THE EFFECT OF HIV-1 SUBTYPES ON HIV TRANSMISSION AND DISEASE PROGRESSION IN RAKAI DISTRICT, UGANDA by NOAH KIWANUKA, MBChB, MPH Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Advisor: Prof. Christopher C. Whalen, MD, MS Department of Epidemiology and Biostatistics CASE WESTERN RESERVE UNIVERSITY May, 2008 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of _Noah Kiwanuka_______________________________________ candidate for the Ph.D___degree *. (signed)__Christopher Whalen, MD, MS ______________________ (chair of the committee) __Ajah Sethi, PhD_____________________________________ __Jeffery Albert, PhD___________________________________ __Catherine Stein, PhD__________________________________ __Peter Zimmerman, PhD________________________________ ________________________________________________ (date) _March 20, 2008_______________ *We also certify that written approval has been obtained for any proprietary material contained therein. i Copyright © 2008 by Noah Kiwanuka All rights reserved ii DEDICATION I dedicate this work to: My parents; mom Edith Namuwonge Busuulwa, and dad Livingstone Busuulwa (late), Who did a lot to ensure that I got a good education and encouraged me to aspire for greater achievements My wife Josephine Nalusiba Kiwanuka, You stood by me and lovingly cheered me on And my children Faith Nakamatte, Favor Nakalembe, and any yet unborn May God help you replicate the same academic path iii TABLE OF CONTENTS List of Tables ……………………………………………………………………........v List of Figures ……………………………………………………………………….vii Acknowledgements ………………………………………………………………....viii Abstract ……………………………………………………………………………......x Introduction ……………………………………………………………………………1 Chapter 1: Background, Specific aims, and Significance ………………………..8 Chapter 2: Study Methods ………………………………………………………...38 Chapter 3: A synopsis of the three manuscripts …………………………………62 Chapter 4: HIV-1 subtype and heterosexual transmission of HIV among HIV discordant couples in Rakai, Uganda. …………………65 Chapter 5: Rate of CD4+ T cell loss by HIV-1 subtype among HIV-1 infected adults in Rakai, Uganda. ………………………………….....99 Chapter 6: Effect of HIV-1 subtype on disease progression in persons with incident HIV-1 infections, Rakai, Uganda.…………………….125 Chapter 7: Conclusions, Recommendations, Data limitations and Strengths …148 Appendices: Extra tables and figures ………………………………………………173 Curriculum vitae ……………………………………………………………………..183 iv LIST OF TABLES Chapter 4 Table 4.1: Characteristics of monogamous HIV-1 discordant couples …………….......79 Table 4.2: Comparison of selected characteristics by HIV-1 Subtype …………………82 Table 4.3: HIV transmission rates among couples with HIV-1 subtype data ..…………83 Table 4.4: HIV transmission rates regardless of stage of HIV infection ……..…………85 Table 4.5: HIV transmission rates in latent stage of HIV infection …………………….86 Table 4.6: HIV transmission and couple genital ulceration disease status ……………..87 Table 4.7: HIV transmission rate per-coital act ………………………………………...88 Table 4.8: Rate ratios of HIV transmission per-coital act ..……………………………..90 Table 4.9: HIV transmission incidence rate per 100 person years ..…………………...174 Table 4.10: Rate ratios: models with and without HIV load …..………………………175 Table 4.11: Transmission rate by enrolment status of index positive partner .………..176 Table 4.12: HIV transmission incidence rate/100 py by couple GUD status…………..177 Chapter 5 Table 5.1: Descriptive characteristics of the study population ……………………….110 Table 5.2: Comparison of HIV-1 subtype by selected characteristics ………………..111 Table 5.3: Unadjusted rate of CD4+ T cell loss by selected characteristics ………….114 Table 5.4: Adjusted rate of CD4+ T cell loss, overall and by HIV-1 subtype ………..115 v Table 5.5: HIV-1 subtype differences in rate of CD4+ cell loss………………………117 Chapter 6 Table 6.1: Characteristics of Study Participants; overall and by HIV-1 Subtype …….134 Table 6.2: Progression from HIV-1 Seroconversion to AIDS ………………………...137 Table 6.3: Progression from HIV-1 Seroconversion to death.………………………...140 Table 6.4: Characteristics of study population at enrolment ..…………………….…..181 Table 6.5: Hazard ratios of progression to AIDS.………………………………..……182 vi LIST OF FIGURES Chapter 1 Figure 1.1: The natural history of HIV-1 infection ………………………………….12 Chapter 2 Figure 2.1: The Multi-region Hybridization Assay…………………………………..48 Chapter 4 Figure 4.1: Measurement of the at risk exposure period ……………………………75 Chapter 5 Figure 5.1 Matrix-scatter plot of CD4+ T counts ……………………………….…179 Figure 5.2 Trajectories of CD4+ T cells with fitted linear line..…………………...179 Chapter 6 Figure 6.1. Kaplan-Meier curves of time-to-AIDS by HIV-1 Subtype …………....136 Figure 6.2. Kaplan-Meier curves of time-to-death by HIV-1 Subtype …………….139 vii ACKNOWLEDGEMENTS Ebenezer! I give all the glory and honor to the Lord Jesus Christ for thus far He hath brought me. On the path to completing this training, I have been graciously helped by many wonderful persons – to them all, I extend my sincere appreciation. I thank the Fogarty AIDS International Research and Training Program (AITRP) at Case Western Reserve University for funding my studies. Special thanks go to Prof. Christopher Whalen, Alice Cantini, and Grace Svilar for all the support you have accorded me. I thank Prof. Christopher Whalen, my advisor and committee chair for guiding me very well during my pursuit of this degree. I have learnt a lot from his systematic and first principles based approach to epidemiology, statistical analysis, and research. I appreciate the guidance and advice of other committee members Dr. Jeffrey Albert, Dr. Ajay Sethi, Dr. Peter Zimmerman, and Dr. Catherine Stein. I am very grateful to the Management of Rakai Health Sciences Program for mentoring me in my research career and for allowing me to use the Rakai data for my dissertation. Special thanks go to Professors Ronald H. Gray and Maria J. Wawer of Johns Hopkins University, Baltimore, USA, and David Serwadda, Fred Wabwire-Mangen, and Nelson K. Sewankambo of Makerere University, Kampala, Uganda. I am very grateful to Dr. Merlin Robb and the Management of the Makerere University Walter Reed Project for viii allowing me to use their data for my dissertation. My sincere thanks to Mr. Oliver Laeyendecker, Dr. Thomas C. Quinn, and Dr. Steven Reynolds of the National Institute of Allergy and Infectious Diseases, and the laboratory staff of Rakai Health Sciences Program for helping me with the lab work. I am very grateful to Joseph Ssekasanvu and John Baptist Bwanika, Data Managers in Rakai Health Sciences Program, for being there for me all the time I needed help with the datasets. I extend my special thanks to my wife Josephine Nalusiba Kiwanuka for encouraging me and for enduring all the time I was taken away from the family by the academic program. My sincere appreciations go to all my siblings particularly Joseph Mabirizi and Dr. Jackson S. Musuuza for being a great source of encouragement. I also thank Pastor Charles Musisi and all the members of Kalisizo Pentecostal Church, Uganda, and Pastor Abel Oriri and Heights Fellowship Church, Cleveland, USA, for praying with me. I thank Dr. Achilles Katamba, my friend and schoolmate from elementary school to Case Western Reserve University, for encouraging me to pursue this training. I am deeply indebted to the people of Rakai district, Uganda, whose participation in the research activities has given rise to important epidemiological and public health findings on the HIV/AIDS pandemic. ix The Effect of HIV-1 Subtypes on HIV Transmission and Disease Progression in Rakai District, Uganda Abstract by NOAH KIWANUKA Objectives: To determine whether HIV transmission and disease progression differ by HIV-1 subtype. Methods: The effect of HIV-1 subtypes on HIV transmission and disease progression was investigated using data from community-based cohort studies in Rakai district, Uganda. HIV-1 subtype was determined by genomic sequencing of the gag and gp41 viral regions and by the Multi-region Hybridization Assay (MHA). HIV viral loads were determined by Roche Amplicor v1.5, and CD4+ T cell counts by BD Facscalibur system. Adjusted measures of association were estimated using Poisson regression model, linear mixed effects model, and Cox proportional hazards model. Results: Adjusting for age, viral load, and genital ulcers (GUD), HIV transmission was higher for subtype A relative subtype D (Rate ratio, 1.98; 95% CI, 1.17 - 3.34). Age (<30 years) of the positive partner (RR, 3.98; 95%CI, 1.90 - 8.35), GUD (RR, 1.83; 95% CI, x 1.16 - 2.89), and viral load (RR, 2.17; 95% CI, 1.60 - 2.94) were significant risk factors for HIV transmission. Regardless of subtype, the rate of CD4+ T cell loss (cells/μL per year) was -31.6 (95%CI; -44.6, -18.6), adjusted for age, baseline CD4 counts, and viral load. Adjusted rate of CD4+ cell loss was -8.8 (95%CI; -35.1, 17.4) for subtype A, -34.3 (95%CI; -64.0, -4.6) for R, -37.5 (95%CI; -54.1, -20.8) for subtype D, and -77.5 (95%CI; -136.6, -18.4) for M. The median time from seroconversion to AIDS was shorter for subtypes D, R and M (6.5, 5.6, and 5.8 years, respectively), compared with A (8.0 years; p =0.022). Relative to subtype A, adjusted hazard ratios (95% CI) of progression to AIDS were 2.13 (1.10 - 4.11) for subtype D, 2.16 (1.05 - 4.45) for recombinants, and 4.40 (1.71-11.3) for infection with multiple HIV strains. Conclusions: In Rakai, subtype A has a higher rate of HIV transmission than subtype D or inter-subtype recombinants. However, subtype A has a slower rate of progression to AIDS compared to subtype D, recombinants, and multiple HIV strains. Determination of HIV-1 subtype may be important in trials of preventive and therapeutic HIV-1 vaccines and in the management of HIV infected individuals. Key words: HIV-1 subtypes, HIV transmission, disease progression Word count: 347 xi INTRODUCTION The human immunodeficiency virus (HIV) is a genetically diverse virus and its genetic diversity appears to affect its pathogenicity and natural history.