Laboratory of Immunobiología-Molecular, Hospital General Universitario Gregorio Marañón, Madrid, Spain
Received 5 July 2005; returned 15 August 2005; revised 8 September 2005; accepted 9 September 2005
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Abstract |
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Objectives: To study whether mutations in the HIV-1 protease gene can reliably predict virological responses to salvage therapy with lopinavir/ritonavir in HIV-infected children.
Patients and methods: We carried out a prospective study in 56 HIV-infected children. PI-associated resistance mutations were determined by genotypic testing and were scored according to the IAS-USA guidelines 2005.
Results: Children with a lopinavir mutation score (LMS) 6 had a negative association for achieving viral load (VL) control [undetectable viral load (uVL)
400 copies/mL] and maintaining uVL for at least 6 months. Moreover, children with protease-associated mutations (PRAMs)
2 had a negative association for achieving VL control but not for maintaining uVL for at least 6 months. The relative proportion (RP) to uVL was 0.32 (CI95%: 0.16; 0.33; P = 0.002) in children with I54V (46% of total) and 0.48 (CI95%: 0.24; 0.97; P = 0.041) in children with V82A/F (52% of total). Children with I54V and V82A/F had higher prevalence of lopinavir-associated resistance mutations and showed RP of 0.36 (CI95%: 0.17; 0.76; P = 0.007) for achieving uVL.
Conclusions: LMS and PRAMs in HIV-infected children were associated with virological failure in pre-treated HIV-infected children on salvage therapy with lopinavir/ritonavir. Moreover, I54V and V82A/F led to the poorest virological response.
Keywords: HIV-1 , viral load , salvage therapy , resistance
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Introduction |
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To address the predictive value of PI mutations on viral load (VL) response to salvage therapy, we have carried out a study with PI-experienced HIV-infected children treated with lopinavir/ritonavir and followed up for a long period of time (24 months).
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Patients and methods |
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The study involved a multicentre prospective cohort of 56 PI-experienced HIV-infected children on salvage therapy with lopinavir/ritonavir followed at 12 Spanish hospitals. The inclusion criteria were: (i) VL > 5000 copies/mL at baseline; (ii) at least 6 months of follow-up; (iii) older than 1 year of age; (iv) being previously treated with antiretroviral therapies (ART) and having records of virological failure with PI and/or non-nucleoside analogue (NNRTI); (v) starting salvage HAART (highly active antiretroviral therapy) with lopinavir/ritonavir; (vi) no CD4+ cell count restrictions. This study was conducted according to the declaration of Helsinki and was approved by the Ethics Committees of all hospitals involved.
The children were monitored at least every 3 months with physical examinations and blood sample collection for serial measurements as previously described.13 Laboratory markers of HIV-1 infection (T cells subsets, VL and genotypic HIV-1 drug resistance) were measured as previously described.13 There was not a uniform approach regarding antiretroviral treatment in the background regimen given together with lopinavir/ritonavir. Instead, each paediatrician administered the appropriate antiretroviral therapy (ART) regimen and changed the drugs according to his/her interpretation of the data and international guidelines.13
HIV-1 VL
VL was measured in plasma using the Amplicon Monitor assay (Amplicon Monitor; Roche Diagnostic Systems, Brandenburg, NJ, USA). The limit of quantification was 400 copies/mL.
Genotypic analysis of HIV-1 isolates
Baseline plasma samples for viral genotype were collected between day 60 (60 days prior to initiation of lopinavir/ritonavir treatment) and day 1. Genotypic HIV-1 drug resistance was determined from plasma-associated HIV-1 RNA using the TruGene HIV-1 Resistance Kit (Visible Genetics, Toronto, Canada). The complete HIV-1 protease gene was analysed using Gene Objects software (Visible Genetics). Drug resistances were defined according to the IAS-USA consensus statement.14
Statistical analysis
The ANOVA test was used to compare between the means of two groups. The Fisher exact test was used for all other comparisons between groups. Differences in the characteristics among number of mutations in children were analysed using a non-parametric test (MannWhitney). All P values were two-tailed, and the threshold of significance was set at 0.05.
The outcome variables examined were the time necessary for achieving VL 400 copies/mL and for maintaining VL
400 copies/mL for at least 6 months. These variables were analysed by the KaplanMeier method. Cox regression analyses were performed to assess the relative proportion (RP) achieving VL
400 copies/mL according to the presence of the PI mutation, and adjusted by baseline characteristics (%CD4+, VL and age at baseline, and additional new drugs in the salvage regimen at baseline). Also, we performed a logistic regression analysis to determine the odds ratio (OR) of VL control (to maintain VL
400 copies/mL for at least 6 months).
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Results |
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Table 1 shows the baseline characteristics of HIV-infected children enrolled in this study. Approximately 50% of children had an AIDS diagnosis. Also, 42/56 of children had CD4+ T cells 15% whereas 25/56 had VL
50 000 copies/mL. Throughout the follow-up period, no child progressed to a new AIDS-defining condition or death. Twenty-nine percent of HIV-infected children did not show any PRAMs whereas 59% had <6 LMS.
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At baseline, children had a median of 5 lopinavir mutations (range: 0; 10). The most frequent PI mutations were L63A/P, A71T/V, L10I and L90M. In addition, all children had a median of 4.5 (range: 0; 8) NRTI mutations and 0 (range: 0; 3) NNRTI mutations. The most frequent reverse transcriptase (RT) mutations were M41L, D67N, Q151M, G190A and T215F.
Children with LMS 6 presented a negative association with achieving VL
400 copies/mL and with maintaining virological suppression (VL
400 copies/mL) for at least 6 months (Table 3). Moreover, children with PRAMs
2 had a negative association with achieving VL
400 copies/mL but not with maintaining virological suppression (VL
400 copies/mL) for at least 6 months (Table 3).
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Association of I54V and V82A/F mutations and its impact on virological response
Twenty-four children showed I54V and 20 of these showed V82A/F. When we analysed the virological impact of the I54V and V82A/F mutations combined, we have shown that children with I54V and V82A/F had a high prevalence of other lopinavir mutations (L10I, K20R, L24I, V32I, L33F, M46L, L63P, A71V/T, G73S, V82A/F, I84V and L90M) (Figure 1a). Children with both mutations had a median of 7 lopinavir mutations (range: 4; 10) in contrast to a median of 4 (range: 0; 8) in children without the two mutations (P < 0.001). Moreover, children with I54V and V82A/F mutations also showed a high prevalence of RT mutations (Figure 1b and c). Thus, they had a median of 5 NRTI mutations (range: 2; 8) in contrast to a median of 4 (range: 0; 9) in children without I54V and V82A/F (P = 0.532). Also, HIV-infected children with I54V and V82A/F showed a median of 2 (range: 0; 2) in contrast to a median of 0 (range: 0; 3) NNRTI mutations in children without the two mutations (P = 0.018).
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Children with I54V and V82A/F had the lowest probability of achieving VL 400 copies/mL (Table 3). I54V and V82A/F PI had a negative association with achieving undetectable VL and with having virological suppression (VL
400 copies/mL) for at least 6 months.
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Discussion |
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Previous studies have shown that the 16-mutation LMS was inversely associated with virological suppression.2,7,9,13,16,17 Algorithms for interpreting viral genotypes have suggested that the efficacy of lopinavir/ritonavir treatment is significantly reduced by the presence of six or more mutations (www.hivdb.stanford.edu). However, one other study found that this scoring system may lead to under-appreciation of lopinavir-associated resistance.18 Mutations at positions 46, 54 and 82 showed a great resistance to lopinavir. The threshold number of lopinavir mutations found by Delaugerre et al.16 was 4, but we found a threshold of 6 as determinant of the virological outcome in agreement with other studies in children13 and adults.6,7,9,19 The differences could be because the children in the Delaugerre et al. report were less heavily treated than the children in our study.
The number of PRAMs necessary to confer resistance to lopinavir seems to be quite low (2 or more).20 We have found that only PRAMs 2 in plasma of children had a negative association with virological response to salvage therapy with lopinavir/ritonavir. However, this association was not found for maintaining virological suppression for at least 6 months.
Our data clearly indicate that I54V and V82A/F mutations were associated with virological failure. These data differ from another study in children that found an association of the PI mutations L10I/F, M46I, I54V/L, A71V/I, V82A/F/T/S and L90M with virological failure.16 Thus, in our study undetectable VL was observed more commonly in children without I54V and V82A/F PI mutations; and VL values were stable between 3 and 15 months in most of these children. In contrast, viral replication was not well controlled in children with I54V and V82A/F as indicated by the low OR for achieving undetectable VL for at least 6 months of follow-up. Children with I54V and V82A/F had been heavily pre-treated and had a high overall number of mutations against lopinavir, NRTIs and NNRTIs. We also found that HIV-infected children with I54V and V82A/F also had a high prevalence of other lopinavir mutations.11
Moreover, we did not find any influence of previous ART at baseline and additional new drugs in the salvage regimen at baseline on the virological response because we did not find statistical significance in Cox regression analysis for achieving undetectable VL and for maintaining undetectable VL for at least 6 months (data not shown). Besides, with regard to the mutations in the HIV-1 protease gene, only VL at baseline showed statistical significance in Cox regression analysis.
In conclusion, our study indicates that both LMS and PRAMs in plasma of children were associated with virological failure to salvage therapy with lopinavir/ritonavir. Despite there being several studies, the exact pattern of lopinavir/ritonavir resistance is not yet known and the beneficial role for lopinavir/ritonavir in salvage antiretroviral therapy remains under debate. However, we have shown that I54V and V82A/F led to the worst virological response. Further studies are necessary on specific mutation patterns associated with a low response to lopinavir/ritonavir in HIV-infected children.
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Transparency declarations |
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Footnotes |
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Participants are listed in the Acknowledgements section.
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Acknowledgements |
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Participating hospitals and personnel staff of the Spanish Group of Paediatric HIV Infection. MADRID: Hospital 12 Octubre: J.T. Ramos. Hospital Gregorio Marañón: J.L. Jiménez, A. Martinez-Colom, J.M. Bellón, M.D. Gurbindo, M.L. Navarro, S. Resino, M. Gonzalez-Rivera, L. Diaz, M.A. Muñoz-Fernández. Hospital La Paz: M.I. Isabel de José, B. Larru. Hospital Carlos III: P. Martín-Fontelos, M.J. Mellado. SEVILLA: Hospital Virgen del Rocio: J.A. León Leal. BARCELONA: Hospital S.Juan de Dios: C. Fortuny. Hospital Valle de Hebrón: J.M. Bertrán, L. García. Hospital del Mar: A. Mur. ALICANTE: Hospital S Juan: R. González-Montero. BILBAO: Hospital de Cruces: I. Pocheville, C. Gutierrez. PALMA DE MALLORCA: Hospital Son Dureta: J. Dueñas. VALENCIA: Hospital La Fe: A. Orti, M.C. Otero, F. Asensi. ZARAGOZA: Hospital Clínico: M. Gracia, L. Ciria.
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