral (ARV) therapy [4–6].
Numerous studies have described the association between multiple mutations and multidrug resistance in HIV-1 isolates in adults [3, 7–10] but knowledge regarding this issue in the population of HIV infected children is limited. We previously reported the occurrence of mutations in the protease (PR) gene in a small group of HIV infected children treated with the protease inhibitor (PI) nelfinavir . HIV-1 mutants have been isolated both from both, ARV treated and untreated HIV infected Brazilian children  Several studies demonstrated that the failure of ARV therapy  and clinical disease progression  in HIV infected children is associated with a high frequency of primary mutations in the reverse transcriptase (RT) gene.
Cognizance of the impact of resistance mutations on the subsequent response to ARV therapy is of importance in the optimization of treatment regimens, especially for HIV-1 infected pediatric patients. The few studies published on the subject of HIV mutagenesis in children do not address the prime question regarding the extent to which HIV-1 mutations impact the clinical, virological, and immunological parameters of HIV disease in the infected children.
We designed a cross-sectional study to assess the impact of the development and association of drug resistant mutations in the HIV-1 genome on the clinical, virological, and immunological presentation of HIV infection in children who were infected at birth.
An Institutional Review Board approved study was conducted at the Robert Wood Johnson Medical School's Pediatric Infectious Disease Clinic between 1999 and 2004. A total number of 42 HIV infected children were enrolled, and 41 (97.6%) of those who completed the required virological, immunological and HIV genotype testing were included in the analysis.
Demographic and treatment related information (gender, age of study entry, length and ARV treatment regimens) was recorded for each subject. In order to provide more prognostic information, plasma viral load (PVL) and CD4+ T- lymphocyte counts were tested simultaneously [15, 16] using HIV-1 RNA assay flow cytometry [17, 18]. Resistance mutations in the HIV genomes, both the RT and the PR genes were examined in the plasma viral RNA by reverse transcription and relevant genome segment amplification (HIV GENOSURE™). The presence of resistance-related mutations was classified in accordance with the recommendations of the International AIDS Society-USA  and International Expert Panel on HIV Antiretroviral Drug Resistance , and the 2005 update by Johnson et al  regarding drug resistance mutations in HIV-1 was also used.
The mutations associated with protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) as well as the individual point (single amino acid changes) mutations were analyzed. Individual drug specific resistance and cross-resistance to one or more drugs of the same class was also identified.
The collected clinical and immunologic data were categorized in accord with the classification system for HIV-infected children of less than 13 years of age  and adolescents . PVL cutoff of 10,000 copies/mL for initiation of ARV therapy in adults and adolescents  was used to evaluate viremia level <10,000 copies/mL and ≥ 10,000 copies/mL.
Clinical, immunological, and virological data was statistically analyzed (Statistica 6.0, StatSoft, Tulsa, OK) with respect to the treatment regimens and resistance mutations. The Pearson chi-square or Fisher's exact tests were performed for categorical data and Mann-Whitney test or Analysis of Variance for continuous data. Odds ratio (OR) and 95% confidence interval (95%CI) was calculated for the association using results from the multivariate regression analysis. All the P values presented are two-tailed with significance <0.05
Demographic, clinical, and immunological data in association with the ARV treatment regimens
ARV treatment regimens
(n = 15)
(n = 6)
NRTI + PI
(n = 13)
(n = 7)
Age of study entry (Mean+/-SD, yrs)
Length of treatment (Mean+/-SD, yrs)
Number of Medications
Clinical categories *
Immune categories **
1 (no suppression)
PVL (n, %)
Number of mutations (Mean+/-SD)
Demographic, clinical, and immunological data in association with the resistance mutations in the PR gene of the HIV-1 isolates
PI associated resistance mutations
N = 13
N = 28
Age of study entry (Mean+/-SD, years)
Length of treatment (Mean+/-SD, years)
9 (64.2%) *
10 (76.9%) ***
Clinical categories #
Immune categories ##
1 (no suppression)
PVL (n, %)
1 (7.7%) **
The introduction of combined therapy with reverse transcriptase and protease inhibitors has resulted in a considerable decrease in the HIV-1 related mortality . Although the therapeutic regimens with the combined medications increase the selective pressure against viruses, they also subsequently induce the development of multiple drug-resistant HIV-1 variants . The present study shows that NRTI -associated resistance mutations occur nearly always in HIV-1 infected pediatric patients receiving NRTIs, but NNRTI and PI-associated mutations are less likely to occur in children whose treatment includes NNRTIs and PIs (87.8% vs. 66.7% vs. 46.1%, respectively, P < 0.03). The rate of virus replication ≥ 10,000 copies/mL is significantly associated with the PI medication resistance mutations in the PR region at codons 10, 46, 54, 82 and 90 which are known to affect susceptibility to all currently FDA-approved PIs, and codon 30 (D30N), which may also significantly affect the virological response . In contrast, four or more mutations are required to overcome the activity of PI medications in adults . It is possible that the thymidine-associated mutations (TAMs) recorded in the HIV-1 infected children in the present study may mediate resistance to multiple mutations unrelated to the NRTI treatment [29, 30]. The mutations associated with more than one NRTI may increase viral replication in an additive manner [2, 31, 32] and NNRTI associated mutations may intensify the rebound in the HIV-1 RNA . The observed association between the higher PVL and resistance mutations in the PR region was confirmed after controlling for mutations in the RT regions. The persistent high-level of viral replication seen in children with primary mutations in the PR gene may increase the accumulation of multiple resistance mutations [34–38] and therefore affect the efficacy of drug therapy.
Although high-level virus production is associated with CD4+ T-cell destruction in adults , no such association was identified between viral replication and severity of immune suppression in the children included in the present study. Resino et al  identified the possibility of CD4+ T-cell recovery despite virological failure resulting in the discordant response in children. Moreover, viral load suppression is not indispensable for recovery of CD4+levels .
We have to acknowledge that insufficient drug potency, pharmacological issues, and poor compliance could be responsible for the anti-viral treatment failure , in addition to the drug resistance mutations in the PR region that were identified as a major predictor for the failure in the suppression of viral replication in this study. Moreover, despite the disagreement in the literature [42–44], medically directed treatment interruptions, which were not analyzed in this study, may influence the virologic and immunologic outcome as well as the development of drug-resistant viral isolates. Long-term clinical and immunologic follow up will be required in order to clarify the predictive power of HIV-1 RNA plasma levels in the monitoring of disease progression.
Despite the limitations, this study clearly demonstrates that primary PR resistance mutations significantly increase the likelihood for high viral replication in children with moderate/severe HIV-1 infection. This raises concern regarding early failure in drug susceptibility, which may jeopardize the long-term clinical prognosis of the HIV-1 infected pediatric patient.
human immunodeficiency virus type 1
plasma viral load
T- lymphocyte count
nucleoside reverse transcriptase inhibitor
non-nucleoside reverse transcriptase inhibitor
mutation in the protease gene
thymidine associated mutation.
The authors would like to express their most sincere thanks to Margaretanne Murray for her assistance with this study.
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