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Journal of Clinical Microbiology, December 2002, p. 4512-4519, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4512-4519.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Testing Genotypic and Phenotypic Resistance in Human Immunodeficiency Virus Type 1 Isolates of Clade B and Other Clades from Children Failing Antiretroviral Therapy

Patrícia A. Brindeiro,¹ Rodrigo M. Brindeiro,¹ Cláudio Mortensen,² Kurt Hertogs,³ Veronique De Vroey,³ Norma P. M. Rubini,⁴ Fernando S. Sion,⁴ Carlos A. M. De Sá,⁴ Deisy M. Machado,⁵ Regina C. M. Succi,⁵ and Amilcar Tanuri^1*

Laboratory of Molecular Virology, Department of Genetics, Federal University of Rio de Janeiro,¹ Gaffrée & Guinle University Hospital, Rio de Janeiro,⁴ Applied Biosystems,² Federal University of São Paulo Medical School, São Paulo, Brazil,⁵ Tibotec-VIRCO NV, Mechelen, Belgium³

Received 7 June 2002/ Returned for modification 8 August 2002/ Accepted 17 September 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
The emergence of resistance to antiretroviral drugs is a major obstacle to the successful treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients. In this work, we correlate clinical and virological trends such as viral load (VL) and CD4 counts to genotypic and phenotypic antiretroviral (ARV) resistance profiles of HIV-1 isolates from the B and non-B subtypes found in vertically infected children failing ARV therapy. Plasma samples were collected from 52 vertically HIV-1-infected children failing different ARV therapies. Samples underwent HIV-1 pol sequencing and phenotyping and were clustered into subtypes by phylogenetic analysis. Clinical data from each patient were analyzed together with the resistance (genotypic and phenotypic) data obtained. Thirty-five samples were from subtype B, 10 samples were non-B (subtypes A, C, and F), and 7 were mosaic samples. There was no significant difference concerning treatment data between B and non-B clades. Prevalence of known drug resistance mutations revealed slightly significant differences among B and non-B subtypes: L10I, 21 and 64%, K20R, 13 and 43%, M36I, 34 and 100%, L63P, 76 and 36%, A71V/T, 24 and 0%, and V77I, 32 and 0%, respectively, in the protease (0.0001 P 0.0886), and D67N, 38 and 8%, K70R, 33 and 0%, R211K, 49 and 85%, and K219Q/E, 31 and 0%, respectively, in the reverse transcriptase (0.0256 P 0.0704). Significant differences were found only in secondary resistance mutations and did not reflect significant phenotypic variation between clade B and non-B.

Top Abstract Introduction Materials and Methods Results Discussion References

 
One of the major causes of treatment failure during antiretroviral (ARV) therapy is the emergence of human immunodeficiency virus type 1 (HIV-1) genotypic variants carrying viral protease and reverse transcriptase (RT) mutations conferring resistance to antiretroviral compounds (8). These variants have been studied, and their resistance-conferring substitutions were mapped in the viral pol gene for each ARV drug (7, 15, 16). These substitutions can be classified into primary and secondary mutations. Primary mutations lead to a severalfold decrease in sensitivity to one or more ARV drugs (15, 16). Secondary mutations may not result in a significant decrease in drug sensitivity but are associated with restoration of the original viral fitness in the presence of existing inhibitors (15, 16). These studies have been carried out in detail for clade B viruses, the prevalent HIV-1 subtype in the United States and western Europe, although this is not the main subtype of HIV infections worldwide. There are few data on genotypic resistance in non-B subtypes of HIV-1 (5, 6, 12, 17). The non-B HIV-1 strains usually carry pol gene polymorphisms as genetic fingerprints that can give these viruses a lower susceptibility to the ARV compounds (5, 6, 12, 17, 29). Hence, the genetic polymorphisms of non-B HIV-1 pol may lead to the establishment of specific resistance patterns that differ slightly from those found for B viruses, and may alter the interpretation of genotyping assays.

The concern about the currently limited information about known resistance patterns of non-B HIV strains can be extended to pediatric ARV treatment of AIDS. Recent studies have reported unique virus population dynamics in vertically infected children (17, 29), although very little is known about the infection, disease progression, and genotypic and phenotypic resistance profiles in children harboring clade B drug-resistant HIV-1 variants (9, 13, 19, 21, 25).

In Brazil, non-B virus strains can be found, such as those from clades F (18% prevalence), C (frequently found in the southernmost part of the country), D, and A. There are also B/F, B/C, and B/D mosaics (20, 23, 26). The prevalence of circulating drug-resistant strains of virus in Brazil has been reported only for drug-naive and treated adults (3, 5, 6, 27). The STD/AIDS Program of the Brazilian Ministry of Health estimates that 5,426 children were infected with HIV in Brazil up to April 2001 (2). A large proportion of them are currently undergoing ARV treatment. The present study is a comparative analysis of clinical and virological data with its focus on comparisons of genotypic and phenotypic viral resistance patterns between different clades (B and non-B) found in HIV-infected Brazilian infants failing antiretroviral therapy.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study population. Samples were obtained from HIV-1-seropositive children attending the university hospitals in Rio de Janeiro and São Paulo, Brazil. Plasma samples were collected from 52 vertically HIV-infected children (2 to 14 years of age) failing different ARV therapies (19 were receiving dual-nucleoside analog inhibitor [dual-NRTI] therapy only, and 33 were under highly active antiretroviral treatment [HAART], 8 of whom had not received dual-NRTI regimens before being given HAART). The criteria of failure were based on viral-load responses, according to the treatment consensus of the AIDS program from the Brazilian Ministry of Health (2). All samples collected during 1 year (from April 1999 to April 2000) and successfully analyzed were included in this study.

Sample data collection. The CD4 counts and plasma HIV RNA levels (viral load [VL]) were routinely determined for each patient, as determined and supported by the Brazilian Program for STD/AIDS, National Ministry of Health, to facilitate appropriate clinical care. All information concerning each patient (CD4 cell counts, latest and previous viral loads, detailed ARV and other treatment history, and clinical classification of HIV infection progression according to the criteria of the Centers for Disease Control and Prevention, Atlanta, Ga.) was provided by the AIDS clinics of the two hospitals in Brazil. Most of this information was cross-analyzed in conjunction with the genotypic and phenotypic data.

Plasma HIV RNA levels were measured by reverse transcription-PCR (RT-PCR) (Cobas Amplicor; Roche Diagnostics, Nutley, N.J.) or nucleic acid sequence-based amplification technology (Nuclisens; Organon Teknika, Boxtel, The Netherlands).

Virus isolation and HIV-1 pol genotyping. Isolation of virus from plasma samples and analysis of HIV-1 pol sequences were performed with the Prt/5' RT HIV-1 genotyping system (Applied Biosystems, Foster City, Calif.). Briefly, HIV-1 RNA was isolated from plasma and reverse transcribed with random hexamer primers and Moloney murine leukemia virus RT. An HIV-1 DNA fragment including the regions encoding protease (amino acids 1 to 99) and RT (amino acids 1 to 310) was amplified by PCR using TaqGold in a single 40-cycle reaction. The amplified DNA was then purified and sequenced with six or seven primers and BigDye terminator reagents with an ABI model 377 automated DNA sequencer (Applied Biosystems). Sequences were edited, aligned, translated into amino acids, and analyzed for the presence of amino acid polymorphisms.

Phylogenetic analysis. The genetic subtypes were determined by phylogenetic tree analysis. The nucleotide sequences from clinical isolates and the sequences of reference strains representing the different genetic subtypes, based on the protease and RT genes, were aligned by the CLUSTAL multiple sequence alignment programs (28). Phylogenetic trees were constructed by the neighbor-joining distance method (24). Evolutionary distances were estimated using the Kimura two-parameter method. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 2.0 (18). An interior-branch test for the nodes was also performed with MEGA version 2.0, using representative standards belonging to different subtypes obtained from the Los Alamos National Laboratory database. The simian immunodeficiency virus cpz sequence was used as the outgroup.

HIV-1 pol phenotyping. The detection of HIV-1 phenotypic resistance to Food and Drug Administration (FDA)-approved ARV compounds was performed using a recombinant virus assay technology (Virco, Mechelen, Belgium). The HIV-1 RNA was extracted from plasma samples, and a 2.2-kb fragment containing the entire HIV-1 pol (protease and RT) coding sequence was amplified by nested RT-PCR. The pool of pol coding fragments was then cotransfected into CD4⁺ T lymphocytes (MT4) with the pGEMT3PRT plasmid carrying the defective pol HIV genomic cDNA. Homologous recombination leads to generation of chimeric virus containing pol sequence derived from patients' viruses. The susceptibility of chimeric virus to all the FDA-approved antiretroviral compounds was determined by an MT4 cell 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-based cell viability assay in an automated system as previously described (14).

Statistical analysis. All statistical treatments were performed using the Analyse-it v. 1.62 for Microsoft Excel statistics package. Statistical analysis of differences between mutation frequencies and also between phenotypic categories (resistant and wild-type isolates) was carried out using Fisher's two-tailed exact test on 2 x 2 contingency tables, with = 0.05. The sample means are always shown together with the standard error of the mean (mean ± SEM).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Phylogenetic analysis of samples. Among the 52 samples successfully analyzed, there were 35 B-subtype samples and 10 non-B. Seven samples with mosaic pol structures were also found, with four F/B and three B/F genotypes, for the protease and RT genes, respectively (Table 1). Of the 10 non-B samples, 8 (15.4% of the total 52) clustered with clade F of HIV-1 group M. One clade C sample and one clade A sample were also found. All subtypes were confirmed by HIV env gp41IDR sequencing and phylogenetic analysis (data not shown), and mosaic B/F and F/B pol samples clustered with B or F clades for this env region, with no specific significant correlation with each pol mosaic arrangement. For subsequent analysis of resistance mutations, each target gene for therapy (protease or RT) was considered separately to avoid misinterpretation due to the presence of these mosaic genotypes.

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TABLE 1. Classification of samples according to subtypes

Treatment data. Three types of ARV regimens could be observed at the time of sample collection for genotyping: 19 patients (15 infected with B viruses and 4 infected with non-B viruses) were receiving dual-NRTI regimens with no experience of HAART, 25 patients (14 infected with B viruses, 5 infected with non-B viruses, and 6 infected with mosaic viruses) were receiving HAART with a history of dual-NRTI regimens, and 8 patients (6 infected with B viruses 1 infected with a non-B virus, and 1 infected with a mosaic virus) had received only HAART (Table 2). There were no differences between subtypes for the different regimens used, the average duration of the most recent and previous regimens used, or the average number of different regimens used during the clinical history of each patient. The ARV drug usage in the regimens was essentially the same for patients infected with the different subtypes. Saquinavir was not used in children because no compound formulation was available for pediatric use.

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TABLE 2. Treatment data for infant HIV-1 infections with different viral subtypesa

Clinical data for infants infected with different subtypes were not significantly different, including age and VL (the two were collected at the time of blood sampling for genotyping) (Table 3). Strikingly, when the CD4 counts were compared between children infected with subtype B and non-B viruses, higher values were found in clade B (P = 0.0208; one-tailed heterocedastic Student's t test).

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TABLE 3. Clinical data of infant HIV-1 infections with different viral subtypesa

Resistance mutations in the protease gene. The overall frequency of protease genotypes resistant to protease inhibitors (PIs) was 44.23%, with no difference among subtype B and non-B viruses. The most common resistant protease genotypes were those carrying primary mutations to positions 54 and 82 (10 genotypes), 54, 82, and 90 (4 genotypes), and 30 (3 genotypes) in the protease protein.

Frequencies of relevant resistance-associated mutations (7) were compared among the subtype groups in the protease region of viruses (Table 4). The prevalence of known drug resistance mutations among B and non-B clades in infected children revealed significant differences: L10I/R/V/F, 21.1 and 64.3% (P = 0.0102), K20M/R, 13.2 and 42.9% (P = 0.0592), M36I, 34.2 and 100% (P < 0.0001), L63P, 76.3 and 35.7% (P = 0.0178), A71V/T, 23.7 and 0% (P = 0.0886), and V77I, 31.6 and 0% (P = 0.0262), respectively in B and non-B samples. Although nelfinavir was commonly used in children infected with non-B virus, this group of subtypes did not present any D30N or N88D, in contrast to the frequencies of 7.9 and 2.6%, respectively, found in samples from infants infected with the B subtype who were given initial HAART with nelfinavir (Table 5). All L90M resistance mutations were found in nelfinavir-experienced children (three for subtype B and one for the non-B group) for whom this drug was a second-choice PI in HAART after ritonavir. The most frequent primary resistance-related mutations in infant samples were the substitutions at codons 82 and 54 (with no significant difference of frequency between clades for both mutations), as expected since most patients receiving HAART were given ritonavir as the first-choice PI.

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TABLE 4. Frequency of resistance conferring mutations in the protease and RT genes

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TABLE 5. Genotypic profiles of viral isolates for each patient

Resistance mutations in the RT gene. Of 52 RT genotypes sequenced 50 (96.15%) were resistant to NRTIs and/or non-NRTIs (NNRTIs). The most common RT genotypes found to be resistant to NRTIs were those carrying primary mutations at positions 184 (16 genotypes), 184 and 215 (16 genotypes), 70 and 184 (5 genotypes), and 70, 184, and 215 (4 genotypes) in the RT. The most common RT genotypes resistant to NNRTIs had primary substitutions at positions 181 (3 genotypes), 103 and 181 (2 genotypes), and 106 and 181 (2 genotypes) in the RT.

The frequencies of resistance mutations were also assessed for the RT region in isolates from infant patients (Table 4). All significant differences in the mutation profiles of B and non-B clades were related to resistance to zidovudine: D67N (38.5 and 7.7%; P = 0.0704), K70R (33.3 and 0%; P = 0.0256), R211K (48.7 and 84.6%; P = 0.0461) and K219Q/E (30.8 and 0%; P < 0.0371); three of these (D67N, K70R, and K219Q/E) were more frequent in the B clade. Although 100% of patients in the B group and 90% of patients in the non-B group were receiving zidovudine, only 46.2 and 53.8% of their samples, respectively, presented the zidovudine primary resistance mutation, T215Y/F. Inversely, the prevalence of the resistance mutation M184V to lamivudine was equally high for both groups of clades (86 and 90%).

Phenotyping of viral isolates. Genotypic results were confirmed by a phenotyping assay for the non-B samples analyzed in this study. Fifteen samples belonging to clade B were taken randomly, along with all non-B samples and mosaics, for the Antivirogram phenotyping assay (Table 6). Again, the results were analyzed separately for each target gene for therapy (protease or RT) to avoid misinterpretation due to the presence of mosaic genotypes. No phenotypic difference could be observed between isolates from groups B and non-B. Instead, the fold increase of mean 50% inhibitory concentrations (IC₅₀) obtained for each drug to which the isolates were resistant was in accordance with the genotypic prediction of resistance, based on each isolate genotypic profile and the published guides to interpretation of genotyping (7).

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TABLE 6. Phenotypic profiles (Antivirogram) of samples from different subtypes

Three samples showed primary resistance mutations to NNRTI (K103N and Y181I for TVGG38, Y181C for TVSP11, and V106A for TVSP19), without any history of previous patient exposure to this class of inhibitors. These mutations found in virus isolates were able to confer in vitro resistance to NNRTIs as revealed by the phenotypic assay.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, clinical and treatment data for infected children were homogeneous across clades, except that the CD4 cell counts were higher in patients infected with B virus than in those infected with non-B virus.

We were able to find a strong correlation between the therapeutic failure in children receiving ARV and the presence of primary resistance mutations in the pol genotypes of their virus isolates. Of 52 pol genotypes analyzed, 50 (96.15%) carried primary mutations conferring resistance to at least one class of antiretroviral drug among those used in therapy for each patient analyzed. In detail, we found a higher frequency of RT genes carrying primary mutations (96.15%) than protease genes with this kind of substitution (44.23%). No difference was found in the frequency of these mutations between B and non-B subtype isolates. Only two isolates did not present any primary mutation to ARV resistance (TVGG34 and TVSP09); this is probably related to patient nonadherence to the therapy.

From the genotypic analysis of the protease region, we could detect subtype differences only among secondary resistance mutations, such as in residues 10, 20, 36, 63, 71, and 77. The variation found in the frequency of these mutations is probably a consequence of the natural occurrence of polymorphisms as genetic fingerprints in non-B isolates. Although there is no evidence of direct correlation between the variation in secondary resistance substitutions present in different clades and their phenotypic resistance profiles (5, 7), we speculate that these non-B genetic variations play an important role in augmenting the viral fitness of isolates carrying primary resistance mutations. Such differences lead to alternative paths to resistance through the genetic barrier for non-B clades and may influence the progression to resistance during drug therapy of infant HIV-1 infection. These findings are in accordance with the analyzed data reported in a recent study that compared the genotypic variation between the prevalent clades, B and C, in Israel (12), as well as in other studies reporting genotypic differences between clades only in the secondary resistance mutation patterns (4, 6, 10, 11, 12, 17, 27). A high frequency of ritonavir and nelfinavir resistance mutations was detected, in contrast to the low prevalence found by others in infants (9).

The RT gene mutation patterns between B and non-B subtypes were basically the same and varied only in residues related to zidovudine resistance, which occurred more frequently in B subtype isolates (except for R211K), as observed elsewhere (12). The primary substitution T215Y/F was found frequently in both B and non-B clades, and the difference in the zidovudine resistance genetic profile occurred mostly in other residues involved in conferring zidovudine resistance (RT residues 67, 70, 211, and 219). This may suggest that different mechanisms were used among clades to achieve the increased pyrophosphorolysis activity and processivity seen in RT enzymes highly resistant to AZT (1).

In some infants, we found viruses harboring mutations conferring resistance to NNRTIs, although some of the infants were not exposed to this class of drugs, as observed by others (12). This may be related to the capacity of viruses to carry these mutations without changing their RT activity. These results confirm the usefulness of the genotypic test for guiding the rescue therapy of pediatric AIDS patients failing HAART in Brazil.

There was no evidence from our results of different patterns of genotypic and phenotypic resistance to ARV compounds of viral isolates from children failing ARV therapy in comparison with the widely studied patterns of isolates from adults (7, 12, 17, 21, 27).

We suggest that HIV-1 protease and RT variability among different clades might influence the efficacy of ARV treatments. These results call for careful investigations implementing well-controlled genotyping and phenotyping clinical trials with follow-up of patients infected with non-B subtype virus before starting large therapy programs in countries where the majority of the HIV-1 epidemic is driven by non-B-subtype strains.

 
* Corresponding author. Mailing address: Universidade Federal do Rio de Janeiro, C.C.S., Instituto de Biologia, Depto. de Genética, bloco A, sala A2-121, 2° andar, Cidade Universitária, Ilha do Fundão, Rio de Janeiro, RJ, CEP: 21944-970, Brazil. Phone: (55 21) 2562-6384. FAX: (55 21) 2562-6384. E-mail: atanuri{at}biologia.ufrj.br.

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Journal of Clinical Microbiology, December 2002, p. 4512-4519, Vol. 40, No. 12
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.12.4512-4519.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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