The HIV epidemic is increasing in Equatorial Guinea (GQ), West Central Africa, but few studies have reported its HIV molecular epidemiology. We aimed to describe the HIV-1 group M (HIV-1M) variants and drug-resistance mutations in GQ using sequences sampled in this country and in Spain, a frequent destination of Equatoguinean migrants.
We collected 195 HIV-1M pol sequences from Equatoguinean subjects attending Spanish clinics during 1997-2011, and 83 additional sequences sampled in GQ in 1997 and 2008 from GenBank. All (n = 278) were re-classified using phylogeny and tested for drug-resistance mutations. To evaluate the origin of CRF02_AG in GQ, we analyzed 2,562 CRF02_AG sequences and applied Bayesian MCMC inference (BEAST program).
Most Equatoguinean patients recruited in Spain were women (61.1%) or heterosexuals (87.7%). In the 278 sequences, the variants found were CRF02_AG (47.8%), A (13.7%), B (7.2%), C (5.8%), G (5.4%) and others (20.1%). We found 6 CRF02_AG clusters emerged from 1983.9 to 2002.5 with origin in GQ (5.5 sequences/cluster). Transmitted drug-resistance (TDR) rate among naïve patients attended in Spain (n = 144) was 4.7%: 3.4% for PI (all with M46IL), 1.8% for NRTI (all with M184V) and 0.9% for NNRTI (Y188L). Among pre-treated patients, 9/31 (29%) presented any resistance, mainly affecting NNRTI (27.8%).
We report a low (<5%) TDR rate among naïve, with PI as the most affected class. Pre-treated patients also showed a low drug-resistance prevalence (29%) maybe related to the insufficient treatment coverage in GQ. CRF02_AG was the prevalent HIV-1M variant and entered GQ through independent introductions at least since the early 1980s.
Citation: Yebra G, de Mulder M, Holguín Á (2013) Description of HIV-1 Group M Molecular Epidemiology and Drug Resistance Prevalence in Equatorial Guinea from Migrants in Spain. PLoS ONE 8(5): e64293. https://doi.org/10.1371/journal.pone.0064293
Editor: William A. Paxton, University of Amsterdam, The Netherlands
Received: February 7, 2013; Accepted: April 10, 2013; Published: May 22, 2013
Copyright: © 2013 Yebra et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS 09/00284). AH is supported by FIS and Agencia Laín Entralgo. This study is included in the Subprograma de Inmigración y Salud, CIBERESP (Spain). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The human immunodeficiency virus type 1 group M (HIV-1M), responsible for the HIV pandemic, originated from a zoonotic transmission from chimpanzees into humans in Cameroon . For decades, the virus evolved and diversified into different lineages in the Congo River basin, where the highest viral diversity circulates . Today, HIV-1M is sub-divided into 9 pure subtypes (A-D, F-H, J and K) and recombinants between them (http://www.hiv.lanl.gov/content/sequence/HIV/CRFs/CRFs.html): 58 circulating recombinant forms (CRFs) and multiple unique recombinant forms (URFs).
Equatorial Guinea (GQ), a Spanish colony until 1968, is a small country located in West Central Africa between Cameroon and Gabon. The estimated number of people living with HIV in GQ (20,000 in 2009) increases annually, and antiretroviral treatment (ART) only reached 48% of them in 2011 . Despite its location, close to the epicenter of HIV-1 pandemic, only two local studies ,  have so far reported the HIV molecular epidemiology in GQ. Ortiz et al. included 76 HIV-1M env gene (gp41) sequences obtained in 1999 from general HIV-infected population. Djoko et al. evaluated 41 HIV-1M pol sequences sampled in 2008 from military personnel in Malabo −the capital city. Both studies revealed CRF02_AG as the prevalent HIV-1M variant, as happens in the neighboring countries .
The latter also reported a rate of transmitted antiretroviral drug resistance (TDR) of 4.2%. This rate represents the percentage of naïve patients infected with HIV variants carrying drug resistance mutations, and threatens the effectiveness of ART. Recent studies report an increasing TDR prevalence in low- and middle-income countries following ART scale-up . This rise especially affects sub-Saharan Africa, driven by the resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) , . The TDR rate in Cameroon is around 8–9% ,  and lower than 5% in Gabon . HIV molecular epidemiology studies are crucial in these areas to prevent the widespread transmission of drug resistance, especially those lacking systematic surveillance as GQ.
We aimed to describe the circulating HIV-1M variants and the presence of antiretroviral drug resistance mutations in Equatorial Guinea. To achieve this, we applied phylogenetic methods to a combination of viral sequences sampled in Spain (frequent destination of Equatoguinean migration) and publicly available sequences sampled in the sub-Saharan country.
Materials and Methods
All the HIV-infected subjects coming from Equatorial Guinea with available HIV-1M pol sequences, and attended in Spanish HIV/AIDS clinics (mainly in Madrid) were collected. They were 195 adult and pediatric patients sampled between 1997 and 2011. Most sequences were previously reported –, while 37 were unpublished. In most cases (136, 69.7%) they included the complete protease (PR, 297nt) and partial reverse transcriptase (RT, 860nt of mean length). In 58 (29.7%) and 1 (0.5%) cases, only the PR or the RT sequence was available, respectively. In addition, all the HIV-1 pol sequences (n = 83) sampled in Equatorial Guinea and available in the GenBank (www.ncbi.nlm.nih.gov/genbank) were retrieved. They belonged to two sequences batches sampled in 1997 (not published) and 2008 . GenBank accession numbers of the sequences included are provided in Accession S1.
HIV-1M variants were re-classified by phylogenetic analysis of the 278 pol sequences, including recombinants not available at publishing time. Representative sequences of the HIV-1M 9 subtypes and 49 CRFs downloaded from Los Alamos HIV sequence database (LANL-DB, http://www.hiv.lanl.gov) were used as references. Sequences were aligned manually, and initial neighbor-joining trees were built using MEGA5 (http://www.megasoftware.net) under the Kimura two-parameter model with 1,000 bootstrap re-sampling. Subtypes were further confirmed in approximately maximum-likelihood (ML) phylogenetic trees constructed under the general time reversible model of nucleotide substitutions with gamma-distributed heterogeneity rate (GTR+Γ) using FastTree v2.1.3 (http://www.microbesonline.org/fasttree). The topology robustness was tested by likelihood-based local branch support. In sequences not ascribed to any known subtype or CRF, recombination analyses were performed using SimPlot v3.5.1 and Recombination Detection Program (RDP, v3alpha44).
In the CRF02_AG subset, we searched monophyletic clusters (associations of ≥3 samples) that involved only sequences from GQ in ML trees that included all CRF02_AG sequences from LANL-DB (n = 2,652) constructed as described above with FastTree v2.1.3.
The time to the most recent common ancestor (TMRCA) of these Equatoguinean CRF02_AG clusters was estimated using Bayesian Markov chain Monte Carlo (MCMC) inference with the program BEAST v1.6.1 (http://beast.bio.ed.ac.uk). A lognormal distributed prior was placed on the estimated number of nucleotide substitution per site per year (mean = 2.5 × 10−3; standard deviation = 0.5). The MCMC chain ran for 108 generations, sampling estimates every 10,000th generation. The uncorrelated lognormal molecular clock model and the SRD06 nucleotide substitution were selected. These parameters were chosen based on previous estimates for CRF02_AG datasets . The tree samples were used to generate a maximum clade credibility (MCC) tree, after a 10% burn-in, using TreeAnnotator v1.6.1. Those Equatoguinean CRF02_AG clusters with a high clade support in both ML and Bayesian trees (≥95%) were chosen as definitive.
For HIV sequences from antiretroviral-naïve patients, the TDR prevalence was defined following the World Health Organization mutation list  using the Calibrated Population Resistance tool (http://cpr.stanford.edu). For antiretroviral-exposed patients, substitutions considered in the 2011 International AIDS Society-USA (IAS-USA) mutation list  were manually identified.
This study was part of a project approved by the review board of the Hospital Ramón y Cajal Clinical Research Ethical Committee. It was designed to protect the rights of all subjects involved under the appropriate local regulations. To maintain subject confidentiality, a unique ID number was assigned to each specimen, and written consent obtained for each patient by clinicians. For the minors involved in the study, written consent was provided by their parents or legal representatives.
Equatoguinean HIV-infected Patients in Spain
The main characteristics of these patients are shown in Table 1. Most subjects (61.1%) of this cohort were women, and were infected with HIV due to heterosexual risk practices, in concordance with the general situation in sub-Saharan Africa . Sorting patients according to the sample periods shown in Table 1, we observed a significant increase in the proportion of men: 13.6% in 1997–2001, 44.6% in 2002–2006 and 53.5% in 2007–2011 (P = 0.0002). We found no apparent trends in the remaining analyzed features across time. The epidemiological information was not available in the 83 sequences retrieved from GenBank and sampled in Equatorial Guinea.
HIV-1M Variants Circulating in Equatorial Guinea
Table 2 shows the distribution of HIV-1M variants among the 278 pol sequences from Equatoguinean patients, where the recombinant CRF02_AG (47.8%) was predominant. Pure subtypes infected 122 (43.9%) patients, and other CRFs were found in 19 (6.8%) cases. Four sequences were classified as URF (URF_JU, URF_BH, URF_02D and URF_BG) using RDP and SimPlot. Of note, the frequency of subtype B was significantly higher in Equatoguinean subjects sampled in Spain than in those sampled in EQ (9.7% vs. 1.2%, P = 0.012).
The new analysis of the sequences permitted to update their classification, especially among recombinants. We re-classified as CRF02_AG 21 sequences initially assigned to subtype G when they were sampled , , 1 CRF11_cpx reported as subtype J , 1 CRF13_cpx and 1 CRF18_cpx also reported as subtype G  and 1 CRF22_01A1 described as subtype A . The recombination analyses revealed that 2 sequences isolated in 1999 and 2000 and reported as pure subtypes G and J  were URF_BG and URF_JU, respectively.
CRF02_AG Cluster Analysis
In the general ML tree that included the 133 CRF02_AG sequences of the study population and 2,652 worldwide CRF02_AG sequences (Figure 1), we found 7 monophyletic clusters including exclusively Equatoguinean CRF02_AG sequences with >95% of statistical support. The geographic origin of the 2,652 CRF02_AG sequences from LANL-DB is described in the legend of Figure 1. The further analysis with BEAST included a 92 sequence alignment: the CRF02_AG involved in the putative Equatoguinean clusters (n = 39), their closest relatives among the CRF02_AG from LANL-DB according to the ML tree (n = 15), and reference CRF02_AG sequences with diverse sampling dates (n = 38) in order to include at least one sequence per year from 1990 when available. The sampling times of the sequence dataset included in the BEAST analysis are shown in the supplementary Table S1.
Tree constructed under the GTR+Γ model of nucleotide substitution using FastTree v2.1.3 (see Methods) from an 1130nt alignment. Branch colors indicate the origin of the sequences (with the same color code in the map). In blue, sequences from Equatoguinean patients (n = 133). In orange, CRF02_AG sequences from Spain (n = 158). In green, CRF02_AG sequences from Western Africa: Nigeria (n = 391), Mali (n = 235), Senegal (n = 185), Ghana (n = 180), Burkina Faso (n = 97), Benin (n = 77), Togo (n = 58), Liberia (n = 4), the Gambia (n = 2) and Guinea-Bissau (n = 2). In red, CRF02_AG sequences from Central Africa: Cameroon (n = 711), Gabon (n = 72), and Democratic Republic of the Congo (n = 7). In black, CRF02_AG sequences from other regions. The 6 monophyletic clusters from Equatorial Guinea further assessed using BEAST are highlighted in yellow and labeled in accordance to Table 3.
The Bayesian MCC tree (Figure S1) showed that 6 of the 7 putative clusters were supported by a posterior probability of ≥0.95 (Table 3). Therefore, these 6 clusters were considered as definitive due to their high statistical support. They included 33 Equatoguinean sequences (mean = 5.5 sequences/cluster). All but one (cluster VI) clusters included sequences sampled in both Spain and GQ. The dates of their most recent common ancestors (MRCA) ranged from 1983.9 to 2002.5. Five of the 6 clusters were included among sequences sampled in Western Africa, and one (cluster V) was related to sequences sampled in Central Africa (Figure 1).
HIV-1M Drug Resistance
Among the 195 sequences collected in Spain from Equatoguinean subjects, 148 (75.9%) corresponded to antiretroviral-naïve patients and 31 (15.9%) to treatment-experienced subjects, with unknown treatment experience in 16 (8.2%) patients. We found at least one transmitted drug-resistance mutation (TDR) in 7 of the 148 (4.7%) naïve patients following the World Health Organization mutations list . TDR was 6.7% (6/90) in women vs. 1.8% (1/56) in men (P = 0.2). Table 4 shows the TDR rate according to the antiretroviral class affected. In 5 (3.4%) subjects, the mutations affected PR inhibitors (PI), with M46I/L substitution in all cases. Another 2 (1.8%) presented the M184V mutation to nucleoside reverse transcriptase inhibitors (NRTI). One of the latter also presented a drug-resistance mutation to non-nucleoside reverse transcriptase inhibitors (NNRTI). No triple-class resistance was found. Among the 31 ART-experienced patients, 9 (29%) presented any drug-resistance mutation according to the IAS-USA list . The highest rate (27.8%) was found for NNRTI-resistance due to mutation K103N in 3 of the 7 cases. The PI-resistance mutations found were M46L (4 cases), and L90M and I84V (3 cases each). For NRTI-resistance, the mutation M184V was found in all 3 cases. We observed a fluctuation in TDR rates in the three periods shown at Table 1∶9.7% (3/31) in 1997–2001, 2.5% (2/81) in 2002–2006 and 5.5% (2/36) in 2007–2011, probably due to the small sample size in each one.
Table 5 shows the main epidemiological and virological features of the 7 drug-naïve and the 9 drug-experienced patients carrying viruses harboring resistance mutations. Of note, most naïve (6/7; 85.7%) and pre-treated (6/9 cases; 66.7%) patients carrying drug-resistant viruses were adult women. They were mainly infected by CRF02_AG (2/7 and 4/9, respectively) and subtype B (2/7 and 3/9 cases, respectively).
Regarding the sequences retrieved from GenBank and obtained in Equatorial Guinea, the antiretroviral status was only available for the 41 samples described in Djoko et al., which were obtained from naïve HIV-infected population in Malabo. Among them, TDR was 4.9% according to the WHO mutations list.
Despite being located in the River Congo basin, where the HIV epidemics originated , only two works ,  have provided data about the circulation of different HIV strains and drug resistance in Equatorial Guinea. Here, we expand and complete the information available about the HIV Equatoguinean epidemics combining the use of HIV-1M pol sequences from immigrants in Spain (the main host developed country for Equatoguinean migration) and from patients sampled in Equatorial Guinea. This is the larger study covering transmitted drug resistance in this country and the first one analyzing general HIV-infected population and dating the introduction of CRF02_AG.
CRF02_AG Recombinant is Prevalent in Equatorial Guinea
We confirm that recombinant CRF02_AG, the prevalent HIV-1M variant in Western Africa , accounts for a half of infections in Equatorial Guinea (Table 2). This is also the most frequent non-B variant in Spain among adult  and pediatric  HIV-infected population. The sequences’ re-analyses identified CRF02_AG isolates classified as subtype G when they were described using only protease sequences . We also found CRF11_cpx, CRF13_cpx, CRF18_cpx and CRF22_01A1 sequences initially described as pure subtypes , . These complex variants are found in Spain among immigrants and rarely in autochthonous population , . Recombinants might be underestimated in studies performed in periods that lacked reference sequences published afterwards. Thus, periodic re-assessments of HIV classification with updated CRFs might improve the subtyping. In addition, modern computer tools permit to identify previously unnoticed unique recombinant forms when phylogenetic analyses are inconclusive.
CRF02_AG Introduction in Equatorial Guinea
The Bayesian MCMC analysis using BEAST revealed 6 monophyletic, highly statistically supported clusters of Equatoguinean CRF02_AG sequences. This could indicate that this variant entered Equatorial Guinea through several independent introductions occurred at least since the early 1980s, and not through a single introduction event. A previous study proposed that CRF02_AG was introduced in Equatorial Guinea from Cameroon , but our results showed that only one of the 6 fell within Central African clades, being the rest related to Western African clades. In many cases, sequences from both regions were interspersed in the analysis (Figure 1), which suggests a frequent circulation of CRF02_AG between countries, as others have also indicated . In both Western and Central Africa the presence of this recombinant, originated at least in the early 1970s , has increased in the last years .
On the other hand, the observed clustering could also be explained by specific transmission networks, although the available epidemiological information collected in the patients’ clinical records does not point to the presence of any of these networks. In addition, as previously commented, all clusters but one (cluster VI) included both CRF02_AG sequences sampled in Spain and in Equatorial Guinea, which minimizes the chance of a local transmission network. Despite these explanations, this alternative possibility cannot be completely ruled out.
Low Rate of Resistance among HIV-1M-infected Equatoguineans
We observed a TDR rate of 4.7% among the 148 antiretroviral-naïve Equatoguineans attending Spanish HIV/AIDS clinics, a figure close to the limit between low and moderate drug-resistance (5%) according to the World Health Organization . This rate is very similar to that found in the only TDR study performed in Equatorial Guinea (4.9%), which screened military personnel in Malabo . Both are lower than in other sub-Saharan countries, including the neighboring Cameroon, with a TDR around 9% , . Since ART still reaches only 48% of the HIV-infected population in need of therapy in GQ , a lower emergence of resistance than in countries with an older ART scale-up is expected .
The highest TDR rate was found for PI (3.4%), led by the presence of the substitution M46I/L in all cases. This was also the most frequent PI-resistance mutation among pre-treated patients, and it was previously reported among antiretroviral-naïve military personnel living in Equatorial Guinea . This high prevalence contrasts with the rare administration of PI in Equatorial Guinea, where the first line treatment includes the NNRTI efavirenz . For transmitted NRTI-resistance, the low presence found (with the predominance of change M184V) agrees with the study of Djoko et al., although they only found the substitution D67N as transmitted NRTI-resistance mutation .
Studies in Uganda ,  have related the expanded use of NNRTI as peripartum prophylaxis among HIV-infected pregnant women to a higher population-wide prevalence of NNRTI-resistance mutations. Thus, the low presence of transmitted NNRTI-resistance mutations in Equatoguineans compared with other sub-Saharan populations  could result from the infrequent administration of this prophylaxis in Equatorial Guinea (only in 19% of HIV-infected pregnant women in 2010) .
In treated Equatoguinean patients attended in Spain, we found a presence of drug-resistance mutations rate of 29%, with NNRTI as the most affected drug class (27.8%). This lower prevalence than in other sub-Saharan antiretroviral-exposed cohorts  could reflect a poor adherence as observed among sub-Saharans living in Spain , who present great rates of follow-up losses. This has also been reported in Africa , caused by the low accessibility of antiretroviral drugs, as happens in Equatorial Guinea, with an antiretroviral coverage of 48% in 2011. In Equatorial Guinea, the use of antiretroviral drugs started in a regular basis in 2005. Thus, previous sporadic treatments and interrupted exposition to drugs could have caused the appearance of resistance mutations that jeopardizes the success of a future systematic antiretroviral programme. Unfortunately, in most cases the information on specific drug exposure for the pre-treated patients was unavailable, which prevent us from a further interpretation of these data.
HIV Infection Place
The phylogenetic analyses showed that HIV-1M sequences sampled in Spain and/or Equatorial Guinea were interspersed, which would suggest that most of the 195 Equatoguinean patients with clinical follow up in Spain were infected in their country of origin. However, we cannot rule out that some of them could have been infected in Spain. This hypothesis would be supported by the overrepresentation of subtype B (the predominant variant in Spain  but infrequent in Central Africa) among samples taken in this host country versus those taken in Equatorial Guinea; and by the higher prevalence of transmitted PI-resistance in these subtype B-infected Equatoguineans sampled in Spain (where treatment is universally available) than in other variants of the study cohort.
Conversely, an evidence for an infection happened in Africa would be the different mutation pattern found in Equatoguineans sampled in Spain than in a study of general, naïve patients  (autochthonous and African and non-African foreigners) living in Spain between 1996 and 2010. Thus, the PI-resistance mutation M46L, present in all subtype B viruses with TDR (Table 5), was rare (<1%) among the subtype B-infected population living in Spain, where L90M was predominant. In addition, if the Equatoguinean patients had been infected in Spain, higher rates of NRTI- and, especially, NNRTI-resistance would have been expected.
In conclusion, the rising HIV drug-resistance transmission in sub-Saharan Africa following antiretroviral rollout highlights the need of periodical surveillance studies to monitor and prevent the resistance emergence, essential information to design or continue the implementation of ART programmes. These studies should be performed among infected people living in the study region. However, in countries lacking surveillance reports (such as Equatorial Guinea), our approach describing the HIV molecular epidemiology of low-income regions from a developed host country can provide any knowledge about the HIV epidemic in specific areas.
Maximum clade credibility tree of the 92 CRF02_AG pol sequences included in the BEAST analysis. The definitive clusters are highlighted in yellow and labeled in accordance to Table 3. The asterisk indicates nodes supported by a posterior probability of ≥0.95. The horizontal axis is expressed in calendar years. Branch colors indicate the origin of the sequences: blue, sequences from Equatoguinean patients (n = 39); green, sequences from Western Africa (n = 21); red, sequences from Central Africa (n = 17); black, other regions (n = 15).
Sampling dates of the 92 CRF02_AG pol sequences included in the BEAST analysis. n, number of sequences.
Conceived and designed the experiments: GY ÁH. Performed the experiments: GY MdM. Analyzed the data: GY MdM ÁH. Wrote the paper: GY ÁH.
- 1. Keele BF, Van Heuverswyn F, Li Y, Bailes E, Takehisa J, et al. (2006) Chimpanzee reservoirs of pandemic and nonpandemic HIV-1. Science 313: 523–526.
- 2. Vidal N, Peeters M, Mulanga-Kabeya C, Nzilambi N, Robertson D, et al. (2000) Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in Central Africa. J Virol 74: 10498–10507.
- 3. UNAIDS (2012) Equatorial Guinea National Progress Report. Available: http://www.unaids.org/en/dataanalysis/knowyourresponse/countryprogressreports/2012countries/ce_GQ_Narrative_Report.pdf. Accessed 28 January 2013.
- 4. Ortiz M, Sanchez I, Gonzalez MP, Leon MI, Abeso N, et al. (2001) Molecular epidemiology of HIV type 1 subtypes in Equatorial Guinea. AIDS Res Hum Retroviruses 17: 851–855.
- 5. Djoko CF, Wolfe ND, Vidal N, Tamoufe U, Montavon C, et al. (2010) HIV type 1 pol gene diversity and genotypic antiretroviral drug resistance mutations in Malabo, Equatorial Guinea. AIDS Res Hum Retroviruses 26: 1027–1031.
- 6. Hemelaar J, Gouws E, Ghys PD, Osmanov S (2011) Global trends in molecular epidemiology of HIV-1 during 2000–2007. AIDS 25: 679–689.
- 7. Hamers RL, Wallis CL, Kityo C, Siwale M, Mandaliya K, et al. (2011) HIV-1 drug resistance in antiretroviral-naive individuals in sub-Saharan Africa after rollout of antiretroviral therapy: a multicentre observational study. Lancet Infect Dis 11: 750–759.
- 8. Gupta RK, Jordan MR, Sultan BJ, Hill A, Davis DH, et al. (2012) Global trends in antiretroviral resistance in treatment-naive individuals with HIV after rollout of antiretroviral treatment in resource-limited settings: a global collaborative study and meta-regression analysis. Lancet 380: 1250–1258.
- 9. Aghokeng AF, Kouanfack C, Laurent C, Ebong E, Atem-Tambe A, et al. (2011) Scale-up of antiretroviral treatment in sub-Saharan Africa is accompanied by increasing HIV-1 drug resistance mutations in drug-naive patients. AIDS 25: 2183–2188.
- 10. Aghokeng AF, Vergne L, Mpoudi-Ngole E, Mbangue M, Deoudje N, et al. (2009) Evaluation of transmitted HIV drug resistance among recently-infected antenatal clinic attendees in four Central African countries. Antivir Ther 14: 401–411.
- 11. Ceccarelli L, Salpini R, Moudourou S, Cento V, Santoro MM, et al. (2012) Characterization of drug resistance mutations in naive and ART-treated patients infected with HIV-1 in Yaounde, Cameroon. J Med Virol 84: 721–727.
- 12. Mintsa-Ndong A, Caron M, Plantier JC, Makuwa M, Le Hello S, et al. (2009) High HIV Type 1 prevalence and wide genetic diversity with dominance of recombinant strains but low level of antiretroviral drug-resistance mutations in untreated patients in northeast Gabon, Central Africa. AIDS Res Hum Retroviruses 25: 411–418.
- 13. Toro C, Jiménez V, Rodríguez C, Del Romero J, Rodés B, et al. (2006) Molecular and epidemiological characteristics of blood-borne virus infections among recent immigrants in Spain. J Med Virol 78: 1599–1608.
- 14. Yebra G, de Mulder M, Pérez-Elías MJ, Pérez-Molina JA, Galán JC, et al. (2011) Increase of transmitted drug resistance among HIV-infected sub-Saharan Africans residing in Spain in contrast to the native population. PLOS ONE 6: e26757.
- 15. de Mulder M, Yebra G, Martín L, Prieto L, Mellado MJ, et al. (2011) Drug resistance prevalence and HIV-1 variant characterization in the naive and pretreated HIV-1-infected paediatric population in Madrid, Spain. J Antimicrob Chemother 66: 2362–2371.
- 16. Holguín Á, de Mulder M, Yebra G, López M, Soriano V (2008) Increase of non-B subtypes and recombinants among newly diagnosed HIV-1 native Spaniards and immigrants in Spain. Curr HIV Res 6: 327–334.
- 17. Yebra G, Holguín Á, Pillay D, Hué S (2013) Phylogenetic and demographic characterization of HIV-1 transmission in Madrid, Spain. Infect Genet Evol 14: 232–239.
- 18. Bennett DE, Camacho RJ, Otelea D, Kuritzkes DR, Fleury H, et al. (2009) Drug resistance mutations for surveillance of transmitted HIV-1 drug-resistance: 2009 update. PLOS ONE 4: e4724.
- 19. Johnson VA, Calvez V, Gunthard HF, Paredes R, Pillay D, et al. (2011) 2011 update of the drug resistance mutations in HIV-1. Top Antivir Med 19: 156–164.
- 20. UNAIDS (2011) 2010 Report on the global AIDS epidemic Available: http://www.unaids.org/documents/20101123_GlobalReport_em.pdf.Accessed 28 January 2013.
- 21. Holguín Á, Álvarez A, Soriano V (2002) High prevalence of HIV-1 subtype G and natural polymorphisms at the protease gene among HIV-infected immigrants in Madrid. AIDS 16: 1163–1170.
- 22. Holguín Á, Álvarez A, Soriano V (2005) Differences in the length of gag proteins among different HIV type 1 subtypes. AIDS Res Hum Retroviruses 21: 886–893.
- 23. Yebra G, de Mulder M, Martín L, Rodríguez C, Labarga P, et al. (2012) Most HIV type 1 non-B infections in the Spanish cohort of antiretroviral treatment-naive HIV-infected patients (CoRIS) are due to recombinant viruses. J Clin Microbiol 50: 407–413.
- 24. Faria NR, Suchard MA, Abecasis A, Sousa JD, Ndembi N, et al. (2012) Phylodynamics of the HIV-1 CRF02_AG clade in Cameroon. Infect Genet Evol 12: 453–460.
- 25. Véras NM, Santoro MM, Gray RR, Tatem AJ, Lo Presti A, et al. (2011) Molecular epidemiology of HIV type 1 CRF02_AG in Cameroon and African patients living in Italy. AIDS Res Hum Retroviruses 27: 1173–1182.
- 26. WHO (2012) World Health Organization HIV drug resistance report. Available: http://www.who.int/hiv/pub/drugresistance/report2012/en/index.html. Accessed 28 January 2013.
- 27. Ndembi N, Hamers RL, Sigaloff KC, Lyagoba F, Magambo B, et al. (2011) Transmitted antiretroviral drug resistance among newly HIV-1 diagnosed young individuals in Kampala. AIDS 25: 905–910.
- 28. Hamers RL, Sigaloff KC, Wensing AM, Wallis CL, Kityo C, et al. (2012) Patterns of HIV-1 drug resistance after first-line antiretroviral therapy (ART) failure in 6 sub-Saharan African countries: implications for second-line ART strategies. Clin Infect Dis 54: 1660–1669.
- 29. Pérez-Molina JA, Rillo MM, Suárez-Lozano I, Casado Osorio JL, Cobo RT, et al. (2010) Do HIV-infected immigrants initiating HAART have poorer treatment-related outcomes than autochthonous patients in Spain? Results of the GESIDA 5808 study. Curr HIV Res 8: 521–530.
- 30. Billong SC, Fokam J, Nkwescheu AS, Kembou E, Milenge P, et al. (2012) Early warning indicators for HIV drug resistance in Cameroon during the year 2010. PLOS ONE 7: e36777.