Involvement of the Genetic Diversity of HIV-1 in the Virological Treatment Failure of First Line Antiretroviral in Kinshasa

Background: Genetic diversity of human immunodeficiency virus affects the treatment and the emergence of resistance. Some subtypes would develop resistance more frequently than others. The aim of this study is to determine the rate of virological treatment failure and the involvement of genetic diversity and different mutations in this failure in Kinshasa. Methods: Of the 153 Antiretroviral-naive patients who were included in the cohort, 138 patients have been received for the appointment of the 6 month. Clinical parameters were recorded on individual patient charts. The determination of Viral Load (VL) was done at the Laboratory of Molecular Biology. Clinical and biological parameters of the 6 month were compared with those taken at baseline of the cohort to determine the evolution of patients under treatment. Results: At the consultation of the 6 month, 138 patients (90.2%) had returned out of the 153 included. Eighty-one (58.7%) patients were women and 57 (41.3%) men. The age of patients is between 18 and 65 with an average of 37 years. Ten deaths (6.5%) and 5 (3.3%) lost have been reported. One hundred twenty-five patients (90.5%) were in clinical stage 3 and 13 (9.5%) in clinical stage 4. The median CD4 T cells is 560 cells mm. The median VLs of patients was 0.90 log10 RNA copies/ml. Of the 34 patients in virological failure, 8 (23.5%) are minimal failure, 23 (67.7%) in moderate failure and 3 (8.8%) in severe failure. According to the Pearson’s test, VLs at 6 months were highly correlated with that of inclusion, with V75 and K70 mutations for NRTIs, with V108 mutation for NNRTI well as the virological failure of treatment. Conclusion: Our results confirmed the hypothesis that high Viral Load at the start of the treatment is a poor prognosis for the development of therapy. Transmitted mutaHow to cite this paper: Ntambwe Kamangu, E., Lunganza Kalala, R., Lelo Mvumbi, G., Vaira, D. and Hayette, M.-P. (2017) Involvement of the Genetic Diversity of HIV-1 in the Virological Treatment Failure of First Line Antiretroviral in Kinshasa. World Journal of AIDS, 7, 23-33. https://doi.org/10.4236/wja.2017.71003 Received: May 4, 2015 Accepted: May 11, 2015 Published: February 24, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access E. Ntambwe Kamangu et al. 24 tions are involved in treatment failure.

tions are involved in treatment failure.

Backgrounds
The Human Immunodeficiency Virus (HIV) has a genetic diversity that is equal to the complexity of its management [1].The classification of types, groups, subgroups, sub-groups and different recombinant forms (CRFs-Circulating Recombinant Forms) or mutant allowed better understanding the virus, its geographical distribution and the evolution of the epidemic [1]- [7].It also helped direct the management of patients infected by HIV [1]- [7].Group M (Major) is the dominant group in Central Africa [1]- [7].The distribution of this group in Africa and the Democratic Republic of Congo (DRC), in particular, is very heterogeneous; it follows a complex and specific algorithm [1]- [7].This distribution is very dynamic, progressive and unpredictable; it will continue to diversify as long as the virus circulates [1]- [7].There is a very large genetic diversity of this group M in the DRC and particularly in Kinshasa [1]- [7].The subtype B, which is the dominant one in Western Europe and North America has a prevalence <1% in Kinshasa [1].
The genetic diversity of HIV affects the treatment and the emergence of resistant strains [8].Some subtypes would develop resistance more frequently than subtypes A and B [9]; this could cause some natural nucleotide polymorphisms on specific codons [10] [11].
Treatment failure includes a variety of situations, whether virological failure resulting from a persistent viral replication 6 months after starting treatment (Viral load > 200 RNA copies/ml or 2.30 log 10 RNA copies/ml), an immunological failure with persistent immunodeficiency (CD4 count < 200 cells/mm 3 ) or clinical failure that usually associated virological failure and immune deterioration [12].In 2012, 16% of patients in the first line antiretroviral therapy (ART) were estimated in treatment failure in Kinshasa [13].
The aim of this study is to determine the rate of virological treatment failure and the involvement of genetic diversity and different mutations in this failure in Kinshasa.

Study Population
At baseline, 153 patients naïve to Antiretroviral Therapy (ART) were selected for follow-up with different support centers in Kinshasa.The inclusion criteria for subjects in the cohort were: (i) being diagnosed HIV-1 positive according to national guidelines [14], (ii) being over the age of 18 years at the inclusion in the cohort, (iii) being eligible for ART in the monitoring center and (iv) be naive to ART.Viral Loads (VL), the different HIV-1 strains and the different mutations associated with resistance to ART were determined for all patients at baseline [15] [16].The 6 th month of ART, only 138 patients (90.2%) of the cohort have been received on the 153 included in the first day.

Clinical and Biological Monitoring Parameters
Clinical parameters were collected on individual patient charts in their respective centers as well as the survey forms.The determination of the VL was done in the laboratory of Molecular Biology of the Faculty of Medicine University of Kinshasa (UNIKIN) using the same "in-house" assays used at the inclusion [16] [17] [18] [19].The count of CD4+ lymphocytes was done by flow cytometry.

Comparison of Variables
In order to determine the evolution of patients under treatment, clinical and biological parameters of the 6 th month were compared with those taken at baseline of the cohort [15] [16] [17] [18].For compliance, clinical parameters are evaluated according to the classification of the World Health Organization (WHO); VLs were determined using the same "in-house" assays under the same conditions.Correlation tests were done between profiles at baseline and those at the 6 th month, VLs at baseline and mortality, VLs at baseline and treatment failure, and the prevalence of acquired mutations and failures treatment.

Statistics
Pearson correlation test was used to analyze the data.The value of p < 0.06 is considered to accommodate the size of our sample.

Operational Definition
The virological failure is defined as a persistent VL higher than 200 RNA copies/ml (2.30 log 10 RNA copies/ml) 6 months after the start of treatment [20].
Three definitions are presented for virological failure: the minimal failure (200 < VL < 5000 or 2.30 log 10 < VL < 3.70 log 10 RNA copies/ml), moderate failure (5000 < VL < 30 000 or 3.70 log 10 < VL < 4.48 log 10 RNA copies/ml) and severe failure (VL > 30 000 or VL > 4.48 log 10 RNA copies/ml) [20] [21].The patients lost are those who did not return for their appointment the 6 th month and have not been found by the community relay service from the centers.

Epidemiological Data
At the 6 th month follow-up, 138 patients (90.2%) had returned out of the 153 patients included at baseline.Eighty-one (58.7%) patients were women and 57 (41.3%) male (Table 1); with a sex ratio M/F of 0.70 (p < 0.06).The age of patients ranges between 18 and 65 years with an average of 37 years (Table 1).The

Clinical and Biological Data
At 6 th months, 5 patients were lost from the relays service (2 women and 3 men) and 10 patients were reported dead (8 women and 2 men) (Table 2).One hundred twenty-five patients (90.5%) were in clinical stage 3 and 13 (9.5%) in clinical stage 4 according to the WHO classification.The rate of CD4 T cells, which were made for 113 patients (71%) were between 98 and 1050 cells/mm 3 with 52 patients (46.02%) having CD4 counts greater than 500 cells/mm 3 and a median value of 560 cells/mm 3 (Table 1).The median VLs of patients was 0.90 log 10

Discussion
This study aimed to determine the rate of virological treatment failure and the involvement of genetic diversity and the different mutations in this failure in Kinshasa.It is the continuation of different studies published with the same population [15] [16] [17] [18].
After 6 months of ART, 10 cases (6.5%) deaths and 5 cases (3.3%) lost were recorded in the cohort of patients followed.For the case of death, the majority (8 patients) are women, while for the patients lost there are men (3 men).Of the 138 patients (90.2%) returned for their 6 th month follow-up, 81 (58.7%) patients were women and 57 (41.3%) men; thus giving a sex ratio M/F of 0.70.Different other studies have also published M/F sex ratios that tend to feminize HIV infec-tion among adults in Kinshasa and in DRC [13]- [18] [22] [23].
According to the recommendations of the World Health Organization (WHO), 125 patients (90.5%) were in clinical stage 3 and 13 (9.5%) in clinical stage 4 after 6 months of treatment.While at baseline of the cohort, 91.5% of patients were in clinical stage 3 and 8.5% in clinical stage 4 [16].This difference is not significant.Clinically, not many signs of improvement in the condition of different patients during different visits have been recorded.
Count CD4 T cells was made for 113 patients (71%).The minimum and maximum values of CD4 T cells were 98 to 1050 cells/mm 3 , respectively.The median CD4 cell count was 560 cells/mm 3 with 52 patients (46.02%) with CD4 counts above 500 cells/mm 3 .It was not possible to determine the immunological failure based on CD4 count due to the irregularity of this parameter in the patients.However, for all the patients who have the results at baseline and at the 6 th month, none were in immunological failure because the CD4 values have increased for all.The median increment of the differences in CD4 count at month 6 compared to baseline was of 247.5 cells/mm 3 .This brings up the issue of the monitoring of People Living with HIV/AIDS (PLHIV) in our environment [23] [24].
The median VLs of the patients was 0.90 log 10 RNA copies/ml.In the past, virological failure was estimated at 14.6% in 2010 [25] and 16% in 2012 [13] for the city of Kinshasa, taking into account 3 clinics that were among the recommended centers for treatment at the time [24].The difference in numbers is in the inclusion criteria of patients and selection centers, and the criteria for determining the processing failure.Indeed, the virological failure was redefined as a VL > 200 RNA copies/ml (2.3 log 10 RNA copies/ml) in 2013 [20] as opposed to a VL > 1000 copies of RNA copies/ml (3.0 log 10 RNA copies/ml) in previous years [21].In this study, 2 centers that met the criteria according to WHO's recommendations were randomly selected by district of Kinshasa [24].Eight treatment centers participated in this study.Hence for Kinshasa, according to the updated criteria, the rate of virological failure is estimated at 24.6% for 2014.
According to the Pearson's test, the VLs in the 6 th month were highly correlated with that of baseline (R 2 = 0.641, p < 0.000), with the K70 codon mutation for NRTI (R 2 = 0.558, p < 0.000), with the V75 for NRTI (R 2 = 0.448, p < 0.000), with the V108 for NNRTI (R 2 = 0.413, p < 0.000), and with the virological treatment failure (R 2 = 0.947; p < 0.000).Various studies have shown that a high VL (CV > 5.00 log 10 ) before starting treatment is a badprognosis for treatment, where the patient is doomed to failure [26] [27].Some studies have implicated the K103N and Y181C mutations for resistance and failure to treatment [28] [29].But in our case, the mutated codons that are responsible for treatment failure are: K70, V75 for NRTI and V108 for NNRTI.This presents a profile of different resistance mutations to Kinshasa specific to the different variants; this corresponds to what has been published in Africa for non-B subtypes [30].

Limitation of Study
Due to different constraints, the determination of mutations 6 th month after the beginning of treatment was not done.However, this does not remove the pertinence of the results.They related to the involvement of transmitted mutations in the treatment failure and responded to the prerogatives and actual questions on mutations.

Conclusion
Our results confirmed the hypothesis that high Viral Load at start of treatment is a poor prognosis for the therapeutic development of the patient.Correlations between virological failure, acquired mutations and viral load at baseline reinforce the importance of the usefulness of genotyping tests and viral load in early treatment to improve treatment and for adequate therapy.

Table 1 .
Characteristics of patients.
At baseline, 153 patients naïve to Antiretroviral Therapy (ART) were selected for follow-up.Ninety-one (60.1%) patients were women and 62 (39.9%) men with a sex ratio M/F of 0.68 (p < 0.06).The age of patients is in the range 18 to 65 years with a mean of 37 years.The age group most represented at the begin-

Table 2 .
Cases of death and patients lost.