Sustained virological failure in Cameroonese patient infected by HIV-1 group N evidenced by sequence-based genotyping assay

Cameroon is a country of broad HIV-1 genetic diversity, where HIV-1 groups M, O, P and N are circulating [1]. HIV-1 genotyping commercial assays for antiretroviral drug resistance mutations (DRMs) were developed based basically on subtype B HIV-1 group M [2], representing less than 1% of HIV in Central Africa [1]. We report the unique history of the 30-year-old woman HIV-1-infected patient #MVE, at WHO clinical stage 2 and treated by first-line WHO antiretroviral therapy including tenofovir, lamivudine and efavirenz from December 2011 (CD4+ T cell count, 124 cells/μl) [3]. In July 2012, plasma HIV-1 load assessed by an Abbott m2000rt Real Time HIV-1 assay (Abbott Laboratories, Chicago, Illinois, USA) and CD4+ T cell count were 7455 copies/ml and 221 cells/μl, respectively. Despite adequate adherence to treatment (assessed by pharmacy refill rates), the HIV-1 load remained elevated at 1969 and 41 655 copies/ml after 13 and 18 months of treatment, respectively, demonstrating virological failure [3]. Successive CD4+ T cell counts during the first 18 months of treatment remained always below 350 cells/μl. DRM genotyping was carried out on plasma sample in protease and reverse transcriptase genes firstly by Trugene HIV-1 Genotyping assay (Siemens Healthcare Diagnostics, Tarrytown, New York, USA) made in routine in Douala. Genotyping was replicated with ViroSeq HIV-1 genotyping system, version 2.0 (Celera Diagnostics, California, USA) and in-house consensus technique of the ANRS (http://http://www.hivfrenchresistance.org). Unexpectedly, no sequence could be obtained either by ViroSeq or ANRS genotyping assays and only the Trugene HIV-1 Genotyping assay succeeded in amplifying a 925 nucleotides sequence. The MVE amino acid translated sequence showed an unresolved poly N segment (15 residues X). We carried out the genetic analysis of the MVE sequence in parallel by the software Genotyping (http://http://www.ncbi.nlm.nih.gov/projects/genotyping/formpage.cgi) [4] and HIV BLAST (http://http://www.hiv.lanl.gov/content/sequence/BASIC_BLAST/basic_blast.html) [5]. By Genotyping, the MVE sequences before and after the poly N stretch harboured 87 and 91% homology, respectively, with the HIV-1 group N isolate 02CM-SJGddd. By HIV BLAST, the poly N-deleted MVE sequence was identified as HIV-1 group N (85% homology with HIV-1 02CM-SJGddd) (Fig. 1).Fig. 1: Phylogenetic tree showing the relationship of the poly N-deleted MVE sequence with HIV-1M, HIV-1N, HIV-1 O, HIV-1 P, SIVcpz and SIVgor reference sequences was inferred by using the maximum likelihood method on the basis of Hasegawa-Kishino-Yano + G model conducted in MEGA6.The tree with the highest log likelihood is shown. Reference sequences [HIV-1 groups or SIVcpz, SIVgor, countries (Fr, France; CM, Cameroon; NG, Nigeria)], year, strain, accession number) used are the following: #N, CM, 1995, YBF30, AJ006022; #N, CM, 1997, YBF106, AJ271370; #CPZ, EK505, DQ373065; #CPZ, CM, 98, CAM5, AJ271369; #N, CM, 1999, YBF116, AJ564923; #N, CM, 2002, DJO0131, AY532635; #N, CM, 2004, 04CM, 1015, 04, DQ017382; #N, CM, 2004, 04CM, 1131, 03, DQ017383; #N, CM, 2006, U14842, GQ324958; #N, CM, 2002, SJGddd, GQ324959; #N, CM, 2006, U14296, GQ324962; #N, FR, 2011, N1, FR, 2011, JN572926; #O, CM, 1994, BCF06, AB485666; #GOR, CM, 2004, SIVgorCP684con, FJ424871; #P, FR, 2006, RBF168, GQ328744; #M, CRF02, NG, x, IBNG, L39106.Lastly, DRM interpretation was carried out by Trugene GuideLines Rules 17.0 (Siemens Healthcare Diagnostic) and ANRS AC-11 resistance group algorithm (V24-2014). By Trugene algorithm, numerous DRMs conferring resistance to first-line WHO antiretroviral molecules, including lamivudine, tenofovir, nevirapine and efavirenz, were evidenced (DRM: K65R, K103N, M184V, Y188C, H221Y); possible resistances to atazanavir and saquinavir were detected (DRM: L10V, K20R, L33I, M36L), although the patient never took protease inhibitors. By the ANRS algorithm, a similar DRM pattern was predicted with slight differences from the Trugene GuideLines Rules, including resistance to rilpivirine (mutation H221Y), a new second-generation nonnucleosidic reverse transcriptase inhibitor not yet introduced in Cameroon, although no mutation was associated with protease inhibitors resistance. After 20 months (December 2013), the treatment was switched for new regimen (zidovudine, lamivudine and lopinavir, ritonavir-boosted). After 4 months, the HIV-1 load dropped to 4758 copies/ml and CD4+ T cell count was at 339 cells/μl. From the first discovery of HIV-1 group N isolate in 1998 [6], around 20 cases of HIV-1 group N infections have been reported primarily in patients originating from Cameroon where the virus remained rare however [6–8]. We were surprised that, despite the high viral load, we could not amplify the protease and RT genes with ViroSeq HIV-1 genotyping system, version 2.0 and ANRS consensus primers, as previously reported in a patient infected by HIV-1 group N [9]. The knowledge of belonging to HIV-1 group N in the patient could be noteworthy because of its potential impact on antiretroviral molecules choice when a therapeutic switch is needed. The patient showed four successive viral loads always above the threshold of virological failure (1000 copies/ml) [3], and persisting low CD4+ T cell counts. High polymorphism in protease gene observed in the patient can be at the origin of primary resistance to certain protease inhibitors, as frequently observed with non-B HIV-1 group M [1,10]. The present case emphasizes the need to carefully assess the therapeutic options in case of virological failure, at best by carrying out resistance mutation genotyping, because of a possible incomplete response to the standard therapeutic regimen recommended by the WHO, and frequent high polymorphism in the protease gene limiting the choice of new molecules in second-line treatment [10]. Finally, in a context of high genetic diversity, such as in Cameroon, the fact that some genotyping assays cannot amplify HIV-1 despite high viral load should prompt the need to investigate in more detail whether the patient is infected with a divergent HIV-1 strain from rare HIV-1 groups such as N, O or P, either by serotyping or using primers that can amplify a wide range of HIV-1 variants. Acknowledgements Informed consent was obtained from the study patient for antiretroviral drug resistance genotyping. We thank Dr Anne Beyou Zuliani, Siemens Healthcare Diagnostics, France, Dr Constance Delaugerre and Pr François Simon, virology laboratory, hôpital Saint-Louis, Paris, France, for helpful discussions. F.T., H.M., H.P., L.B. conceived and designed the study. H.P. and F.T. performed the experiments. H.P., F.T., P.R., C.C., L.B. analysed the data. F.T. and H.M. contributed to patient data collection. H.P., P.R., L.B. wrote the article. All authors read and approved the final version of the manuscript. Conflicts of interest There are no conflicts of interest.