Five subtype B clusters ensued (see Fig. 1). Cluster A contains 10 sequences (M20, M33, M34, M35, M40, M50, M51, M52, M64, and M65) supported by significant bootstrap values in env (98) and pol (89), except M33, which branches with a bootstrap value of 83 in pol but only 60 in env. Cluster B contains 7 sequences (M11, M17, M21, M24, M25, M55, and M61) supported by a bootstrap value of 94 in env and 95 in pol. Cluster C contains 3 sequences (M57, M58, and M63) supported by a bootstrap value of 100 in pol and 86 in env. Cluster D contains 3 sequences (M47, M68, and M70) supported by a bootstrap value of 100 in pol and 93 in env. Cluster E contains 3 sequences (M67, M77, and M78) supported by a bootstrap value of 100 in both phylogenies.
Known Epidemiologic Relationships in the Cohort
Epidemiologic linkage was known a priori for some patients. Nine heterosexual couples (M02/M08, M11/M21, M25/M55, M20/M52, M35/M50, M64/M65, M57/M58, M42/M44, and M14/M23), 1 homosexual couple (M68/M70), and 1 group of 1 man and 2 women (M45/M48/M54) had a known direct link. Patients M11 and M25 were also epidemiologically related, thus linking couples M11/M21 and M25/M55.
ML analyses confirmed that sequences recovered from patients with a known epidemiologic relationship shared close genetic relatedness, were topologically associated, and clustered with bootstrap values greater than 80, except for sequences from the group M45/M48/M54. Sequences from epidemiologically related couples fell within cluster A (M20/M52, M35/M40, and M64/M65), cluster B (M11/M21 and M25/M55), cluster C (M57/M58), and cluster D (M68/M70) as well as in isolated branches (M14/M23 and M42/M44). Sequences from couple M02/M08 were B/F1 recombinants.
Comparison of Envelope and Polymerase Phylogenies
Env and pol phylogenies yielded the same overall results, and relationships in one of the studies were reproduced in the other. The env tree, however, tended to present somewhat lower bootstrap values. Examples are the lower bootstrap figures for the common branch of clusters A (including sequence M33), C, D, and E as well as for couple M42/M44. In contrast, couple M14/M23 was strongly supported in env. Nonclustered sequences in env (eg, samples from patients M12, M39, M49, and M76, and from inhabitants of a neighboring county; see Fig. 1A) were invariably nonclustered in pol.
Demographic, Social, Clinical, and Behavioral Associations Within Phylogenetic Clusters
Samples from 29 (46%) patients yielded clustered sequences (Table 1). On univariate analysis, these patients were found to be younger, more likely to have a known epidemiologic link within the cohort, and to always have lived in Miracema. Multivariate logistic regression analysis identified having a known direct epidemiologic relationship (odds ratio [OR] = 4.46, 95% confidence interval [CI]: 3.27 to 5.68; P = 0.014) and having always lived in Miracema (OR = 5.48, 95% CI: 4.30 to 6.65; P = 0.0044) as independent predictors of belonging to a cluster.
Genotypic Resistance to Reverse Transcriptase and Protease Inhibitors
Two B/F1 recombinants and 7 subtype B sequences recovered from treatment-experienced patients harbored mutations known to confer reduced drug susceptibility (see Fig. 1B). No evidence of transmission of resistant variants was recorded. The presence of these mutations did not seem to obscure or artificially create phylogenetic relationships.
Current epidemiologic data show the spread of the AIDS epidemic toward small counties and the innermost parts of Brazil.1 Small Brazilian communities are likely to be challenged to provide care to an increasing number of patients, yet these places generally have a much less comprehensive health infrastructure and lack physicians familiar with the medical management of HIV-1 infection. Patients from these areas may face problems such as confidentiality issues (which can present barriers to counseling, testing, and treatment), long distances to medical facilities, and lack of nongovernmental organization support. The features of HIV-1 infection in these places need to be appropriately studied so as to optimize the institution of adequate control measures, grasp the true magnitude of the problem, improve clinical recognition and management, and better allocate resources.
The extreme nucleotide sequence variation of HIV-1 makes it possible to trace patterns of viral spread between populations, groups, and people.2 There has been considerable discussion, however, on which genomic region is the most informative and on which phylogenetic methodology5,26 is best suited for such investigations. In recent years, genotypic analysis of antiretroviral drug resistance has generated expanding local and public pol data sets. The suitability of this genomic region to epidemiologic investigations is being increasingly demonstrated,27-29 although it remains a controversial issue.30-32 The present Miracema cohort offers, in our opinion, a unique contribution to the subject. Previous investigations on the usefulness of pol involved the study of viral transmission between isolated cases27,28,30 or the preselection of samples on the basis of closest pairwise distances.29 Instead, we had the opportunity to study cross-sectional samples from 63 members of a 78-patient cohort. Our patients were placed at variable degrees of proximity in the transmission chains, and analyses of both regions yielded essentially the same overall results. Caution should be exercised when refined transmission chain analysis is needed, however, because a pattern of parallel evolution may emerge among sequences that harbor a similar set of resistance mutations.33
Caution should also be taken when studying patients who might have been multiply infected. In a C2V3 study, the establishment of epidemiologic linkage between 2 multiply infected IVDUs who had shared infected needles required the analysis of a series of proviral and plasma strains.34 In the Miracema cohort, the only situation in which a phylogenetic relationship between env and pol sequences from linked patients could not be established involved an IVDU (patient M48) and his female sexual partners (M45/M54). Interestingly, studies that found pol suboptimal for phylogenetic analyses also involved IVDU-recovered sequences.3,30
We found a polyphyletic pattern suggesting multiple viral introductions in the region. Subtyping analysis was in accordance with studies that found the prevalent subtypes in southeast Brazil to be B, F1, and B/F1 recombinants.22,35 We also found 29 samples (representing 46% of the cohort) forming 6 clearly defined clusters. Intracluster cases, including those with no known direct epidemiologic link, probably took part in the same chain of viral transmission, suggesting the existence of sexual networks and the emergence of multiple new infections within a relatively short period. Such potential molecular markers of high incidence highlight the urgent need to perform incidence studies in inner Brazil. In general, clusters were supported by a lower bootstrap value in env. This might reflect the continuously evolving nature of C2V3/env and may even indicate that the targeted fragment of pol is better suited for establishing relationships, especially after a given period has elapsed between transmission and sampling. The presence of mutations associated with drug resistance in the Miracema cohort did not seem to obscure or artificially create phylogenetic relationships.
In summary, this molecular epidemiology study of HIV-1 sequences from a small Brazilian county found evidence of multiple clusters of strains sharing close genetic relatedness, suggesting the existence of sexual networks and a high-incidence molecular profile. Our results highlight the need for further investigations to delineate the features of HIV-1 infection in small Brazilian counties better as well as appropriate control strategies to halt epidemic spread toward these areas.
The authors are indebted to the patients for agreeing to participate in this study. They thank the many dedicated health care professionals in Miracema, without whom this study would not be possible. They also thank Laboratório Avançado de Saúde Pública in Salvador, Bahia State, Programa Nacional de DST/AIDS, and Fundação de Amparo à Pesquisa do Estado da Bahia for organizing invaluable international workshops on bioinformatics.
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Keywords:© 2006 Lippincott Williams & Wilkins, Inc.
HIV-1; molecular epidemiology; Brazil; transmission networks; small counties; pol gene