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HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients

Aden Elmi Abar12*, Asma Jlizi2, Houssein Youssouf Darar3, MohamedAliBenHadj Kacem2 and Amine Slim2

Author Affiliations

1 Laboratoire de la Caisse Nationale de Sécurité Sociale, Djibouti, Republic of Djibouti

2 Laboratoire de Microbiologie, CHU Charles Nicolle, Tunis, Tunisie

3 Service des Maladies Infectieuses de l’Hôpital Général Peltier, Djibouti, Republic of Djibouti

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Diagnostic Pathology 2012, 7:138  doi:10.1186/1746-1596-7-138

The electronic version of this article is the complete one and can be found online at: http://www.diagnosticpathology.org/content/7/1/138


Received:24 June 2012
Accepted:18 September 2012
Published:8 October 2012

© 2012 Elmi Abar et al.; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients.

Patients and methods

A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping.

Results

Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C.

Conclusion

The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients.

Virtual slides

The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2051206212753973 webcite

Keywords:
HIV-1 drug resistance; First-line antiretroviral therapy failure; Antiretroviral naive; Djibouti; Virological failure

Background

Since the discovery of human immunodeficiency virus (HIV) as the causative agent in 1983, the HIV-1 pandemic is characterized by an extensive variability and the circulation of numerous genotypes and recombinant forms (CRFs) [1,2]. Subtype C is the most prevalent, estimated to represent 48% infections in the HIV-1 pandemic [3]. Subtype C is predominant particularly in south and east Africa [4]. This strongly contrasts with the situation in central Africa where a marked genetic heterogeneity is observed.

HIV-1 remains a crucial global health problem. Delivery of ART in low and middle income countries has required utilization of a public health approach in which standardized, rather than individualized, regimens are prescribed to very large numbers of HIV-1-infected individuals [5]. At present, the majority of individuals in these countries are initiating first-line therapy with a non-nucleoside reverse transcriptase inhibitor (NNRTI) and two nucleoside reverse transcriptase inhibitors (NRTI) [6].

The introduction of antiretroviral (ARV) therapy in resource-poor settings is effective in suppressing HIV-1 replication and prolonging life of infected individuals. Nevertheless, patients in routine clinical care experience sustain detectable viral replication even under potent combination therapy. Various factors underlie this failure to achieve viral suppression, but resistance to current medications has been increasingly recognized as an important factor. Rational choice of the NRTI component of second-line therapy should be based on patterns of resistance developed during first-line therapy [7].

Djibouti is the smallest country in east Africa. The estimated prevalence of HIV-1 infection was around 2.5% (2009). Since the introduction of antiretroviral therapy in 2004, no data exist on the epidemiology of drug resistance mutations and little was known about HIV-1 genotypes circulating in Djibouti. Data on drug resistance in the routine care setting are urgently required to check ART efficacy. The present study aims to characterize circulating HIV-1 subtypes and to identify drug resistance mutations in patients who first-line therapy failed (>1000 copies/mL) and antiretroviral-naive patients from Djibouti.

Materials and methods

Population study and sample collection

Between October and December 2009, the whole blood samples were collected in Djibouti city from 35 HIV-1 infected patients consulting at three health centers (Hôpital General Peltier, N = 23, Centre Yonis Thoussaint, N = 9 and Centre Paul Faure, N = 3). EDTA (ethylenediaminetetraacetic acid-anticoagulated) plasma specimens were obtained after centrifugation, and stored at −80°C in aliquots for later assessment of HIV RNA viral-load and genotypic drug resistance testing. The ethical aspects of this study were approved by the local committee. For all participants, blood collection was performed after informed consent, under the supervision of local sanitary authorities.

Viral load and CD4

The CD3/CD4+ cells were enumerated by flowcytometry (BD FACSCount System) and the quantification of HIV-1 viral load (VL) was measured using NASBA, NucliSENS®EasyQ Assays (BioMerieux) at the central laboratory of “Hôpital General Peltier” in Djibouti.

The plasma samples were shipped to the Laboratory of Microbiology of “Charles Nicolle” Hospital (Tunis, Tunisia) for drug resistance genotyping tests.

Drug resistance genotyping

For Drug Resistance genotyping, the viral RNA was isolated from plasma samples using DSP virus RNA kit (QIAGEN Inc.) and reverse transcribed to make complementary DNA (cDNA) copies using a cDNA synthesis kit the One Step RT-PCR (QIAGEN Inc). The reverse transcriptase (RT) and protease (PR) genes were amplified according to the ANRS consensus sets of primers: for the RT gene amplification, MJ3 and MJ4 primers were used for a first PCR (RT-PCR), and A(35) with NE1(35) as inner primers for nested PCR. For the protease gene amplification, 5′Prot1 and 3′Prot1 outer primers were used and the 5′Prot2 and 3′Prot2 as inner primers: (ANRS: http://www.hivfrenchresistance.org/ANRS-procedures.pdf webcite).

RT-PCR involved a reverse transcription step at 50°C for 30 minutes, followed by 95°C for 15 minutes before proceeding to 40 cycles of 95°C for 45 seconds, 50°C for 30 seconds and 72°C for 1 minute 30 seconds, with a final extension step of 10 minutes at 72°C. The RT-PCR products were then subjected to nested PCR with the following cycling conditions: a denaturation step of 2 minutes at 95°C, and then 35 cycles with 1 cycle consisting of 30 seconds at 95°C, 30 seconds at 55°C and 30 seconds at 72°C with a final extension at 72°C for 7 minutes.

PCR products (5 μL) were resolved by electrophoresis on a 1.5% agarose gel GellyPhorLE, (Euroclone, UK). DNA bands were stained with RedSafe™ Nucleid Acid Staining Solution [20,000X] (iNtRON Biotechnology, Inc, Korea). They were then visualized in UV light to assess the presence of fragments of the expected length compared with the standard marker SharpMass™50-DNA ladder (Euroclone, Wetherby, UK). The amplification products were purified using QIAquick PCR Purification kit (QIAGEN Inc).

The PCR products were finally sequenced on an ABI3130 Genetic Analyser (Applied Biosystems), and compared with HXB-2 sequence using SeqScape v2.6 software (Life technologies). All sequences were subjected to quality assessment and determination of DR mutation profile (susceptible, intermediate resistance and resistance) using the Stanford Database (available at http://hivdb.stanford.edu/ webcite).

Results

This study includes 35 HIV-1-infected Djiboutian patients enrolled between October and December 2009. Their mean age was 32.3 years. Among these patients, nineteen patients were ART-naïve (54.2%) and 16 were first-line ART failures (45.8%). In ART naïve patients, the median CD4 count was 144 cells/μl and the average viral load was 5.58 log10/ml (388 643 copies/ml). For the patients with first-line ART failure, the median CD4 count was 178 cells/μl, viral loads mean was 5.62 log10/ml and the median time of therapy was 30 months (Table  1). There were 20 females (57%) which were not pregnant at the time of blood collection. Genetic subtyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C (Table  2).

Table 1. Patients characteristics

Table 2. HIV-1 subtypes and distribution of resistance-associated mutations among 19 ART-naïve patients attending three health centers in Djibouti city between October and December 2009

The ART naïve patients showed only NNRTI-associated mutations which were K103N (2 strains) and K101Q (1). No mutations of NRTI were detected. The minor PI mutation L10V was detected in one ART naïve patient [8].

At the time of sampling, all the patients receiving ART were treated by first line. According to the Djiboutian National Program for fighting against AIDS (PNLS) recommendations [9], the first-line treatment consists in 2 NRTI + 1NNRTI. The NRTI backbones were stavudine, zidovudine, and lamivudine. The most used drug combination (12 Patients (75%)) was AZT/3TC/EFV, while the 6 other patients received either AZT/3TC/NVP, D4T/3TC/EFV or D4T/3TC/NVP. The NRTI-associated mutations were D67N (2 strains), T69N (1), M184V (6), L210W (2), T215Y (2). The NNRTI-associated mutations were K101Q (1), K103N (4), V179E (1), Y181C (1), Y188L (2), G190A (2) (Table  3). For PI-associated mutations, the minor mutation L10V and other polymorphism were detected.

Table 3. HIV-1 subtypes and distribution of resistance-associated mutations among 16 patients with first-line ART failure attending three health centers in Djibouti city between October and December 2009

Drug susceptibility results were classified into three categories: susceptible, intermediate resistance and resistance. Of the 16 patients with first-line ART failure, 56.25% showed resistance to at least one antiretroviral molecule: two patients (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. We found that the resistance rates to Lamivudine and Nevirapine in the 16 patients with first-line ART failure were 37.5% and 43.75% respectively. Forty four percent (44%) of sequences from viral strains did not show any mutation associated to ARV drug resistance- 7out of 16 [10]. Therefore, therapeutic solutions using second line drugs could be proposed to our patients with therapeutic failure and drug resistance mutations.

Discussion

Minimizing drug resistance to highly active antiretroviral therapy (HAART) is important. First, it maximizes the opportunity for successful second-line and subsequent therapies after viral rebound during first-line treatment; second, it limits the transmission of drug-resistant viruses. We report results of the HIV-1 genetic diversity and prevalence of mutations associated with antiretroviral drugs among ART naïve as well as patients who failed first line therapy. The HAART was introduced in Djibouti in 2004. Djibouti, surrounded by Eritrea, Ethiopia, and Somalia, is an important crossroad for trade and exchange.

Our result revealed a majority of subtype C (~66%). In fact, Subtype C prevalence has been described in the Horn of Africa [11-14] and a similar prevalence was previously reported in Djibouti in 2005 [15,16]. However, our study describes the subtype B in Djibouti for the first time. It is the predominant subtype in the Western world and the clade that has circulated in North America and Europe. The detection of CRF02_AG strains indicates that they are still circulating in Djibouti, the only country in East Africa in which this recombinant virus was found some years ago [17,18]. CRF02_AG recombinant isolates were primarily described in West and Central Africa [19,20]. The discordances observed between RT and PR sequences in 2 isolates (CRF02_AG/C, K/C) are probably attributable to the high variability of the genome of HIV in particularly intra subtype recombination within group M subtype C [21].

For the ART naïve patients, the low rate of CD4 and the high rate of VL can be explain by the fact that 80% of patients infected by HIV in Djibouti was already detected at an advanced stage of the epidemic according to WHO classification [22,23]. Among the 19 strains, 2 were resistant only to non-nucleoside reverse transcriptase inhibitors (NNRTIs) while one strain showed a minor mutation to PI. Detection of RT and protease mutations associated with drug resistance in individuals who have not been exposed to antiretroviral therapy is thought to result from either transmission from a treated individual.

Concerning first-line ART failures, 38% of 16 strains sequenced in this group had the M184V mutation. This mutation is selected by lamivudine (3TC), emtricitabine (FTC) or abacavir (ABC) use and confers high-level resistance to 3TC (Figure  1) [24]. Patients receiving incompletely suppressive 3TC regimens usually develop M184V as their first mutation [25,26]. Accumulation of the other mutations observed included thymidine associated mutations (including M41L, D67N, K70R, L210W, T215Y/F, K219Q) results in increasing resistance to AZT, Tenofovir, D4T, Abacavir, and DDI [27,28]. Two multidrug resistance mutation profiles in general, confer high level drug resistance to all NRTIs. The most common occurring nucleotide excision mutations (NEMs) that confer resistance by enhanced excision [29] were at position 215. The T215Y mutation was observed in 13% of the sequences while T215F was detected in 6%. Clinical studies have shown that the NEMs, particularly mutations at position 215, interfere with the clinical response to zidovudine, stavudine, abacavir, didanosine, and most dual NRTI combinations [30]. Mutations conferring resistance to NNRTIs were also seen, with one strain showing resistance only to NNRTIs. The NNRTI resistance-conferring mutations included K103N (25%), Y188L (12.5%), Y181C (6%), G190A (6%), H221Y (6%), M230L (6%). Our data suggest the non-epidemic circulation of resistant viruses, and the absence of multi-class drug resistant viral strains.

thumbnailFigure 1. Frequencies of resistance levels to nucleoside (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors. The horizontal bars indicates the frequency of susceptible (white), intermediate resistance (grey) and resistant (black) samples to NRTI and NNRTI reverse transcriptase inhibitors.

It is important to appreciate that resistance at failure is only one factor to consider when choosing an initial HAART regimen. Coformulation, simplicity of administration, price, drug interactions (particularly with tuberculosis therapy), toxicity and adverse events are all important considerations and will differ between patient populations. We noticed seven patients (~44%) did not show resistance-conferring mutations and the therapeutic failure may have been due to other factors [31-35]. Information on treatment from their clinic folders allowed us to confirm that for five of these seven cases the ART failure profile was due to poor adherence and for the remaining two cases the detectable viral level was due to coinfection with tuberculosis and malnutrition [36-38].

The escalating rates of transmission of drug resistant virus observed in the past few years, coupled with the poorer response to treatment in persons with drug resistant virus, are being considered as a basis for a recommendation that resistance tests should be performed routinely for persons with new HIV infection [39]. This evaluation, both at the baseline and in follow-up, will be crucial to assess the impact of therapy regimens on resistance development and also their adequation.

Conclusion

This study reveals primary mutations that confer resistance to NRTI, NNRTI in HIV-1 non-B and B subtype strains, isolated from ART naïve as well as patients who failed first line therapy in Djibouti. It also illustrates the importance of preliminary surveys of drug-resistance mutations in resource-poor countries. Therefore, we suggest the use of resistance testing to check the prevalence of the drug resistance mutation that arises following failure of the first line regimen. This will help in establishing guidelines for second line regimens in Djibouti. This allows the selection of adapted antiretroviral regimens in these regions. Our results provide a basis for repeated epidemiological studies to measure the population effects of HIV treatment programs over time.

Abbreviations

ART: Antiretroviral therapy; PR: Protease; RT: Reverse Transcriptase; ARV: Antiretroviral.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

AEA: wrote the manuscript, has made substantial contributions to acquisition of data, or analysis and interpretation of data. AEA and AJ: participated in data analysis and interpretation. HYD and MA BHK: revised the manuscript. AS: proposed the idea, revised the manuscript and save final approval of the version to be published. All authors read and approved the final manuscript.

Acknowledgments

We thank Ali Abdillahi, Nima Ahmed, Mohamed Miganeh, Houssein Yonis and Yacin Ahmed for technical assistance from Djibouti. This work was performed at the National HIV Reference Laboratory, Charles Nicolle Hospital, Tunis, Tunisia. The expert assistance, Assistant Salma ABID, Mme Majda BEN NASR of the laboratory staff is gratefully acknowledged.

References

  1. Robert S, Michael Gottlieb S, Robert S, Howard Schanker M, Joel D, Weisman DO, Peng Thim F, Robert Wolf A, Andrew S: Pneumocystis carinii Pneumonia and Mucosal Candidiasis in Previously Healthy Homosexual Men — Evidence of a New Acquired Cellular Immunodeficiency.

    N Engl J Med 1981, 305:1425-1431. PubMed Abstract | Publisher Full Text OpenURL

  2. Barre-Sinoussi F, et al.: Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS).

    Science 1983, 220:868-871. PubMed Abstract | Publisher Full Text OpenURL

  3. Hemelaar J, Gouws E, Ghys PD, Osmanov S: Global trends in molecular epidemiology of HIV-1 during 2000–2007.

    AIDS 2011, 25:679-689. PubMed Abstract | Publisher Full Text OpenURL

  4. Thomson MM, Perez-Alvarez L, Najera R: Molecular epidemiology of HIV-1 genetic forms and its significance for vaccine development and therapy.

    Lancet Infect Dis 2002, 2:461-471. PubMed Abstract | Publisher Full Text OpenURL

  5. Gilks CF, Crowley S, Ekpini R, et al.: The WHO public-health approach to antiretroviral treatment against HIV in resource-limited settings.

    Lancet 2006, 368(9534):505-510. PubMed Abstract | Publisher Full Text OpenURL

  6. World Health Organization: Antiretroviral therapy for HIV infection in adults and adolescents: recommendations for a public health approach. WHO, HIV Department, Geneva, Switzerland; 2006.

    Available at: http://www.who.int/hiv/pub/guidelines/artadultguidelines.pdf webcite. Accessed 12 December 2007

    OpenURL

  7. Gazzard BG, British HIV: Association guidelines for the treatment of HIV-1-infected adults with antiretroviral therapy 2008.

    HIV Med 2008, 9:563-608. PubMed Abstract | Publisher Full Text OpenURL

  8. Vergne L, Peeters M, Mpoudi-Ngole E, Bourgeois A, Liegeois F, Toure-Kane C, Mboup S, Mulanga-Kabeya C, Saman E, Jourdan J, Reynes J, Delaporte E: Genetic diversity of protease and reverse transcriptase sequences in non-subtype B human immunodeficiency virus type 1 strains: evidence of many minor drug resistance mutations in treatment-naive patients.

    J Cardiovasc Manag 2000, 38:3919-3925. OpenURL

  9. Guide d’utilisation des Antirétroviraux (ARV) chez l’adulte et l’adolescent en République de DJIBOUTI

    2008.

    http://www.aidstar-one.com/sites/default/files/treatment/national_treatment_guidelines/Djibouti_adult_guidelines_2008.pdf webcite

  10. Jlizi A, Ben Ammar A, El Gaaied A, Slim F, Tebourski M, Ben Mamou T, Ben C, et al.: Profile of drug resistance mutations among HIV-1-infected Tunisian subjects failing antiretroviral therapy.

    Arch Virol 2008, 153:1103-1108. PubMed Abstract | Publisher Full Text OpenURL

  11. Abecasis AB, Vandamme AM, Lemey P: Quantifying differences in the tempo of human immunodeficiency virus type 1 subtype evolution.

    J Virol 2009, 83:12917-12924. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  12. Nyombi BM, et al.: Diversity of human immunodeficiency virus type 1 subtypes in Kagera and Kilimanjaro regions Tanzania.

    AIDS Res Hum Retroviruses 2008, 24:761-769. PubMed Abstract | Publisher Full Text OpenURL

  13. Abebe A, Lukashov VV, Pollakis G, Kliphuis A, Fontanet AL, Goudsmit J, Rinke TF, de Wit TF: Timing of the HIV-1 subtype C epidemic in Ethiopia based on early virus strains and subsequent virus diversification.

    AIDS 2001, 15:1555-1561. PubMed Abstract | Publisher Full Text OpenURL

  14. Abebe A, Lukashov VV, Rinke De Wit TF, Fisseha B, Tegbaru B, Kliphuis A, Tesfaye G, Negassa H, Fontanet AL, Goudsmit J, Pollakis G: Timing of the introduction into Ethiopia of subcluster C′ of HIV type 1 subtype C.

    AIDS Res Hum Retrovir 2001, 17:657-661. PubMed Abstract | Publisher Full Text OpenURL

  15. Maslin J, Rogier C, Caron M, Grandadam M, Koeck JL, Nicand E: Antiretroviral drug resistance among drug-naive HIV-1-infected individuals in Djibouti (Horn of Africa).

    Antivir Ther 2005, 10:855-859. PubMed Abstract OpenURL

  16. Maslin J, Rogier C, Berger F, Khamil MA, Mattera D, Grandadam M, Caron M, Nicand E: Epidemiology and genetic characterization of HIV-1 isolates in the general population of djibouti (Horn of Africa).

    J Acquir Immune Defic Syndr 2005, 39:129-132. PubMed Abstract | Publisher Full Text OpenURL

  17. Faria NR, Suchard MA, Abecasis A, Sousa JD, Ndembi N, Bonfim I, Camacho RJ, Vandamme AM, Lemey P: Phylodynamics of the HIV-1 CRF02_AG clade in Cameroon.

    Infect Genet Evol 2012, 12(2):453-460. PubMed Abstract | Publisher Full Text OpenURL

  18. Hemelaar J, et al.: The origin and diversity of the HIV-1 pandemic.

    Trends in Molecular Medicine 2011, 20:1-11. OpenURL

  19. Carr JK, Salminen MO, Albert J, Sanders-Buell E, Gotte D, Birx DL, McCutchan FE: Full genome sequences of human immunodeficiency virus type 1 subtypes G and A/G intersubtype recombinants.

    Virology 1998, 247:22-31. PubMed Abstract | Publisher Full Text OpenURL

  20. Cornelissen M, van Den Burg R, Zorgdrager F, Goudsmit J: Spread of distinct human immunodeficiency virus type 1 AG recombinant lineages in Africa.

    J Gen Virol 2000, 81:515-523. PubMed Abstract | Publisher Full Text OpenURL

  21. Rousseau CM, Learn GH, Bhattacharya T, Nickle DC, Heckerman D, Senica C, Christian B, Philip J, Goulder R, Bruce D, Walker Photini K, Korber BT, Mullins JI: Extensive Intra-subtype Recombination in South African HIV-1 Subtype C Infections.

    J Virol 2007, 81(9):4492-4500. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  22. Darar HY, Kibangou N, Mbuzenakamwe MJ, Goulan MA, Ibrahim IM: Réponse au traitement antirétroviral chez les patients infectés par le VIH-1 suivis au Centre Yonis Toussaint de Djibouti [Abstract].

    5éme Conférence Francophone VIH/SIDA, Maroc 2010, 493(77):215.

    28–31 March 2010

    PubMed Abstract | Publisher Full Text OpenURL

  23. Kibangou N: Prise en charge des patients sous traitement ARV à Djibouti entre 2004 et 2009 [Abstract].

    5éme Conférence Francophone VIH/SIDA, Maroc 2010, 578(88):237.

    28–31 March 2010

    PubMed Abstract | Publisher Full Text OpenURL

  24. Johnson VA, Vincent C, Günthard HF, Roger P, Deenan P, Robert S, Wensing AM, Richman DD: 2011 update of the drug resistance mutations in HIV-1.

    Top Antivir Med 2011, 19:156-164. PubMed Abstract | Publisher Full Text OpenURL

  25. Geretti AM: Clinical implications of HIV drug resistance to nucleoside and nucleotide reverse transcriptase inhibitors.

    AIDS Rev 2006, 8:210-220. PubMed Abstract OpenURL

  26. Theys K, Deforche K, Libin P, Camacho RJ, Van Laethem K, Vandamme AM: Resistance pathways of human immunodeficiency virus type 1 against the combination of zidovudine and lamivudine.

    J Gen Virol 2010, 91:1898-1908. PubMed Abstract | Publisher Full Text OpenURL

  27. Van Vaerenbergh K, Van Laethem K, Albert J, Boucher CA, Clotet B, Floridia M, Gerstoft J, Hejdeman B, Nielsen C, Pannecouque C, Perrin L, Pirillo MF, Ruiz L, Schmit JC, Schneider F, Schoolmeester A, Schuurman R, Stellbrink HJ, Stuyver L, Van Lunzen J, Van Remoortel B, Van Wijngaerden E, Vella S, Witvrouw M, Yerly S, De Clercq E, Destmyer J, Vandamme AM: Prevalence and characteristics of multinucleoside-resistant human immunodeficiency virus type 1 among European patients receiving combinations of nucleoside analogues.

    Antimicrob Agents Chemother 2000, 44:2109-2117. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  28. Coakley EP, Gillis JM, Hammer SM: Phenotypic and genotypic resistance patterns of HIV-1 isolates derived from individuals treated with didanosine and stavudine.

    AIDS 2000, 14:F9-F15. PubMed Abstract | Publisher Full Text OpenURL

  29. Shafer RW: Genotypic testing for human immunodeficiency virus type 1 drug resistance.

    Clin Microbiol Rev 2002, 15:247-277. PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  30. Emanuele B, Ana PC M, Victor Moreschi N, Camila DA S, Karla A, Saito AO, Mecatti MC, Santos MG, Pimentel CR, Larissa LG S, Cruz CR, de Noronha L: Histomorphometric and immunohistochemical analysis of infectious agents, T-cell subpopulations and inflammatory adhesion molecules in placentas from HIV-seropositive pregnant women.

    Diagn Pathol 2011, 6(101):101.

    24 October 2011

    PubMed Abstract | Publisher Full Text | PubMed Central Full Text OpenURL

  31. Eholie SP, N’Dour CT, Cisse M, Bissagnene E, Girard PM: Observance of antiretroviral treatments African specificities.

    Med Mal Infect 2006, 36:443-448. PubMed Abstract | Publisher Full Text OpenURL

  32. Phillips AN, Dunn D, Sabin C, et al.: UK Collaborative Group on HIV Drug Resistance, UK CHIC Study Group. Long term probability of detection of HIV-1 drug resistance after starting antiretroviral therapy in routine clinical practice.

    AIDS 2005, 19:487-494. PubMed Abstract | Publisher Full Text OpenURL

  33. Chesney MA, Ickovics J, Hecht FM, et al.: Adherence: a necessity for successful HIV combination therapy.

    AIDS 1999, 13(A):S271-S278. PubMed Abstract | Publisher Full Text OpenURL

  34. Bangsberg DR, Charlebois ED, Grant RM, et al.: High levels of adherence do not prevent accumulation of HIV drug resistance mutations.

    AIDS 2003, 17:1925-1932. PubMed Abstract | Publisher Full Text OpenURL

  35. Shah B, Walshe L, Saple DG, et al.: Adherence to antiretroviral therapy and virologic suppression among HIV-infected persons receiving care in private clinics in Mumbai, India.

    Clin Infect Dis 2007, 44:1235-1244. PubMed Abstract | Publisher Full Text OpenURL

  36. Ahmed AA, Katlama C, Ghosn J, Guiguet M, Costagliola D: Évaluation de l’observance au traitement antirétroviral au sein d’une cohorte de 200 patients à Djibouti (2005).

    Eastern Mediterranean Health Journal 2007, 13(6):1286-1297. PubMed Abstract OpenURL

  37. Renoux E, Barreh Matan A, Sevre JP, et al.: Tuberculose et infection VIH: expérience du programme national de lutte antituberculeuse de Djibouti: 1990–1996.

    Med Trop 2002, 62:171-176. OpenURL

  38. Acina E, Morand C, Mahdi A, Morand JJ: La malnutrition en République de Djibouti: un problème majeur de santé publique.

    Revue de l’ISERST 1991, 5:11-15. OpenURL

  39. Little SJ, Holte S, Routy JP, et al.: Antiretroviral-drug resistance among patients recently infected with HIV.

    New Engl J Med 2002, 347:385-394. PubMed Abstract | Publisher Full Text OpenURL