Abstract
The prevalence of mutations that confer resistance to protease inhibitors and to nucleoside and nonnucleoside reverse transcriptase inhibitors in 49 blood samples from drug-naïve human immunodeficiency virus type 1-infected blood donors living in Rio de Janeiro state, Brazil, in 1998 was evaluated genotypically and phenotypically.
The new generation of drugs targeting the reverse transcriptase (RT) and protease (PR) genes of the pol region of human immunodeficiency virus type 1 (HIV-1), such as nucleoside reverse transcriptase inhibitors (NRTIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs), and protease inhibitors (PIs), has revolutionized treatment of infected individuals (11, 18). However, not all patients respond to highly active antiretroviral treatment (HAART), and many develop drug resistance, one of the most serious obstacles to sustained suppression of HIV-1 (16, 21, 28) The emergence of amino acid mutations associated with resistance to RTIs and PIs has been extensively characterized (14, 23), and these substitutions can be classified into primary and secondary mutations. Primary mutations lead to a severalfold decrease in sensitivity to one or more antiretroviral (ARV) drugs (14, 23). 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 (14, 23).
The transmission and dissemination of drug-resistant strains have major public health implications, including disrupting the efficiency of established ARV treatment for HIV-1-infected patients and of prophylaxis against both accidental exposures and perinatal HIV-1 transmission. Prevalences of primary resistance mutations for any drug observed among recent seroconverters range from 5 to 11% in Switzerland (32; S. Yerly, E. Race, S. Vora, P. Rizzardi, J. P. Chave, M. Flepp, P. Vernazza, A. Telenti, M. Battegay, A. L. Veuthey, B. Hirschel, L. Perrin, and the Swiss HIV Cohort Study, Program Abstr. 8th Conf. Retroviruses Opportunistic Infections, abstr. 754, 2001), 4 to 17% in France (9, 24; M. L. Chaix, M. Harzic, B. Masquelier, I. Pellegrin, L. Meyer, D. Costagliola, C. Rouzioux, and F. Brun-Vezinet, Program Abstr. 8th Conf. Retroviruses Opportunistic Infections, abstr. 755, 2001), 13% in Germany (10), 14% in the United Kingdom (27), 2 to 26% in North America [1, 2, 5, 17, 22; S. J. Little, S. Holte, and J. P. Routy, abstract from 5th Int. Workshop HIV Drug Resist. Treatment Strategies, 4 to 8 June 2001, Scottsdale, Ariz. Antivir. Ther. 6(Suppl. 1):21, 2001], and 23 to 26% in Spain (4).
Information on PI- and RTI-associated mutations in HIV-1-infected drug-naïve individuals in Brazil is limited to specimens collected before 1996, when free access to HAART was established for AIDS patients by the Brazilian Ministry of Health (3, 26). These specimens have shown a low prevalence of mutations related to NRTIs, NNRTIs, and PIs. In this paper, we describe the genetic diversity of HIV-1 PR and RT sequences and the prevalence of mutations linked to PI and RTI resistance in treatment-naïve HIV-1-infected blood donors living in the state of Rio de Janeiro, Brazil, in 1998.
Of the 18,667 blood units collected from January through December 1998 at blood banks distributed throughout the state of Rio de Janeiro, 56 were rejected based on their HIV-1 positivity by enzyme immunoassay (Prism HIV-1/HIV-2 [Abbott, North Chicago, Ill.] and Vironostika HIV Uni-Form plus O [Organon Teknika BV, Boxtel, The Netherlands]). After confirmation by Western blot analysis, the viral RNA was isolated from plasma, and PR and RT genes were RT-PCR amplified and sequenced as previously described (26). This study was approved by the Brazilian Ethics Committee (CONEP) as an anonymous unlinked study.
Phylogenetic analysis was performed using the neighbor-joining method as previously described (25). Of 44 samples positive for both PR and RT sequences, 40 had concordant subtype assignments in these regions, including 39 (89%) subtype-B specimens and 1 (2%) subtype-C specimen (Table 1). The remaining four samples revealed discordant subtypes for the PR and RT regions, which may represent recombinant viruses. Finally, of the five specimens with HIV-1 subtype assignment for only one viral region, two PR and two RT sequences were of subtype B and one RT sequence was of subtype F. These results indicate that subtype distributions in our sampling reflect the proportions previously observed in Brazil (7, 25, 29).
TABLE 1.
Presence of PR and RT inhibitor-associated mutations in subtype-B and non-B Brazilian HIV-1 strains collected in 1998
| Samplesa | Subtypesb
|
Genotype(s)c at resistance-related position(s)
|
||
|---|---|---|---|---|
| PR | RT | PR | RT | |
| 01 | B | B | WT | 135T |
| 02 | B | B | 77I 82I | 135T |
| 06 | B | B | WT | 135T |
| 07 | B | B | 63A 77I | 135T |
| 11 | B | ND | 77I 82I | NA |
| 12 | B | B | 63P 77I | 1061 135T |
| 14 | B | B | 63P 71T | 135T |
| 15 | B | B | 36I 63P 71V 77I | WT |
| 16 | B | B | 63P 71T | 135T |
| 18 | B | B | 63P | 44G |
| 19 | B | B | 63S | 135T 219P |
| 24 | F | B | 36I | WT |
| 33 | ND | B | NA | WT |
| 36 | B | B | 63V 77I | 135T |
| 51 | B | B | 36I | 44G 219R |
| 52 | U | B | 63S 10I36I | 135T |
| 53 | B | B | 63P | WT |
| 56 | F | U | WT | 135T |
| 57 | B | B | 63P | 135T |
| 58 | U | B | 10V36I | 106L 135T |
| 64 | B | B | 63H | WT |
| 66 | B | B | 63P 71T | 75L 98S 135T |
| 67 | B | B | 63P | 135T |
| 68 | B | B | 63P | WT |
| 69 | B | B | 77I | 135T 219R |
| 70 | B | B | 63P | 135T 151K |
| 71 | B | B | WT | 106I 135T |
| 73 | B | B | 77I | 135T 219T |
| 74 | B | B | 36L | 135T |
| 75 | B | B | 63P 71T | WT |
| 76 | B | B | 63P | 118I 135T |
| 77 | B | B | 63P | 135T |
| 79 | B | B | 63P 71T 77I | 135T |
| 80 | B | B | 63P 77I | 135T 215P |
| 81 | B | B | 36I 63P | 98S 135T |
| 83 | ND | F | NA | 135V |
| 84 | B | B | 63P | WT |
| 85 | B | B | 36I | 135T |
| 86 | ND | B | NA | WT |
| 87 | B | ND | WT | NA |
| 91 | B | B | 63P 71T | 135T |
| 94 | C | C | 36I | WT |
| 96 | B | B | 63P | 135T |
| 97 | B | B | 63P | 44G 135T |
| 98 | B | B | 77I 82I | 106I 135T |
| 99 | B | B | 63P | 106I 215P |
| 102 | B | B | 63P 77I | 106I 135T 215S |
| 106 | B | B | 63P | 135T 215S |
| 107 | B | B | 63P | 135T |
Each sample number represents a “98BRVM” strain; for example, the first sample listed is strain 98BRVM01. Boldfaced samples either are non-B subtypes or recombinants or have at least one unclassifiable region.
ND, PCR negative; U, unclassifiable.
WT, wild type; NA, not analyzed. Boldfaced genotypes represent secondary mutations observed with U.S. FDA-approved ARV agents.
To examine the frequency of primary (D30N, M46I, G48V, I50V, V82A, -F, or T [V82A/F/T], I84V, and L90 M) and secondary (L10I/R/V, K20M/R, L24I, V32I, L33F, M36I, M46I/L, I47V, I54L/M/V, L63P, A71V/T, G73S, V77I, V82I, and N88D) PI-associated substitutions within the PR region, we analyzed these 20 amino acid sites, which are associated in vivo with resistance in HIV-1 subtype B to PIs approved by the U.S. Food and Drug Administration (FDA) (14, 23). This analysis revealed a lack of primary substitutions in 46 PR sequences. However, secondary mutations were found in 39 (85%) of the 46 viral sequences at six positions. L63P was the most common secondary substitution (54%; 25 of 46), followed by V77I (26%; 12 of 46), M36I (20%; 9 of 46), A71V/T (15%; 7 of 46), V82I (6%; 3 of 46), and L10I/V (4%; 2 of 46). Single amino acid mutations were identified in 23 (50%) of the 46 PR sequences, whereas 34% of sequences harbored dual (30%; 14 of 46), triple (2%; 1 of 46) and quadruple (2%) PI-associated substitutions (Table 1).
To search for changes over time in the PI-associated mutations among the subtype-B infections in Brazil, PI-associated substitutions found in 41 PR sequences collected in 1998 were compared with those found in 38 sequences collected from drug-naïve individuals in 1987 to 1994 (19, 20, 26) (Fig. 1). There were no primary PI-associated mutations in PR sequences in either collection, and PI secondary mutational patterns were similar in the two sets. However, the frequency of isolates harboring PI-associated secondary resistance mutations was higher (0.025 < P < 0.05 by a one-tailed χ2 test) in the 1998 data set (85%) than in the 1987-to-1994 collection (66%). This finding possibly reflects a natural evolution of the PR gene over time rather than selection of HIV-1 strains harboring PI-associated substitutions resulting from PI treatment in Brazil.
FIG. 1.
Percentages of PR subtype-B sequences with PI-associated secondary amino acid mutations at two different time points. The number of sequences with the indicated mutation is given inside or directly above each bar. Statistical significances of the differences (by a one-tailed Fisher or χ2 test) are given above the bars. Total∗, total numbers of sequences with at least one mutation.
The results presented in Table 1 indicated that none of the 47 RT sequences from the 1998 collection harbored primary mutations associated with resistance to FDA-approved NRTIs (14, 23) and only one had the secondary mutation V118I (2%; 1 of 47).
Analysis of FDA-approved NNRTI-associated mutations indicated that neither primary nor secondary substitutions (14, 23) were present in 47 RT sequences analyzed.
Our findings are in agreement with the low proportion of drug-naïve subjects infected with viruses harboring primary drug-resistant mutations in Venezuela and Argentina (8, 15) but in contrast with reports of the presence of HIV-1 strains carrying primary mutations in drug-naïve individuals in North America [1, 2, 5, 22, 31; Little et al., Antivir. Ther. 6(Suppl. 1):21, 2001] and Europe (4, 10, 24, 27, 32; Chaix et al., Program Abstr. 8th Conf. Retroviruses Opportunistic Infections, abstr. 755, 2001; Yerly et al., Program Abstr. 8th Conf. Retroviruses Opportunistic Infections, abstr. 754, 2001) who are infected with subtype-B viruses. A few factors may contribute to these differences, such as the length of time under drug selective pressures and the time period between primary infection and sampling.
In addition to specific mutations known to contribute to RTI resistance, a few unusual changes were observed in RT sequences at both the NRTI resistance-associated sites (E44G [6.4%], K70I [2.2%], V75L [2.2%], Q151K [2.2%], T215P/S [8.5%], and K219P/R/T [8.5%]) and the NNRTI resistance-associated positions (A98S [4.2%] and V106/I/L [13%]) (Table 1). Of note, recent data indicate that the T215S substitution, which was found in two of our RT sequences, is responsible for a more-rapid emergence of zidovudine (AZT) resistance in vitro, and it is speculated that this selection could also take place in vivo (J. G. Garcia-Lerma, S. Nidtha, K. Blumoff, H. Weinstock, and W. Heneine, Program Abstr. 5th Int. Workshop HIV Drug Resistance Treatment Strategies, abstr. 21 and 22, 2001).
Sixteen isolates that represented distinct phylogenetic subtypes and a variety of genotypic PI- and RTI-associated mutation patterns were selected for in vitro evaluation of susceptibility to the PIs (saquinavir [SQV], ritonavir [RTV], indinavir [IDV], nelfinavir [NFV], and amprenavir [APV]), NRTIs (AZT, lamivudine [3TC], didanosine [ddI], zalcitibine [ddC], stavudine [d4T], and abacavir [ABV]), and NNRTIs (nevirapine [NVP], delavirdine [DLV], and efavirenz [EFV]) by using VIRCO's Antivirogram assay (13). This phenotypic analysis revealed moderate resistance to NFV in only one strain (98BRVM15), which harbored quadruple PI-associated secondary mutations: 36I 63P 71V 77I. Three strains (98BRVM15, 98BRVM16, and 98BRVM85) revealed moderate resistance (4.0- to 6.5-fold) to ddI. Finally, strain 98BRVM58 showed levels of resistance above the biological cutoff value for NVP (8.0-fold) (Table 2). The levels of resistance above cutoff values for ddI and NVP observed in these samples could not be easily explained by the respective viral genotypes, which lacked primary resistance mutations. It is likely that mutations L283I, Y318F, and P236L (codons that were outside the RT fragment analyzed in this study), in combination with other, as yet unidentified NNRTI mutations, are responsible for moderate resistance. For example, the dual mutations I135T L283I were recently found to be associated with intermediate resistance to NVP and DLV (6). Our study showed that isolate 98BMVM58, which has shown resistance to NVP, had the I135T substitution. Also, it is possible that the presence of leucine instead of valine at codon 106 could contribute to the NVP resistance of this strain (Table 2). Observed discrepancies between genotype and phenotype, especially with regard to intermediate resistance to NNRTIs, have been reported previously (1, 17, 30, 31).
TABLE 2.
Phenotypic and genotypic profiles of selected samples
| Samplesa | Fold resistanceb to the following drug (susceptibility cutoffc):
|
PR genotyped | RT genotype | PR/RT subtypee | |||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| AZT (4.0) | 3TC (4.5) | ddI (3.5) | ddC (3.5) | d4T (3.0) | ABV (3.0) | NVP (8.0) | DLV (10.0) | EFV (6.0) | IDV (3.0) | RTV (3.5) | NFV (4.0) | SQV (2.5) | APV (2.5) | ||||
| 01 | 2.3 | 0.3 | 1.3 | 1.2 | 1.4 | 0.7 | 1 | 2.2 | 1.2 | 1 | 0.6 | 2.2 | 0.7 | 0.7 | WT | 135T | B |
| 06 | 1 | 0.3 | 0.7 | 0.8 | 0.5 | 0.4 | 1.1 | 2.9 | 0.8 | 0.7 | 0.3 | 0.5 | 0.4 | 0.6 | WT | 135T | B |
| 12 | 2.1 | 0.9 | 0.7 | 1.4 | 1.1 | 1.8 | 3.1 | 2 | 1.1 | 0.5 | 0.7 | 2.9 | 0.7 | 1.4 | 63P 77I | 106I 135T | B |
| 15 | 1.2 | 1.6 | 4.2 | 4 | 1.9 | 1.9 | 0.7 | 3.5 | 3.7 | 2.9 | 2.4 | 7.3 | 1.9 | 1.1 | 36I 63P 71V 77I | B | |
| 16 | 3.1 | 0.7 | 4 | 1.3 | 1.2 | 1.3 | 3.2 | 3.6 | 3.9 | 0.5 | 2 | 3.2 | 0.9 | 2.4 | 63P 71T | 135T | B |
| 52 | 1.1 | 0.6 | 1.1 | 0.8 | 0.7 | 0.7 | 1.8 | 2.5 | 0.8 | 0.7 | 0.8 | 1.1 | 0.9 | 0.6 | 10I 36I 63S | 135T | U/B |
| 56 | 1.7 | 0.7 | 1.3 | 1.1 | 0.5 | 0.8 | 4.3 | 2.9 | 1.3 | 0.6 | 0.7 | 1.2 | 0.7 | 0.6 | WT | 135T | F/U |
| 58 | 2.6 | 0.5 | 1 | 1 | 1.3 | 1.3 | 11.9 | 5.3 | 1.8 | 0.7 | 2.2 | 0.9 | 0.9 | 0.6 | 10V 36I | 106L 135T | U/B |
| 64 | 0.8 | 1.1 | 0.3 | 0.4 | 0.9 | 0.7 | 1.5 | 1.2 | 0.8 | 0.7 | 1.2 | 0.6 | 0.4 | 0.6 | 63H | B | |
| 75 | 1.2 | 0.6 | 1.8 | 2.6 | 0.8 | 1 | 3.3 | 6.6 | 0.6 | 1.2 | 3.3 | 2.6 | 1.2 | 1.5 | 63P 71T | B | |
| 79 | 0.7 | 0.4 | 1.2 | 1.1 | 0.9 | 0.8 | 0.6 | 1.3 | 2 | 1.7 | 1.1 | 1 | 1.1 | 0.5 | 63P 71T 77I | 135T | B |
| 84 | 1.9 | 0.7 | 1.1 | 1.2 | 3.1 | 2.1 | 3.3 | 3.9 | 2.1 | 1.2 | 2.8 | 2.8 | 0.4 | 1.2 | 63P | B | |
| 85 | 1.6 | 1.4 | 6.5 | 2.4 | 2.2 | 1.5 | 1.8 | 4.3 | 3.9 | 1.2 | 3.5 | 2.6 | 0.7 | 0.9 | 36I | 135T | B |
| 91 | 1.6 | 0.7 | 1.2 | 0.8 | 0.6 | 0.4 | 0.5 | 2.1 | 0.6 | 0.9 | 1.4 | 3.9 | 0.9 | 0.6 | 63P 71T | 135T | B |
| 94 | 1.8 | 0.7 | 1 | 0.7 | 1.2 | 1.3 | 4.4 | 9.3 | 2.9 | 0.8 | 0.9 | 0.7 | 0.5 | 0.7 | 36I | C | |
| 107 | 2 | 0.6 | 1 | 0.7 | 0.5 | 0.6 | 3.5 | 2.9 | 1.2 | 0.6 | 1.5 | 0.9 | 0.5 | 0.6 | 63P | 135T | B |
Each sample number represents a “98BRVM” strain; for example, the first sample listed is strain 98BRVM01. Boldfaced samples either are non-B subtypes or recombinants or have at least one unclassifiable region.
Relative to the wild-type control (HXB2 micromolar IC50). Values above the biological cutoff for the drug are boldfaced.
Specific biological cutoff for normal susceptible range (see reference 12).
WT, wild type. Boldfaced genotypes represent secondary mutations observed with U.S. FDA-approved ARV agents.
Non-B subtypes or different subtype assignments for PR and RT are boldfaced. U, unclassifiable.
This study provides the most comprehensive evaluation to date of both the genetic diversity and the PR and RT drug-resistant patterns in treatment-naïve HIV-1-infected individuals after the introduction of free access to HAART in Brazil in 1996. Our findings showing a lack of resistant strains circulating in drug-naïve subjects do not support the need for genotyping before ARV therapy initiation.
Nucleotide sequence accession numbers.
The nucleotide sequences of the HIV-1 PR and RT genes sequenced in this study have been assigned GenBank accession numbers AF413811 to AF413912.
Acknowledgments
This work was supported by CAPES, CNPq, FAPERJ, and Fogarty International Center/AIDS International Training and Research Programs.
We express our gratitude to Warren D. Johnson, Jr., for encouragement and support.
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