Evolutionary trend of drug resistance associated mutations in the protease region of HIV-1 polymerase gene in Cameroon

Atah Sielenou Youpa Jude (atahjude@yahoo.com)
Physiologic Sciences and Biochemistry, University of Yaounde 1
June, 2013


Human Immunodeficiency Virus (HIV) is a major public health concern worldwide. Much research attention has been paid to the virus and its resultant syndrome, the Acquired Immunodeficiency Syndrome (AIDS) since the discovery of the virus in the 1980s. Although sub-Saharan Africa is only 10% of the world population, more than 67% (22.6 million) of those living with HIV infection worldwide live in this region. The prevalence of the infection in Cameroon in 2011 was reported to be 4.3% in the Cameroonian population. This fall from 5.4% reported in the past is due to antiretroviral therapy (ART) and sensitisation campaigns, especially highly active ART (HAART). However, HIV drug resistance HIVDR has been reported in Cameroon, with an increasing number of patients treated with first-line regimens showing resistance. Such resistant patients are usually placed on second-line regimens. In this project, we propose to survey resistance to these second-line drugs in order to provide information for better patient management.

We carried out a cross-sectional descriptive study from November 2012 to April 2013 using sequences of the pol gene generated from patients in Cameroon and available in the Genbank at Los Alamos. These sequences were downloaded from the HIV sequence database of the Los Alamos National Library (LANL).The sequences were divided into two main groups: Group A made up of sequences generated before 2004; and Group B made up of sequences from 2004 to date. Group B was subdivided into three groups constituting three time periods (2004-2007; 2008-2010; 2011-2013). The sequences were then aligned and trimmed using MEGA version 4.0.2, which is a Molecular Evolutionary and Genetic Analysis tool used for genotypic and phylogenetic studies. They were then retrieved and submitted to the Stanford University HIV Drug Resistance database for mutational analysis using the CPR 6.0 a tool used for population level surveillance of HIVDR. Individual drug resistances were obtained by submitting these mutations to the Mutational Analysis for RT and PR tool still at the Stanford HIV drug resistance database. The results from CPR 6.0 analysis were copied on Microsoft Excel 2007 sheets for statistical analysis. Tables, Graphs and evolutionary trends were then established.

From 1286 HIV-1 sequences containing the protease region collected from Cameroonian patients, 1253 (97.4%) sequences were processed for the analysis, based on inclusion and exclusion criteria. The general trend of resistance to PIs was 2.9% before 2004, 2% between 2004 and 2007, 1.3% between 2008 and 2010, and 2% between 2011 and 2013. Aggregated analysis showed that HIV-1 Group M represents about 96% of the entire HIV-1 pandemic in Cameroon, against 4% for HIV-1 Group O. A wide genetic diversity was found, with increasing prevalence of CRF02_AG. The overall rate of CRF02-AG was 70%, followed by CRF01_AE (6%), J (5%), D (4%), G (4%), F (3%), A (3%).The analysis showed a high rate of variability between the two expert algorithms commonly used for HIV-1 drug resistance analysis and interpretation, with similar discordance and concordance rates obtained in terms of drug resistance associated mutations overtime.
The trends in PI resistance reflect the use of unboosted PI before 2004, and the gradual exclusion of unboosted PI, coupled to the inclusion of boosted PI in second line regimens in our national guidelines since 2004. Since PI boosted with ritonavir (PI/r) are drugs with very high genetic barriers, which can sustain susceptibility even in the presence of some drug-resistant mutants, the current re-emergence of PI/r resistance (2%) with the use of PI/r is likely due to the increasing number of patients switching on second line regimens, which in turn would be accompanied by increasing risks of resistance to PI/r drugs.
The high prevalence of HIV group O infections may have some public health and/or individual patients monitoring implications, because group O is known to be naturally resistant to NNRTIs, a drug class commonly and widely used in first line ART in Cameroon. The high prevalence of CRF02_AG (60% before 2004, 78% in 2004-2007, and 66% at present) could be due to its cytopathic effect during its replication cycle. These data confirm previously published reports on the HIV situation in Cameroon.
The discordance observed between the Stanford HIV database and ANRS are probably due to differences in terms of the population of sequences used.

Conclusion and Recommendations:
From the foregoing, we conclude that:our hypothesis that "The frequency of HIV-1 mutations associated with resistance to PIs in Cameroon is increasing overtime" was tested and confirmed, that resistance to PI/r is slowly increasing in Cameroon, that PI resistance was higher with the use of unboosted PI, that HIV-1 Group M is the main genotype in the HIV population of Cameroon, that CRF02-AG is the main circulating recombinant form, that there is a significant discordance rate between the main expert lists used for the analysis and interpretation of HIV-1 drug resistance associated mutations, that current drug regimens used in Cameroon seem to be suitable since HIV-1 group M is the most abundant genotype but the most common major PI mutations circulating in Cameroon show a low to moderate level of resistance to at least one of PI drugs mainly used in Cameroon.