The classification of HIV strains in groups, subtypes, sub-subtypes, and circulating recombinant forms (CRFs) allows one to draw a broad picture of the distribution of HIV strains.1,2 Group M predominates however, all HIV-1 groups in Africa, subtypes, and at least 7 of the 15 known CRFs cocirculate, but their geographic distribution within the continent is heterogeneous.3 The highest genetic diversity is observed in West Central Africa; all HIV-1 groups cocirculate in Cameroon, and the western part of the Democratic Republic of Congo (DRC) harbors the highest number of cocirculating HIV-1 group M variants, which are also characterized by high intrasubtype diversity, a high proportion of unique recombinants, and a relatively substantial number of strains that cannot be classified into the current subtypes.4-7 Therefore, it is thought that the initial diversification of group M may have occurred within or near the DRC.8,9 In addition, the earliest case of HIV-1 infection has been documented in a serum sample from 1959 in Kinshasa, the capital city of the DRC.10 Despite the fact that HIV has been spreading for a longer period in this region of Africa, HIV seroprevalence rates are much lower than those observed in East Africa and southern Africa.11-13
Although the government has endorsed a national policy for HIV/AIDS prevention and control in the DRC since 1984, mismanagement and corruption have led to severe deterioration of the socioeconomic situation, with the breakdown of the health system.14 In addition, since 1998, the country has experienced an internal armed conflict involving 6 foreign armed forces. As a consequence of the war, 2 million Congolese are internally displaced; unemployment and poverty have increased; the health and education sectors have deteriorated; and informal economies, including those associated with sex work, have expanded.15
The purpose of our studies in the DRC was to study the HIV epidemic against the background of political instability and civil war. We thus conducted a seroepidemiologic survey in 2002,13 and we genetically characterized the HIV-positive samples identified during this serosurvey and compared the distribution of HIV-1 variants with those described in 1997 to see whether changes in subtype distribution had occurred.
MATERIALS AND METHODS
Study Population and DNA Isolation
A total of 288 HIV-1-positive samples collected during an epidemiologic survey conducted in February 200215 were genetically characterized. The samples were obtained from 4 cities in the DRC: Kisangani in the north, Mbuji-Mayi in the south, the capital city Kinshasa in the west, and Lubumbashi in the extreme southeast. After providing informed consent, the participants were interviewed according to a standardized questionnaire developed for this study to collect social and demographic information such as age, nationality and travel within or out of the DRC. Blood samples were collected in EDTA anticoagulant tubes. Buffy coat and plasma samples were stored at −20°C and shipped on dry ice for further genetic characterization. DNA was extracted using the Qiagen DNA isolation kit (Qiagen SA, Courtabeuf, France).
Genetic Subtyping by Sequence and Phylogenetic Tree Analysis
Sequencing of Part of the Envelope (V3-V5 region)
The genetic subtype in the envelope was determined by direct sequencing of the V3-V5 region. Outer primers were designated from complete envelope or full-length genome sequences of previously characterized samples from the DRC6: env1 (5′-GTCTATTATGGGGTACCTGTGTGG-3′, position 6336-6359 on HxB2) and env2 (5′-GCGCCCATAGTGCTTCCTGCTGC-3′, position 7819-7797 on HxB2). Previously described ES7 and ES8 primers were used as inner primers. The amplified DNA was purified (QiaQuik gel extraction kit; Qiagen SA) and directly sequenced using Big-Dye Chemistry (Applied Biosystems, France) according to the instructions of the manufacturer. Electrophoresis and data collection were performed on an Applied Biosystems 3100 Genetic Analyzer.
Nucleotide sequences were aligned using CLUSTAL W16 with minor manual adjustments, bearing in mind the protein sequences. Regions that could not be aligned unambiguously because of length or sequence variability were omitted from the analysis. Phylogenetic trees using the neighbor-joining method and the bootstrap approach were implemented with CLUSTAL W. Genetic distances were calculated with the Kimura 2-parameter method.17 The new sequences were aligned with known representatives of the different subtypes, sub-subtypes, and CRFs described in West and Central Africa.18-24 Samples that could not be classified into the known subtypes and/or CRFs were further analyzed to see whether they cluster with unclassified sequences from our previous survey in 1997.6
Phylogenetic analysis was conducted for each sequence individually. Different trees were then constructed for each group of new sequences thought to cluster together, and, finally, a general tree was obtained to visualize the results. The clustering of each new sequence should be concordant among all trees.
The distribution of HIV-1 genetic variants from the 2002 survey was compared with that obtained in 1997 using the Fisher exact test (STATA 7.0 software; Stata Corporation, College Station, TX).
Accession Numbers of the New Sequences
The sequences have been submitted to the European Molecular Biology Laboratory (EMBL) genbank under the following accession numbers: AJ877578 to AJ877908.
HIV-1 env Genetic Subtypes in the Democratic Republic of Congo in 2002
Among the 288 HIV-positive samples studied, 144 were from Kinshasa, 43 were from Mbuyi-Mayi, 78 were from Lubumbashi, and 23 were from Kisangani. Samples were mainly obtained from tuberculosis patients (n = 113 [39% of samples tested]; 49 from Kinshasa, 5 from Kisangani, 28 from Lubumbashi, and 31 from Mbuyi-Mayi) and from outpatients with clinical signs of AIDS (n = 79 [27% of samples tested]; 39 from Kinshasa, 13 from Kisangani, 27 from Lubumbashi, and 0 from Mbuyi-Mayi). Only a limited number of samples were from women attending antenatal clinics (n = 25; 3 from Kinshasa, 5 from Kisangani, 5 from Lubumbashi, and 12 from Mbuyi-Mayi) or from female sex workers (FSWs; 37 from Kinshasa only). An additional 34 samples represented miscellaneous population groups: 19 sexually transmitted disease (STD) patients (7 from Lubumbashi and 12 from Kinshasa), 8 blood donors (4 from Lubumbashi and 4 from Kinshasa), and refugees residing in Lubumbashi but originating from the northeastern part of the country (n = 7). All 288 samples were from adults (mean age: 29.4 ± 9.6 years); only 1.3% were not of Congolese nationality, and only 2.1% had traveled outside the DRC.
Table 1A shows exact numbers and percentages of the different subtypes and CRFs in the 4 study sites in the DRC, and Figure 1 visualizes the heterogeneous subtype distribution in the 4 regions studied. The genetic heterogeneity in the cocirculating subtypes and CRFs is high: 6 in Kisangani, 7 in Mbuyi-Mayi, 9 in Lubumbashi, and 11 in Kinshasa. In addition, 16 (5.6%) samples could not be classified into a known subtype and/or CRF. Subtype A predominates in Kisangani, Kinshasa, and Mbuyi-Mayi, whereas subtype C is largely predominant in Lubumbashi. In Mbuyi-Mayi, the other southern city, subtype C is the second important variant. Subtype D increases from south to north, subtype G is present at different levels in the 4 cities and represents up to 21.7% of cases in Kisangani, and subtype H is more frequently present in the north and west. All subtype F viruses belonged to sub-subtype F1. None of the new strains clustered with the tentative subtype L viruses and CRFs that have been documented to play a major role in the epidemic in other parts of Africa represented only minority or anecdotic variants in the DRC.
The phylogenetic trees of the V3-V5 envelope sequences illustrate the high degree of intrasubtype diversity. The genetic distances were calculated, and mean intrasubtype diversity was as follows: 19.8 (range: 13.4-7.2) for subtype A, 18.4 (12.0-25.9) for subtype C, 19.03 (14.0-24.1) for subtype D, 14.7 (13.2-24.8) for subtype F1, 20.3 (15.0-28.9) for subtype G, 21.1 (16.0-24.9) for subtype H, and 19.1 (15.7-22.9) for subtype J. Subclusters within certain subtypes were seen (Fig. 2), although they were not supported by high bootstrap values because of the short sequence fragment, for example, subtype C in Lubumbashi (see Fig. 2d) and subtype A in Mbuji-Mayi (see Fig. 2c) and Kinshasa (see Fig. 2b). Some of the samples obtained in 2002 clustered with unclassified samples from the 1997 survey (in red) and could thus represent new HIV subtypes or CRFs specific to the DRC; for example, 2 of the 6 samples clustering with CRF01-AE formed a separate subcluster with a previously described complex CRF01-recombinant virus,25 and a clear cluster constituted by several strains indicated by “X” corresponded to all the unclassified samples found in Mbuji-Mayi and Lubumbashi as well as to 2 of the 11 unclassified viruses in Kinshasa.
Distribution of HIV-1 env Genetic Subtypes From 1997 to 2002: Increase of Subtype C in Kinshasa
In the 1997 survey, 249 HIV-1-positive samples were genetically characterized in the env V3-V5 region.6 Similarly, as in 2002, the samples were predominantly taken among tuberculosis patients (34% of samples tested) and outpatients with clinical signs of AIDS (27% of samples tested) but also from woman attending antenatal clinics, FSWs, and miscellaneous population groups like blood donors and STD patients. At the time of that study, several HIV-1 variants were not yet described, and we reanalyzed all the sequences by including the recently described new subtypes and CRFs. In addition, we sequenced 39 other samples from this survey previously only characterized by heteroduplex mobility assay (HMA) or subtype A-specific polymerase chain reaction (PCR) (n = 28) or which could not be amplified with the primers used at that time (n = 11). Table 1B shows the detailed distribution of the HIV-1 variants in 1997, and results are visualized in Figure 1. Comparison of the mean genetic distances showed a slight increase in intrasubtype diversity over time: from 16.7 to 19.8 for subtype A, from 15.9 to 18.4 for subtype C, from 15.7 to 19.0 for subtype D, from 12.8 to 14.7 for subtype F1, from 16.4 to 20.4 for subtype G, from 17.3 to 21.1 for subtype H, and from 17.1 to 19.1 for subtype J.
We compared the subtype and/or CRF distribution between 1997 and 2002 in more detail in Kinshasa, because the numbers of samples were relatively high and from comparable population groups (n = 145 and n = 144, respectively). As shown in Table 1, the overall proportions of subtype A, D, and G strains did not change but that of subtype H strain decreased; for the remaining variants like F1, J, K, and CRF01, the numbers were too low to allow any conclusion. Conversely, there was a greater proportion of subtype C in 2002, which increased from 2.1% to 9.7%. To analyze more in detail whether this trend was seen in all population groups tested, we compared subtype distribution in population groups with large and comparable numbers of samples characterized (Table 2). The proportion of subtype C infections increased between 1997 and 2002 from 2.2% to 6.1% in tuberculosis patients (P = 0.618), from 0% to 7.9% in outpatients with AIDS (P = 0.256), and from 0% to 18.9% in FSWs (P = 0.013). Overall, the proportion of subtype C infections increased significantly from 1.0% to 10.5% in these 3 population groups (P = 0.004).
In Mbuji-Mayi, the second city that was studied in 1997 and 2002, the comparison was more difficult because of the small sample sizes (n = 60 and n = 43, respectively). In the limited numbers of samples tested, the overall proportions of subtype A and C did not significantly change between 1997 and 2002.
The overall objective of this study was to examine the distribution of HIV-1 variants over time in the DRC against a background of political and economic instability and civil war. We compared the distribution of HIV-1 variants in sentinel population groups from a serosurvey performed in 2002 with that obtained 5 years ago. The study was carried out in 4 major cities in the DRC, 2 (Kinshasa and Mbuji-Mayi) of which were also included in the previous survey. Bwamanda, located in the northern Equateur province, could not be included in the 2002 survey because of the insecure conditions related to the civil war. With the exception of Kisangani, which was under the control of the Rassemblement Congolais pour la Democratie (RCD) and the Rwandan army in the northeastern part of the country, the 3 other sites studied in 2002 are located in the government-held areas. Overall, there were no significant changes in the characteristics of the populations studied compared with those of the previous study.6,13,15
As in 1997, we showed a high overall genetic diversity, according to the region studied, of between 6 and 11 cocirculating HIV-1 variants. Subtype A is the most prevalent variant, except in the extreme south, where subtype C predominates. In addition, 5.6% of the strains could not be classified, and some strains formed a separate cluster with previously unclassified strains from the 1997 survey; however, full-length sequence analysis is necessary to determine to what extent they represent new HIV subtypes or CRFs. Overall, the geographic distribution of HIV-1 variants in the DRC seems to reflect the distribution of variants involved in the local epidemics in neighboring countries, where certain variants predominate and cocirculate. For example, in Kisangani in the northeast, subtypes A and D are relatively well represented, and in countries bordering the DRC in the northeast, such Kenya, Uganda, and Rwanda, the epidemic is also predominated by subtypes A and D in different proportions.26-29 Similarly, subtype C is found mostly in the south of the DRC (Lubumbashi), and the epidemic in countries bordering this part of the DRC, such as Zambia, is predominated by subtype C.30
Between 1997 and 2002, we observed in Kinshasa a significant increase of subtype C from 1.0% to 10.5%. This trend was observed in 3 different but comparable population groups. The increase of subtype C was the most striking in FSWs: 0% in 1997 to 18.9% in 2002. Despite stable seroprevalence over time, which suggests a stable HIV epidemic, our study showed that combining molecular epidemiology with serosurveillance data on sentinel populations can add significant new information on trends in the HIV/AIDS epidemic. All FSWs studied in 2002 were active in commercial sex work for a limited time only; therefore, it can be assumed that subtype C infections were recently acquired. Factors leading to the emergence of subtypes are not precisely known but are most probably a combination of multiple events, such as genetic drift, adaptation to human beings, evasion of the host's immune system, and founder effects in certain population groups. Whether the increase of subtype C is related to population movements during the recent conflict in the DRC cannot be established from this study; it might even be possible that in the absence of this civil war and the consequent forced migrations of populations, other subtypes have increased or subtype C has increased even more.31 Since 1998, however, numerous soldiers from Zimbabwe, Namibia, and Angola have been enrolled by the central government of the DRC to help maintain social coherence in Kinshasa, Mbuyi-Mayi, and Lubumbashi.32 The troops from Zimbabwe, especially, are coming from a region where subtype C is almost the unique variant in the local epidemic and where HIV prevalences are already high (eg, 30% of women attending antenatal clinics).11,33,34 In addition, military personnel constitute a high-risk population. A possible explanation for the increase in subtype C among FSWs could be that as a consequence of the increased unemployment and poverty, clients of FSWs are now more likely foreign soldiers coming from regions where subtype C predominates.
Because so many HIV-1 variants cocirculate in the DRC, it is not easy to observe changes in subtype patterns over a short period. The introduction of new strains could lead to even more complex recombinants or CRFs, possibly with different biologic properties. Similarly, foreign troops and displaced people can become infected with one of the numerous HIV-1 variants that circulate in the DRC and could subsequently introduce new variants and/or recombinants when returning to their respective countries or cities. This would also lead to a more complex epidemic in regions where only a limited number of HIV-1 variants actually cocirculate. This ever-increasing genetic diversity of HIV is thus likely to continue to be a challenge to treatment and vaccine strategies in Africa. Continuous monitoring of HIV variants seems necessary to adapt treatment and vaccine strategies so that they are efficient against local and contemporary circulating HIV variants. Our study showed that genetic characterization of HIV-positive samples from sentinel surveys can provide significant additional information on new trends in the HIV epidemic. Monitoring for subtypes and/or CRFs can thus be implemented, together with seroprevalence studies on sentinel or at-risk populations.
The authors express their gratitude to the Ministry of Health, the National AIDS/STD Program, and the National Ethical Committee for granting permission to perform this survey. The authors also thank the directors and staff of the laboratories in the different regions, especially Antoinette Mayamba, Gisèle Bwalungu, Olivier Disasi, Adolphine Kalume, and Rigobert Kambembo, for providing logistic and technical support in the field and for their kind cooperation.
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