The Rates of HIV Superinfection and Primary HIV Incidence in a General Population in Rakai, Uganda

MAJOR ARTICLE The Rates of HIV Superinfection and Primary HIV Incidence in a General Population in Rakai, Uganda Andrew D. Redd,1 Caroline E. Mullis,2 David Serwadda,4,5 Xiangrong Kong,3 Craig Martens,7 Stacy M. Ricklefs,7 Aaron A. R. Tobian,2 Changchang Xiao,3 Mary K. Grabowski,3 Fred Nalugoda,4 Godfrey Kigozi,4 Oliver Laeyendecker,1,2 Joseph Kagaayi,4 Nelson Sewankambo,4,6 Ronald H. Gray,3 Stephen F. Porcella,7 Maria J. Wawer,3,a and Thomas C. Quinn1,2,a 1 Background. Human immunodeficiency virus (HIV) superinfection has been documented in high-risk individuals; however, the rate of superinfection among HIV-infected individuals within a general population remains unknown. Methods. A novel next-generation ultra-deep sequencing technique was utilized to determine the rate of HIV superinfection in a heterosexual population by examining two regions of the viral genome in longitudinal samples from recent HIV seroconverters (n = 149) in Rakai District, Uganda. Results. The rate of superinfection was 1.44 per 100 person years (PYs) (95% confidence interval [CI], .4–2.5) and consisted of both inter- and intrasubtype superinfections. This was compared to primary HIV incidence in 20 220 initially HIV-negative individuals in the general population in Rakai (1.15 per 100 PYs; 95% CI, 1.1–1.2; P = .26). Propensity score matching (PS) was used to control for differences in sociodemographic and behavioral characteristics between the HIV-positive individuals at risk for superinfection and the HIV-negative population at baseline and follow-up. After PS matching, the estimated rate of primary incidence was 3.28 per 100 PYs (95% CI, 2.0–5.3; P = .07) controlling for baseline differences and 2.51 per 100 PYs (95% CI, 1.5–4.3; P = .24) controlling for follow-up differences. Conclusions. This suggests that the rate of HIV superinfection in a general population is substantial, which could have a significant impact on future public health and HIV vaccine strategies. Human immunodeficiency virus (HIV) superinfection occurs when an HIV-infected individual acquires a new viral strain that is phylogenetically distinct from all detectable viral strains at a previous time point [1]. Inter- and intrasubtype HIV superinfections have been Received 17 January 2012; accepted 9 April 2012; electronically published 6 June 2012. a M. J. W. and T. C. Q. share senior authorship. Correspondence: Andrew D. Redd, PhD, National Institute of Allergy and Infectious Diseases, Rangos Bldg, Rm 527, 855 N Wolfe St, Baltimore, MD 21205 (). The Journal of Infectious Diseases 2012;206:267–74 Published by Oxford University Press on behalf of the Infectious Diseases Society of America 2012. DOI: 10.1093/infdis/jis325 reported in high-risk individuals exposed through sexual or intravenous drug use [1–12]. The rate of HIV superinfection has often been found to be relatively frequent, particularly if multiple genomic sites are examined [6, 9, 13–15]. Other researchers have found no evidence of superinfection in both small- and largescale studies; however, these studies utilized clonal analyses that were likely not sensitive enough to detect the levels of virus observed in some superinfection cases [16–18]. These discrepancies partly reflect differences in the techniques used to identify and verify superinfection [19]. Initial studies of the frequency of superinfection utilized heteroduplex mobility or multiregion hybridization assays followed by selective clonal analysis Rates of HIV Superinfection • JID 2012:206 (15 July) • 267 Laboratory of Immunoregulation, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, and 2Johns Hopkins Medical Institute, and 3Bloomberg School of Public Health, Johns Hopkins University, Baltimore, Maryland, and 4Rakai Health Sciences Program, Kalisizo, and 5School of Public Health, and 6School of Medicine, Makerere University, Kampala, Uganda; and 7Genomics Unit, RTS, Rocky Mountain Laboratories, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana METHODS Superinfection Population The RCCS is a rural, community-based, open cohort of persons aged 15–49 years in Rakai District in southwestern Uganda, which has been described in detail previously [21]. Since 1994, interviews and venous blood samples have been obtained annually from approximately 14 000 consenting adults living in 50 villages. All subjects provided written informed consent for sample storage and testing. The study was approved by the Science and Ethics Committee of the Uganda Virus Research Institute, the Uganda National Council for Research and Technology, Western Institutional Review Board, and the Committee on Human Research at Johns Hopkins Bloomberg School of Public Health. Known seroconverters who had a positive HIV serological result within 2 years of a prior negative test between 1998 and 2004 and who had also provided at least 1 subsequent serological sample prior to 2009 were randomly selected for examination from the RCCS population (n = 203) [21, 22]. To identify HIV superinfection, viral RNA was isolated from serum obtained at the seroconverter’s first HIV-positive time point (baseline) and the latest time point available prior to initiation of antiretroviral therapy (ART), loss to follow-up, or death [22]. Subjects were excluded from analysis if neither genomic region in the baseline sample could be amplified (n = 14). Subjects were also excluded if the follow-up genomic region corresponding to the amplified baseline sample failed to amplify (n = 40). The remaining subjects’ (n = 149) viral RNA extracts were initially amplified by reverse-transcription polymerase chain reaction (RT-PCR) in duplicate; the resulting products were pooled and subsequently amplified in a nested PCR 268 • JID 2012:206 (15 July) • Redd et al strategy using barcoded primers specific for use on the 454 pyrosequencer platform [1]. Laboratory Analysis Briefly, amplicons of p24 (approximately 390 base pair [bp]) and gp41 (approximately 324 bp) were amplified and sequenced as previously described [1]. The Amplicon Library Preparation Method was performed as recommended by the manufacturer (Roche), and all PCR products were purified with the following minor alterations. In an effort to eliminate the capture of primers, the bead-to-target ratio was reduced by incubating 30 μL of AMPure Beads XP (Agencourt; Beckman Coulter Genomics) with 25 μL of PCR product diluted in 25 μL of water. Purified PCR products were quantified using PicoGreen (Invitrogen), and each template was diluted to a 1 × 109 molecules/μL stock. The amplicon pools were made by combining 5 μL of each diluted barcoded template to make a final 1 × 109 molecules/μL stock containing 14 barcoded amplicons. Preparation of templated beads for next generations sequencing (NGS) followed the emPCR Method ManualLib-L-MV (Roche). The 1 × 109 molecules/ (...truncated)