Objectives. To determine the prevalence of drug resistance and to analyze the subtyping in HIV-1 samples from Cuba.
|HIV, AIDS, antiretroviral drugs, drug resistance, Cuba.|
Since 1996, antiretroviral treatment has changed the natural history of HIV type 1 (HIV-1) infection. An increased use of these drugs and the introduction of more potent new mole- cules has improved the prognosis for HIV-infected patients and has reduced the mortality rate associated with AIDS.3
In combination with the fact that HIV-1 cannot be eradicated, the problems that are associated with adherence to treatment can result in therapeutic failures that are frequently associated with the selection of viral resistance mutations that are naturally present even in isolates from patients with no prior exposure to the drugs used to treat HIV infection (1-4). Viral recombination can also be an important factor in the propagation of resistance (5-6).
Genotyping methodologies to monitor HIV-1 resistance mutations have been used to study resistant HIV-1 variants in various populations, and this approach's usefulness has even been demonstrated for evaluating mixtures of mutant viruses and wild type viruses (viruses with no primary resistance mutations), according to both published reports (7-9) and unpublished ones.4
Most of the antiretroviral resistance studies that have been done so far have been carried out in developed countries, where HIV subtype B is predominant. In Cuba one study (10) has reported that there is a predominance of the B subtype, but more recent research has shown that there is a mosaic of subtypes and recombinant viruses (Cuevas MT, et al. High HIV-1 genetic diversity in Cuba [unpublished manuscript]. 2001). A recent survey of subtypes in Cuba identified the presence of three non-B subtype viruses: A, H, and C (11).
In other countries, studies on genetic resistance have also been carried out on isolates of non-B subtypes, and the implications of this information for vaccine development have been assessed (12, 13).
Because no studies concerning the prevalence of HIV-1 resistance to antiretrovirals had been done in Cuba, we decided to investigate that issue as well as to look at the subtyping of the Cuban samples. In addition, we explored the importance of both of these types of information for public health on the island.
MATERIAL AND METHODS
This research in Cuba was part of a pilot study of the WHO-UNAIDS (World Health Organization-Joint United Nations Programme on HIV/ AIDS) to monitor HIV resistance to antiretroviral drugs in Latin America. The UNAIDS study was carried out in Argentina, Brazil, Cuba, and Venezuela, with the Department of Viral Pathogenesis of the National Center of Fundamental Biology of the Carlos III Institute of Health, of Madrid, Spain, serving as the coordinating center for the study.
In this survey we analyzed Cuban samples, which were collected in 1999. Written informed consent was obtained from the subjects, and a standardized questionnaire was used to obtain demographic, clinical, and risk-factor data on each of those persons.
Cuba has the lowest HIV prevalence in the Americas (0.03%), with an estimated number of infections of 1 950 at the end of 1999 in a total population of 11.2 million persons (14). For our study we selected a sample population of 103 patients. We placed each of the 103 into one of four categories that were related to the antiretroviral regimen that each patient had been receiving at the beginning of his or her treatment.
The first category consisted of patients who had never received any antiretroviral drug. There were 27 persons in this "untreated patients" category, or 26.2% of the 103 subjects in the study.
The second category consisted of 29 patients (28.2% of the 103) who had started their treatment with zidovudine (ZDV). In 3 of these 29 patients, the monotherapy treatment was later changed to bitherapy (two-drug therapy), using zidovudine plus another reverse transcriptase inhibitor (RTI). Nine of these 29 patients were not receiving any antiretroviral drug when the sample was obtained.
The third category consisted of 28 patients (27.2% of the 103) who had begun treatment with bitherapy. In 24 of these 28 persons the treatment was ZDV plus another RTI. Two of these 24, after one month of bitherapy, had a protease inhibitor (PI) added to their treatment. Three of these 24 patients were not receiving any antiretroviral drug when the sample was obtained. The remaining 4 patients undergoing bitherapy had been taking one RTI plus one PI for 1 to 3 years. In 2 of these 4 cases, after 1 year of treatment, a second RTI was added. All of these 4 patients were undergoing treatment when the samples were collected.
Finally, the fourth category consisted of 19 individuals (18.4% of the 103) who had started their treatment with triple therapy. Of these 19, 17 of them were receiving two RTIs plus one PI, and 1 of them was receiving one RTI plus two PIs. The last of these 19 patients had received triple therapy for only 6 months during 1998 and was not receiving any antiretroviral drug when the samples were collected.
Out of the 103 patients in our study, 42 of them (40.8%) were classified in the "C" AIDS clinical stage, according to the classification system of the Centers for Disease Control and Prevention of the United States of America (15). Sexual contact had been the predominant route of infection for the 103 study patients, with it being heterosexual contact for 42 of the patients (40.8%) and homosexual contact for 60 of them (58.3%). There was 1 case of perinatal transmission.
For the 103 patients, the mean viral load was 80 000 copies/mL (range, < 200 to 1 800 000), and the mean CD4+ count was 356 cells/mL (range, 10 to 1 284). We performed DNA extraction from 106 peripheral blood mononuclear cells using the Casas method (16). Amplification and direct sequencing of pol gene coding for reverse transcriptase and protease were performed using the ABI PRISM® Dye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, California, United States) with AmpliTaq® DNA polymerase FS (Roche Molecular Systems, Alameda, California, United States). The HIV-1 RNA plasma viral load was measured using Nucleic Acid Sequence Based Amplification (NASBA) technology (Organon Teknika, Boxtel, Netherlands). Genotyping of the envelope was performed by heteroduplex mobility assay (HMA), using an HMA subtyping kit (National Institutes of Health, AIDS Research and Reference Reagent Program, Bethesda, Maryland, United States). In order to corroborate the HMA results, sequencing of env (C2-V3-C3) was done in one-third of each subtype that had been detected by HMA.
The resistance mutation positions were defined following criteria previously established (4).
An analysis comparing the proportions of B and non-B viruses harboring the various mutations was carried out using the RSIGMA BABEL statistical software package (Horus Hardware, Madrid, Spain).
Resistance mutations to RTIs were detected in 25 of the 76 treated patients and in 2 of the 27 untreated patients. (The absence of primary resistance mutations was defined as "wild type.") The data on the resistance mutations we found are shown in Table 1.
Resistance to nucleoside reverse transcriptase inhibitors (NRTIs) was found in 21 of the 76 treated patients and in 2 of the 27 untreated patients (data not shown). The most common of these mutations, among treated patients, was T215Y/F, which was found in 12 isolates. This was followed by K70R in 9 isolates, M184V in 6, and T69D in 4. All these 4 persons had been receiving ZDV when they were enrolled in the study. T215Y/F mutation was also observed in 1 of the untreated patients, and T69D in another one.
We did not find insertion of two amino acids following RT codon 69, nor did we find the Q151M multidrug resistance mutation to RTIs (data not shown). Resistance mutations associated with nonnucleoside RTIs (NNRTIs) were detected in 4 of the 76 treated patients (5.3%) but not in any of the 27 untreated patients (data not shown). The K103N resistance mutation was found in 1 patient who was receiving delavirdine; Y181C in 2 patients, treated with nevirapine (NVP) plus ZDV in 1 case and with only ZDV in the other case; and Y188C in 1 case without NNRTI treatment but with ZDV. The predominant secondary mutations and polymorphisms among the 76 treated patients were: L214F in 35 cases (46% of the 76 treated patients), R211K in 10 of them (13.2%), M41L in 9 (11.8%), D67N in 8 (10.5%), and S68G in 7 of them (9.2%). The respective frequencies of these mutations among the 27 untreated patients were: 33.3%, 7.4%, 3.7%, 3.7%, and 14.8%.
A low prevalence of resistance mutations to PIs was also found: 4 of the 76 treated patients, with L90M in 3 cases, and G48V in 1 other. These 4 patients were all receiving saquinavir (SQV), and all of them had resistance mutations to RTIs.
Secondary resistance mutations to PIs found were: L63P in 55 cases, M36I in 48, V77I in 20, L10V/I in 9, K20R in 8, and A71V/T in 6 samples.
There were 29 treated patients with resistance mutations to RTIs (Table 2). In these 29, the plasma HIV-1 RNA viral load was higher than 1 000 copies/mL in 21 of them, between 200 and 1 000 copies in 1 of them, and undetectable (< 200 copies/mL) in 7 of them. There were also 47 treated patients in whom a wild-type genotype in the reverse transcriptase (RT) sequence was detected; in 10 of those 47 persons, the viral load was higher than 105 copies/mL.
When we performed the genotyping of the envelope by HMA, out of the 103 samples, 81 of them (78.6%) were classified as subtype B, 19 (18.5%) as subtype A, and 3 (2.9%) as subtype C. The sequencing of env that was done in one-third of the samples of each subtype¾27 B, 6 A, and 1 C¾found results that were the same as those with HMA.
In the group of 19 isolates classified by HMA as subtype A, resistance mutations to both RTIs and PIs were detected in 2 of the treated patients, and to RTIs alone in 3 of the treated patients. In this group of patients infected with subtype A viruses, T215Y/F mutation to ZDV was observed in 3 samples and M184V in another 3 cases, 1 of them in association with T215Y/F mutation.
Resistance mutations were not detected in any of the untreated patients infected with subtype A viruses.
This is the first survey providing the prevalence of HIV-1 resistance mutations together with genotyping in Cuba. The low frequency of primary resistance mutations to RTIs and/or PIs that we found among HIV-1-infected patients in Cuba reflects the delay in introducing this antiretroviral therapy to the island.
Overall, the prevalence of RTI and PI resistance mutations are close to the values found in other Latin American countries studied in our laboratory under this UNAIDS Program.5 This is the case of Venezuela (17) and of Argentina,6 indicating that these drugs have been recently introduced in these two countries. The remaining country included in the UNAIDS study, Brazil, shows higher resistance prevalence values, probably due to an earlier and more extended use of the drugs.
All the 13 patients in whom a T215Y/F mutation was found had been treated with ZDV in monotherapy or dual therapy, the predominant therapeutic schemes in Cuba. This T215Y/F mutation has been reported as indicating a poor patient prognosis (18).
The presence of an M184V mutation, which is related to resistance to 3TC and ABC, was found in 6 of the 76 treated patients, and was not found among the untreated patients. This is indicative of the limited use of these drugs in Cuba.
Various studies carried out in developed countries (19-21) have indicated that there is still a low prevalence of HIV-1 strains with multidrug resistance (MDR) to NRTIs. In this study in Cuba, while MDR to RTIs was not detected, it will be important to maintain the surveillance over MDR emergence.
The NNRTIs have had limited use in the HIV-1-infected population in Cuba, which is reflected in the low frequency (5.3%) of resistance mutations to these drugs, as compared to the 17.2% that we found in a study that we conducted in Spain (21). In this Cuba study there were two cases with Y181C and Y188C mutations associated with the use of NNRTIs, corresponding to two patients who had only received ZDV monotherapy, and thus could be considered resistant strains that had been transmitted. The absence of resistance mutations to ZDV in these two patients with prolonged ZDV monotherapy treatment could be due to the suppression of the T215Y mutation by the presence of Y181C, as previously described (22).
Resistance mutations to RTIs were found in 2 of the 27 untreated patients, or 7.4% of them. This percentage is noticeably lower than the 22% to 29% that has been found in studies with untreated patients in some other countries (23-25).
Resistance mutations to PIs were only found in 4 of the 76 treated patients (5.3%); in all those cases it was in association with the use of the corresponding inhibitor. This frequency is still low for these drugs in comparison to that in other studies from some other countries. For example, in our study in Spain (21) we found an overall level of 25.1%, similar to results from other developed countries, according to at least one published study (26) and one unpublished one.4 Nevertheless, the 5.3% level in Cuba is in line with other countries where this type of treatment has been recently introduced, such as Côte d'Ivoire, where a prevalence of 6% was recently reported (27). That same Côte d'Ivoire study reported that resistance among patients infected with non-B subtypes is conferred by mutations similar to those documented for subtype B infections. We also saw the same profile for primary resistance mutations in subtypes A, B, and C in Cuba (Table 1).
With regard to the viral load that we saw in Cuba, it is interesting to note the high percentage of cases presenting undetectable levels of RNA in plasma associated with the presence of resistance mutations. This may indicate that the samples were taken before the viral load had increased. This also suggests the usefulness of performing resistance studies in the follow-up with treated patients, regardless of their low viral load. There is a simple new procedure for nucleic acid extraction and amplification that permits sequencing studies of resistance mutations in patients with an undetectable viral load, thus facilitating treatment decisions (28).
Also in this Cuban study, we found a group of 10 patients in whom the viral load was higher than 105 copies/mL but who did not have mutations associated with resistance (Table 2). This may be indicative of a lack of adherence to treatment during the early treatment phase.
An R211K/L214F natural polymorphism in the RT gene was found in 21 of the 76 treated patients and in 9 of the 27 untreated patients. In addition, a polymorphism in the protease gene at positions 63 was found in 55 out of the total of 103 patients and at position 36 in 48 of the 103.
In this paper we have reported on the first study of the prevalence of antiretroviral drug-associated resistance mutations in Cuba. It is important to note the absence of MDR to NRTIs, and the low prevalence of resistance to RTIs and to PIs, thus allowing the continued use of these drugs for the HIV-1-infected population in Cuba. Our findings may also provide valuable epidemiological information to consider for vaccine design.
The detection of several non-B subtype viruses circulating in Cuba supports the relevance of phylogenetic surveillance studies, given the possibility of spread of these genetic forms and the potential emergence of recombinant viruses.7
Acknowledgements. We would like to express our gratitude to the WHO-UNAIDS Vaccine Initiative for encouragement and financial support (HQ/98/457048 and HQ/98/440913) and to Francisco Parras of the Plan Nacional del SIDA of Spain for his help (Grant VI1236 2). We also thank the following three persons for their help with facilities and with the preparation and review of this manuscript: Saladin Osmanov (WHO-UNAIDS Vaccine Initiative, World Health Organization, Geneva, Switzerland), Francisco Machado-Ramírez (AIDS Research Laboratory, Microbiology Department, Havana, Cuba), and Rafael Nájera (Instituto de Salud Carlos III, Centro Nacional de Biología Fundamental, Área de Patogenia Viral, Madrid, España).
1. Dornadula G, Zhang H, VanUitert B, Stern J, Livornese L, Ingerman MJ, et al. Residual HIV-1 RNA in blood plasma of patients taking suppressive highly active antiretroviral therapy. JAMA 1999;282(17):1627-1632.
2. Nájera I, Richman DD, Olivares I, Rojas JM, Peinado MA, Perucho M. Natural occurrence of drug resistance mutations in the reverse transcriptase of human immunodeficiency virus type 1 isolates. AIDS Res Hum Retroviruses 1994;10:1479-1488.
3. Nájera I, Holguín A, Quiñones-Mateu ME, Muñoz-Fernandez MA, Nájera R, López-Galindez C, et al. pol gene quasispecies of human immunodeficiency virus: mutations associated with drug resistance in virus from patients undergoing no drug therapy. J Virol 1995;69:23-31
4. Hirsch MS, Conway B, D'Aquila RT, Johnson VA, Brun-Vézinet F, Clotet B, et al. Antiretroviral drug resistance testing in adults with HIV infection: implications for clinical management. JAMA 1998;279(24):1984-1991.
5. Richman D. Antiviral drug resistance: issues and challenges. In: Richman DD, ed. Antiviral drug resistance. Chichester and New York: Wiley; 1996. pp. 1-9.
6. Moutouh L, Corbiel J, Richman D. Recombination leads to the rapid emergence of HIV-1 dually resistant mutants under selective drug pressure. Proc Natl Acad Sci USA 1996;93: 6106-6111.
7. Durant J, Clevenbergh P, Halfon P, Delgiudice P, Porsin S, Simonet P, et al. Drug-resistance genotyping in HIV-1 therapy: the VIRADAPT randomised controlled trial. Lancet 1999;353(9171):2195-2199.
8. Rubio A, Leal M, Pineda JA, Caruz A, Luque F, Rey C, et al. Increase in the frequency of mutation at codon 215 associated with zidovudine resistance in HIV-1-infected antiviral-naive patients from 1989 to 1996. AIDS 1997;11(9):1184-1186.
9. Villahermosa ML, Contreras G, Pérez-Alvarez L, Bru F, Medrano L, Delgado E, et al. Evaluation of mixtures of wild-type HIV-1 and HIV-1 with resistance point mutations against reverse transcriptase inhibitors. Antiviral Therapy 1998,3(4):221-227.
10. Rolo FM, Miranda L, Wainberg MA, Gu Z, Lobaina L, Noa E, et al. Envelope V3 region sequences of Cuba HIV-1 isolates. J Acquir Immune Defic Syndr Hum Retrovirol 1995; 9(2):123-125.
11. Gómez CE, Iglesias E, Perdomo W, Rolo F, Blanco M, Lobaina L, et al. Isolates from four different HIV-1 type 1 clades circulating in Cuba identified by DNA sequence of the C2-V3 region. AIDS Res Hum Retroviruses 2001;17(1):55-58.
12. Cornelissen M, van den Burg R, Zorgdrager F, Lukashov V, Goudsmit J. pol gene diversity of five human immunodeficiency virus type 1 subtypes: evidence for naturally occurring mutations that contribute to drug resistance, limited recombination patterns, and common ancestry for subtypes B and D. J Virol 1997; 71(9):6348-6358.
13. Palmer S, Alaeus A, Albert J, Cox S. Drug susceptibility of subtype A, B, C, D and E HIV-1 primary isolates. AIDS Res Hum Retroviruses 1998;14(2):157-162.
14. Joint United Nations Programme on HIV/AIDS, Pan American Health Organization, World Health Organization. Epidemiological fact sheet on HIV/AIDS and sexually transmitted infections. Cuba. 2000 update. Geneva. Switzerland: UNAIDS; 2000.
15. United States of America, Centers for Disease Control and Prevention. 1993 revised classification system for HIV infection and expanded surveillance case definition for AIDS among adolescents and adults. MMWR 1992;41(RR-17):1-19.
16. Casas I, Powell L, Klapper PE, Cleator GM. New method for the extraction of RNA and DNA from cerebrospinal fluid for use in the polymerase chain reaction assay. J Virol Methods 1995;53(1):25-36.
17. Delgado E, León-Ponce M, Villahermosa ML, Cuevas MT, Deibis L, Echeverria G, et al. Analysis of HIV type 1 protease and reverse transcriptase sequences from Venezuela for drug resistance associated mutations and subtype classification: a UNAIDS study. AIDS Res Hum Retroviruses 2001;17:753-758.
18. Wainberg MA, Gu Z, Gao Q, Arts E, Geleziunas R, Bour S, et al. Clinical correlates and molecular basis of HIV drug resistance. J Acquir Immune Defic Syndr 1993;6(Supp 1): S36-S46.
19. Loveday C, Devereux H, Huckett L, Johnson M. High prevalence of MDR mutation in a UK HIV/AIDS patient population. AIDS 1999;13 (5):627-628.
20. Bass E. Gods and monsters: are we breeding a superbug? HIV Plus Issue 7, February/March 2000. Available from: http://ww.aegis.com [Internet site]. Accessed 28 September 2000.
21. Pérez-Alvarez L, Villahermosa ML, Cuevas MT, Delgado E, Manjon N, Vazquez de Parga E, et al. Single and multidrug resistance mutations to reverse transcriptase and protease inhibitors: human immunodeficiency virus type 1-infected patients from two geographical areas in Spain. J Hum Virol 2000;3(3):150-156.
22. Larder B. 3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors. Antimicrob Agents Chemother 1992;36(12):2664-2669.
23. Van Vaerenbergh K, Debaisieux L, De Cabooter N, Declercq C, Desmet K, Fransen K, et al. Prevalence of genotyping resistance among antiretroviral drug-naive HIV-1 infected patients in Belgium. Antivir Ther 2001; 6(1):63-70.
24. UK Collaborative Group on Monitoring the Transmission of HIV Drug Resistance. Analysis of prevalence of HIV-1 drug resistance in primary infections in the United Kingdom. BMJ 2001;322(7294):1087-1088.
25. Alexander CS, Dong W, Chan K, Jahnke N, O'Shaughnessy MV, Mo T, et al. HIV protease and reverse transcriptase variation and therapy outcome in antiretroviral-naive individuals from a large North American cohort. AIDS 2001;15(5):601-607.
26. Quiros-Roldan E, Signorini S, Castelli F, Torti C, Patroni A, Airoldi M, et al. Analysis of HIV-1 mutation patterns in patients failing antiretroviral therapy. J Clin Lab Anal 2001; 15(1):43-46.
27. Adje C, Cheingsong R, Roels TH, Maurice C, Djomand G, Verbiest W, et al. High prevalence of genotypic and phenotypic HIV-1 drug-resistant strains among patients receiving antiretroviral therapy in Abidjan, Cote d'Ivoire. J Acquir Immune Defic Syndr 2001; 26(5):501-506.
28. Villahermosa ML, Thomson M, Vázquez de Parga E, Cuevas MT, Contreras G, Pérez-Álvarez L, et al. Improved conditions for extraction and amplification of human immunodeficiency virus type 1 from plasma samples with low viral load. J Hum Virol 2000,3(1):27-34.
Manuscript received 7 December 2000. Revised version accepted for publication 3 August 2001.
Mutaciones de resistencia genotípica a los fármacos antirretrovíricos en los subtipos B y no B del VIH-1 en Cuba
Objetivos. Determinar la prevalencia de la resistencia a los fármacos y analizar la presencia de mutaciones de resistencia genotípica en los subtipos B y no B del VIH-1 en Cuba.
▲ AIDS Research Laboratory, Microbiology Department, Havana, Cuba.
▲ Instituto de Salud Carlos III, Centro Nacional de Biología Fundamental, Área de Patogenia Viral, Madrid, España. Send correspondence to: Lucía Pérez-Alvarez, Área de Patogenia Viral, Centro Nacional de Biología Fundamental, Instituto de Salud Carlos III, 28220 Majadahonda, Madrid, España; telephone: 34-91-509-7937; fax: 34-91-509-7914; e-mail: email@example.com
3 Palalla F, Moorman A, Chmiel J, Chan C, Holmberg S. Continued low morbidity and mortality among patients with advanced HIV infection and their patterns of HAART usage [conference presentation]. 7th Conference on Retroviruses and Opportunistic Infections, San Francisco, California, United States of America, 30 January-2 February 2000.
4 Little S, Daar E, Keiser P, D'Aquila R, Connick E, Hellmann C, et al. The spectrum and frequency of reduced antiretroviral drug susceptibility with primary HIV infection in the United State [conference presentation]. 7th Conference on Retroviruses and Opportunistic Infections, San Francisco, California, United States of America, 30 January-2 February 2000.
5 Contreras G, Cuevas MT, Manjón N, Vázquez de Parga E, Delgado E, Villahermosa ML, et al. WHO-UNAIDS HIV-1 antiretroviral resistance studies [conference presentation]. 4th International Workshop on Drug Resistance and Treatment Strategies, Sitges, Spain, 12-16 June 2000.
6 Vázquez de Parga E, Ávila M, Cuevas MT, Salomon H, Thomson MM, Osmanov S, et al. Sequencing analysis of HIV-1 reverse transcriptase and protease genes in a cohort of patients from Argentina. The Fifth European Conference on Experimental AIDS Research, Madrid, Spain, 16-19 June 2000.
7 Cuevas MT, Thomson MM, Villahermosa ML, Ruibal-Brunet IJ, Díaz-Torres H, Vázquez de Parga E, et al. High proportion of non-B subtype and intersubtype recombinant viruses among HIV-1 individuals in Cuba [conference presentation]. 101st General Meeting of the American Society for Microbiology, Orlando, Florida, 20-24 May 2001.