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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Keywords:© 2005 Lippincott Williams & Wilkins, Inc.
HIV; Africa; Democratic Republic of Congo; subtypes; circulating recombinant form