Clinical Implications of HIV-1, HSV-2 Co-Infection and Opportunities for Intervention. Darrell Hoi-San Tan MD FRCPC

Transcription

1 Clinical Implications of HIV-1, HSV-2 Co-Infection and Opportunities for Intervention by Darrell Hoi-San Tan MD FRCPC A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy in Clinical Epidemiology Institute of Health Management, Policy and Evaluation University of Toronto Copyright by Darrell Hoi-San Tan, 2012

2 Clinical Implications of HIV-1, HSV-2 Co-Infection and Opportunities for Intervention Abstract Darrell Hoi-San Tan Doctor of Philosophy in Clinical Epidemiology Institute of Health Management, Policy and Evaluation University of Toronto 2012 HSV-2 may have adverse consequences in HIV. I evaluated the impact of HSV-2 co-infection on (highly active antiretroviral therapy)-untreated HIV infection in a systematic review of observational studies (study 1) and a retrospective cohort (study 2). I further evaluated whether HSV reactivation rates in co-infected persons differ by use of suppressive cart (study 3). Study 1 found modest evidence that HSV-2 seropositivity may be associated with accelerated progression to opportunistic infection or clinical AIDS, but not with increased HIV viral load. Some evidence suggests that HSV-2 disease activity is associated with increased HIV viral load and decreased CD4 counts. Study 2 compared rates of CD4 count change by HSV-2 status (Focus HerpeSelect ELISA) among 218 patients with a past period of ART-untreated follow-up using mixed linear regression models. No significant difference in the rate of CD4 count change was observed in HSV-2 seropositives at cells/mm 3 /year (p=0.12) in univariate analysis, and -4.5 cells/mm 3 /year (p=0.68) in analysis adjusted for sex, HSV-1, oral and genital HSV symptoms, immigrant status, and immigrant*time interaction. These findings support the need for carefully designed and executed studies of HSV-2 suppression as an adjunctive management ii

3 strategy for HIV disease, but raise questions regarding the exact mechanism of negative synergy between these viruses and the relative importance of HSV-2 latency and replication in driving these effects. In Study 3, 44 cart-naïve and 41 treated (HIV RNA<50 copies/ml) HIV+ adults with HSV-1 and/or 2 co-infection collected oral, genital and anal swabs daily for 28 days. Negative binomial models were used to quantify the relationship between cart and HSV shedding (Roche LightCycler HSV1/2). Overall HSV shedding was low, at a median (IQR) of 3.6% (0, 14.3%) of days. No relationship was seen between cart and HSV-1 or 2 shedding in univariate (RR=1.55, 95%CI=0.83,2.87) or multivariate analysis adjusted for sex, baseline CD4, recent immigrant status, and time since HIV diagnosis (arr=1.05, 95%CI=0.43,2.58). Null results were also observed for HSV-1 and HSV-2 considered separately. That HSV shedding persists despite cart suggests that trials of anti-hsv drugs for improving HIV outcomes may be warranted in such patients. iii

4 Acknowledgements I am grateful to the CIHR Canadian HIV Trials Network (CTN), the Canadian Institutes of Health Research (CIHR), and the Ontario HIV Treatment Network (OHTN) for providing fellowship funding during my PhD studies. I also wish to thank the Canadian Foundation for AIDS Research (CANFAR) for funding the retrospective cohort study presented in Chapter 3, and the Ontario HIV Treatment Network (OHTN) for funding the prospective cohort study presented in Chapter 4. I am deeply appreciative of the assistance provided by research coordinators Arshia Ali, Brian Boyachuk, Warmond Chan, Rosemarie Clarke, Gloria Crowl, Adriana D Aquila, Allyson Ion, Linda Moran and Munazza Mudassar in identifying and recruiting participants for my studies. I am particularly grateful for the helpful practical advice and camaraderie provided by the TGH Immunodeficiency Clinic research coordinators during this process, who made sure there was seldom a dull moment in the clinic. I also wish to thank Drs. Jason Brunetta, Kevin Gough, Colin Kovacs, Ed Lee, Jonathan Leutkehoelter, Anita Rachlis, Fiona Smaill and Marek Smieja for their collaboration and goodwill in facilitating participant recruitment in their respective clinics. Additional thanks go to Dr. Prakesh Shah and Elizabeth Uleryk for their methodologic assistance during the conduct of the systematic review described in Chapter 2. John Ng, Analiza Aquino, Dr. Tony Mazzulli and the personnel in the serology and virology sections of the Mount Sinai Hospital Microbiology Department were extremely helpful in providing lab testing for my studies, as were Linda Myziuk and Dr. Charu Kaushic who provided details of participant serology results from their laboratories. Sincere thanks go to my the members of my thesis committee, Drs. Rupert Kaul, Kellie Murphy Janet Raboud for their time, encouragement, expertise and wisdom over the course of my studies, and in particular to my supervisor Dr. Sharon Walmsley, who has further provided invaluable guidance and mentorship dating back to the days of my residency training. Finally, I thank my family, Dr. T. K. Tan, Michelle Tan, and Tiffany Tan Kohler, who have always taught me the values of dedication and compassion, encouraged me to be the best that I can be, and celebrated my accomplishments. Most of all I am grateful to my husband and iv

5 partner in life, Mark Duwyn, for his unwavering support and belief in me, and for his patience and love during many years of long hours and hard work. This thesis is dedicated to my two beloved sons, Sebastian and Peyton Duwyn-Tan, who were born around the time that the writing process for this text began, and who have provided two inspiring new sources of meaning and purpose in my life. v

6 Table of Contents Abstract... ii Acknowledgements... iv List of Tables... ix List of Figures... x List of Appendices... xi List of Abbreviations... xii CHAPTER 1: Introduction... 1 OVERVIEW... 1 Natural History of HIV Infection... 2 Natural History of HSV Infections... 4 Diagnosis of HSV Infection... 6 Management of HSV Infection... 9 Epidemiology of HIV, HSV-2 Co-Infection Negative Synergy 1: HSV-2 may be fuelling the HIV epidemic Negative synergy 2: HIV exacerbates HSV-2 infection Negative synergy 3: HSV-2 may exacerbate HIV disease HSV-2 Co-infection in HIV: An Opportunity for Intervention? CHAPTER 2: A Systematic Review of the Impact of HSV-2 on HIV Disease Progression BACKGROUND METHODS Study and participant criteria Exposure Outcome measures Search methods for identifying studies Selection of studies and data extraction Assessment of risk of bias Analysis RESULTS Studies included in the review Assessment of risk of bias vi

7 Studies evaluating the impact of HSV-2 seropositivity Studies evaluating other measures of HSV-2 activity Subgroup and Sensitivity Analyses DISCUSSION CHAPTER 3: A Retrospective Cohort Study Quantifying the Impact of HSV-2 on HIV Disease Progression BACKGROUND HSV-1: An unjustly ignored co-pathogen? Study Objectives METHODS Participants Determination of HSV Serostatus Clinical data Sample size considerations Data Analysis Ethical Approval RESULTS Participant characteristics Herpes characteristics Random effects models Survival analysis DISCUSSION CHAPTER 4: A Prospective Cohort Study on the Impact of HAART on HSV Shedding BACKGROUND HSV Shedding Study Objectives METHODS Eligibility Criteria HSV Serostatus Clinical data Specimen Collection vii

8 HSV Shedding Statistical Analysis Sample size calculation Ethics RESULTS Participants HSV Shedding HSV symptoms and triggers for HSV reactivation HSV Shedding by HAART status Sensitivity Analyses Comparison of HSV-1 and HSV-2 shedding rates DISCUSSION CHAPTER 5: CONCLUSION Reconciling the findings: Does HSV-2 exacerbate HAART-untreated HIV disease or not?. 116 Is acyclovir an antiretroviral drug? Is it appropriate to delay HAART with anti-hsv medications? Does HSV-2 co-infection matter in HAART-treated HIV patients? In which other groups might HIV, HSV-2 co-infection offer opportunities for intervention? 122 Conclusion REFERENCES APPENDIX A: Electronic Database Search Strategies APPENDIX B: Sample Size Considerations for Retrospective Cohort Study viii

9 List of Tables Table 1.1 Operating characteristics of gg-based HSV serology assays Table 2.1 Criteria for assessing the risk of bias in included studies Table 2.3 Impact of HSV-2 Seropositivity on HIV Disease Progression Table 2.4 Impact of HSV-2 Seropositivity on Viral Load Table 2.5 Impact of HSV-2 Seropositivity on CD4 Count Table 2.6 Impact of HSV-2 Activity on Plasma HIV RNA Level Table 2.7 Impact of HSV-2 Activity on Genital Tract HIV RNA Level Table 2.8 Impact of HSV-2 Activity on CD4 Count Table 3.1 HSV type-specific seroprevalence among HIV-infected and HIV-mixed status populations Table 3.2 Baseline characteristics of included participants, by HSV-2 serostatus Table 3.3 Clinical characteristics of herpes among participants reporting a history of herpes Table 3.4 Contingency tables of self-reported herpes symptoms by type-specific HSV serostatus Table 3.5 Phi correlation coefficients for contingency tables of self-reported herpes symptoms by type-specific HSV serostatus Table 3.6 Relationship between participant characteristics and CD4 count Table 3.7 Multivariable model showing relationships between participant characteristics and CD4 count Table 3.8 Relationships between participant characteristics and time to either two consecutive CD4 counts<350 cells/mm 3 or HAART initiation Table 4.1 Comparison of included and excluded patient characteristics Table 4.2 Participant Characteristics Table 4.3 Proportion of all specimen collection days with HSV shedding by anatomic site Table 4.4 HSV Shedding rates by HAART status Table 4.5 Negative binomial model for any HSV shedding Table 4.6 Negative binomial model for HSV-2 shedding Table 4.7 Negative binomial model for HSV-1 shedding ix

10 List of Figures Figure 1.1 Enhanced sexual acquisition of HIV in HSV-2 infected persons Figure 1.2 Negative synergies between HIV-1 and HSV-2 in co-infected persons Figure 2.1 Flowchart of search strategy Figure 2.2 Forest Plot of the impact of HSV-2 seropositivity on time to opportunistic infection/aids - Unadjusted Figure 2.3 Forest Plot of the impact of HSV-2 seropositivity on time to opportunistic infection/aids - Adjusted for stage of HIV disease Figure 4.1 HSV Shedding by HAART Status Figure 4.2 Comparison of HSV-1 and HSV-2 shedding among dually infected participants x

11 List of Appendices APPENDIX A: Electronic Database Search Strategies APPENDIX B: Sample Size Considerations for Retrospective Cohort Study 151 xi

12 List of Abbreviations aor adjusted odds ratio AIDS acquired immunodeficiency syndrome ART antiretroviral therapy ART-CC antiretroviral cohort collaboration CCR5 C-C chemokine receptor type 5 CI confidence interval CMV cytomegalovirus CVL cervicovaginal lavage CXCR4 C-X-C chemokine receptor type 4 DNA deoxyribonucleic acid EBV Epstein-Barr virus ELISA enzyme-linked immunosorbent assay FDA Food and Drug Administration FSW female sex worker GBV-C GB virus-c GUD genital ulcer disease HHV-6 human herpesvirus-6 HIV human immunodeficiency virus HLA human leukocyte antigen hscrp highly sensitive C-reactive protein HSV herpes simplex virus HPTN HIV Prevention Trials Network HR hazard ratio IAS International AIDS Society IC 50 inhibitory concentration 50 ICP infected cell protein IL-6 interleukin-6 IQR interquartile range LBP-2 leader binding protein-2 xii

13 MSM MTCT N/A NA-ACCORD NF-κB NNRTI NR NRTI OI OR PCR PI RNA RR RT SMART STIs START STROBE TK VL VALIDATE VZV WB WHO WIHS men who have sex with men mother to child transmission not applicable North American AIDS Cohort Collaboration on Research and Design nuclear factor kappa B non-nucleoside reverse transcriptase inhibitor not reported nucleoside reverse transcriptase inhibitor opportunistic infection odds ratio polymerase chain reaction protease inhibitor ribonucleic acid relative risk reverse transcriptase Strategies for Management of Antiretroviral Therapy transmitted infections Strategic Timing of Antiretroviral Treatment Strengthening the Reporting of Observational Studies in Epidemiology thymidine kinase viral load VALacyclovir In Delaying Antiretroviral Treatment Entry varicella zoster virus Western Blot World Health Organization Women s Interagency HIV Study xiii

14 CHAPTER 1: Introduction OVERVIEW Highly active antiretroviral (ARV) therapy (HAART) has dramatically reduced the morbidity and mortality associated with the human immunodeficiency virus type 1 (HIV-1, herein referred to as HIV ) infection, and transformed an invariably fatal disease into a manageable, chronic condition. However, the inconvenience, cost, potential long and short-term toxicities, and risk of developing drug-resistant HIV associated with daily, lifelong HAART make the delay of HAART initiation a potentially desirable goal for some HIV-infected individuals. Among individuals already using HAART, adjunctive measures to further improve health are also desirable. Pharmacologic suppression of herpes simplex virus type 2 (HSV-2) with medications like acyclovir may provide a novel strategy for achieving these goals, since existing data, as outlined below, suggest that HSV-2 co-infection exacerbates HIV disease. These research questions are best answered by conducting clinical trials, but there are additional important questions that will inform the design and interpretation of such trials. First, what is the impact of HSV-2 co-infection on HIV disease progression among antiretroviraluntreated, HIV-infected adults? Because HSV-2 co-infection is thought to increase HIV viral load (VL), and because the viral load set point is the major predictor of HIV disease progression as measured by the rate of CD4 count decline, it is hypothesized to accelerate HIV disease progression as well. However, there are few longitudinal studies examining this issue, and the data available have not been synthesized quantitatively. This thesis thus includes a systematic review of observational studies summarizing the impact of HSV-2 on the natural history of ARV-untreated HIV infection in Chapter 2. In addition, a retrospective cohort study examining the impact of HSV-2 co-infection on the rate of CD4 cell count decline among ARVuntreated adults is presented in Chapter 3. The results of these studies inform the design and underlying hypotheses of clinical trials of anti-hsv medications as a strategy for delaying the need for HAART. A second unanswered question is, among adults with HIV, HSV-2 co-infection, is HAARTinduced HIV suppression associated with a decrease in HSV-2 shedding? If so, then the 1

15 2 magnitude of expected benefit from HSV-2 suppressive therapy in HAART-treated co-infected individuals might be attenuated. This dissertation thus also involves a prospective cohort study among HSV co-infected persons using HAART in Chapter 4, to determine whether HAART impacts on HSV shedding. This introductory chapter will review the natural history of both untreated HIV infection and HSV-2 infection, which are important to understanding how HSV-2 suppression might be of benefit, and will provide an overview of HSV diagnosis, HSV management, and the epidemiology of co-infection. Three negative synergies between these viruses, and opportunities that these interactions may provide specifically for the management of HIVinfected persons in the modern era, are then described. Natural History of HIV Infection Following acute infection with HIV, there is typically an initial significant drop in the CD4- positive T lymphocyte count from levels of >1000 cells/mm 3 to approximately cells/mm 3 within six months.[1, 2] The patient then enters a generally asymptomatic period characterized by slow but continual CD4 count decline, the rate of which varies according to the individual s initial average plasma HIV ribonucleic acid (RNA) level (also called the viral load set point ).[3-7] For instance, in large cohort studies, the annual rate of decline is roughly -20 or -35 cells/mm 3 for individuals with viral loads <500 copies/ml, but increases to roughly -75 cells/mm 3 for those with viral loads >30000 or copies/ml.[3, 5] Of note, the biological variability in absolute CD4 counts have led some authors to question the appropriateness of understanding CD4 count decline as a linear process, suggesting instead that the best-fitting models of CD4 counts over time may be closer to linear only after square root or logarithmic transformation.[8] However, because such transformations hinder the clinical interpretation of results, and because other reports do not show an increasing rate of CD4 decline during the course of HIV infection,[9] simple linear models of CD4 count are generally preferred. Thus, the overall annual rate of CD4 count decline in HIV infection can be reasonably estimated at approximately -50 to -60 cells/mm 3 per year. Chapter 1: Introduction

16 3 Because the risk of opportunistic infection rises sharply below CD4 counts of 200 cells/mm 3, guidelines recommend initiating HAART well before this threshold is reached, although at this stage individuals are typically asymptomatic from the perspective of their HIV infection. The specific CD4 count level at which to start therapy in asymptomatic persons has changed over time, and remains controversial, with few well-conducted randomized controlled trial data to guide recommendations. After the advent of HAART in 1996, the tendency was to treat HIVinfected individuals early in the course of disease. However, because of the toxicity, tolerability and pill burden of the antiretroviral agents available in that era, practice later shifted to delaying therapy until CD4 counts of 200 cells/mm 3. With subsequent improvements in antiretroviral drugs, this threshold has gradually increased in industrialized world settings over the last decade, initially to 350 cells/mm 3 and most recently to 500 cells/mm 3 according to some, but not all, published guidelines.[10-12] Evidence in support of this trend includes the CIPRAHT001 trial, which demonstrated a mortality benefit as well as decreased drug toxicities among Haitian HIV-infected patients randomized to starting HAART at CD4 counts in the cells/mm 3 range compared with <200 cells/mm 3.[13] Evidence in favour of the 500 cells/mm 3 threshold comes primarily from observational studies performed by the antiretroviral cohort collaboration (ART-CC) and the North American AIDS Cohort Collaboration on Research and Design (NA-ACCORD), which demonstrated increased risk ratios for acquired immunodeficiency syndrome (AIDS) and death in those delaying HAART until CD4 counts <350 cells/mm 3 compared to cells/mm 3, quantified at 1.28 (95% confidence interval, CI=1.05,1.57) and 1.69 (95%C=1.26,2.26), respectively.[14, 15] Further, the recently completed HIV Prevention Trials Network (HPTN) 052 trial showed a higher incidence of clinical events (death, World Health Organization (WHO) stage IV disease, pulmonary tuberculosis or severe bacterial infection) in individuals randomized to HAART initiation at CD4 counts of cells/mm 3 compared with 250 cells/mm 3, although the types of clinical events observed and the clinical and geographic settings in which they occurred may limit the trial s applicability to many industrialized settings.[16] A randomized comparison of the 350 and 500 cells/mm 3 CD4 count thresholds is underway in the START trial, with results anticipated in Chapter 1: Introduction

17 4 The above considerations apply to asymptomatic individuals with HIV disease. In individuals with clinically manifest HIV disease, including the presence of an opportunistic infection or malignancy and HIV-associated nephropathy, as well as in hepatitis B co-infected individuals who require hepatitis B therapy, HAART initiation is indicated. Therapy should also be initiated in pregnant women regardless of their CD4 count in order to decrease the likelihood of vertical HIV transmission. Once HAART is initiated, treatment must be lifelong, because HIV infection cannot be eradicated, and because treatment interruptions are associated with accelerated disease progression, and increased risks of HIV-associated and non-hiv-associated morbidity.[17] As such, the decision to start HIV therapy must be made carefully, and must take into consideration the patient s willingness and ability to commit to lifelong therapy. Natural History of HSV Infections Both HSV-1 and HSV-2 are enveloped deoxyribonucleic acid (DNA) viruses that cause lifelong, incurable infections. HSV initiates infection upon contact with non-intact skin or mucosal surfaces, classically causing vesicular or pustular ulcerative lesions on skin that heal with crusting, and/or ulcerative lesions on mucosal surfaces that heal without crusting. Symptoms appear roughly 4-7 days after exposure, and may be accompanied by painful, itching and burning sensations as well as tender lymphadenopathy,[18] but importantly, most infected persons never experience symptoms with initial infection. Systemic symptoms such as fever, malaise, headache and myalgias also occur in 40% of men and 70% of women.[18] New symptomatic infections may also manifest as dysuria, cervicitis, and central nervous system complications including urinary retention and aseptic meningitis. In rare cases of initial infection, HSV can be transported via the lymphatics to cause disseminated infection. However, HSV viremia can be detected using polymerase chain reaction (PCR) even in 24% of uncomplicated primary genital infections.[19] After initial infection, the virus is taken up by sensory nerve endings and the nucleocapsid travels in a retrograde fashion down the nerve axon. The virus evades the host immune response through interference with cytotoxic T-lymphocyte mediated apotosis of infected cells.[20, 21] Chapter 1: Introduction

18 5 Through mechanisms that remain poorly understood, it remains dormant in a latent, unintegrated, non-replicating form in the neurons of the dorsal root ganglia and autonomic nervous system, but can reactivate and cause recurrent symptoms with varying periodicity. In contrast to first episode herpes, recurrent symptoms are more often unilateral, and may occur at any anatomic location innervated by branches of the infected axons. However, studies involving intensive sampling of multiple anatomic sites have shown that asymptomatic reactivations often occur at multiple, distinct locations throughout the genital and perianal areas, and are often bilateral.[22] Symptomatic recurrences are often preceded by prodromal symptoms ranging in severity from mild tingling to shooting pain, but systemic symptoms are uncommon. Known triggers for recurrences include trauma, ultraviolet light, fever, menstruation and immunosuppression. In the year after a first clinical episode, recurrences occur in up to 90% of patients with HSV-2, and 20-50% for HSV-1; the median number of these recurrences in the first year is four for HSV-2 and one for HSV-1.[23-25] This frequency also varies according to factors such as the anatomic site of infection (more common in genital than orolabial infection),[23] the clinical severity of the primary episode (more common if this lasted over 35 days),[24] and the presence of immunosuppression.[25] The frequency of symptomatic HSV reactivations generally decreases over time, by about 50% in year 2 after primary HSV-1 and by about 50% at years 3-5 after primary HSV-2, although some patients may experience similar or even higher rates of relapse over time.[24, 26, 27] Findings for asymptomatic reactivations, discussed further below, mirror this trend.[28-30] First episode HSV infections can be classified according to whether pre-existing HSV antibody is present (non-primary infection) or not present (primary infection). This classification may have clinical relevance; for instance, pre-existing anti-hsv-1 antibody attributable to orolabial infection appears to protect against the subsequent acquisition of symptomatic genital HSV-1 infection.[31] Further, although pre-existing antibodies against HSV-1 do not diminish the likelihood of acquiring HSV-2 infection,[32, 33] they do increase the likelihood that incident HSV-2 infection will be asymptomatic by a factor of 2.6 (p<0.001).[33] Similarly, in a study of immunocompetent women, participants were more likely to recognize genital HSV symptoms if Chapter 1: Introduction

19 6 seropositive for HSV-2 only as compared with being seropositive for both HSV-1 and HSV-2 (odds ratio, OR=2.39, 95%CI,=1.30,4.37).[34] Interestingly, pre-existing type-specific antibody may not completely protect against superinfection by another strain of the same type. Indeed, genotypic analyses show that serially obtained HSV isolates from patients with recurrent genital herpes occasionally represent different viral strains, both in the case of HSV-1 [35] and HSV-2,[36-38] although co-infection with multiple strains with different reactivation times may be an alternative explanation for these findings. It is also noteworthy that first clinical episodes of HSV infections may occur despite the presence of pre-existing type-specific antibody.[39] Whether this phenomenon represents intratypic superinfection or reactivation after clinically inapparent primary infection is unknown. Of note, studies using highly sensitive PCR assays have shown that even in the absence of active herpes lesions, infected individuals may show evidence of ongoing viral replication on swabs of the usually affected mucosa or skin.[25, 40, 41] Such individuals are said to be asymptomatically shedding the virus. Studies involving intensive daily sampling of HSV-2 infected adults have shown that roughly 40-50% of shedding episodes in immunocompetent adults and 50-60% in HIV-infected adults last only twelve hours or less,[42, 43] and mathematical modeling further suggests that neuronal release of small numbers of HSV virions into the genital tract is nearly constant.[44] This frequent, low-level viral shedding is difficult to suppress even with high doses of anti-hsv medications.[45] That most HSV reactivations are asymptomatic is consistent with the observation that up to 90% of persons with serologic evidence of HSV-2 initially deny any history of herpes symptoms.[34, 46, 47] Diagnosis of HSV Infection The diagnosis of HSV infection can often be made on clinical grounds, based on the recognition of classic signs and symptoms as described above. However, individual episodes of HSV-1 and HSV-2 are clinically indistinguishable. In addition, clinical diagnosis has imperfect accuracy; in one report describing the predictive value of a clinical diagnosis of genital herpes in a cohort of 2391 initially HSV-2 seronegative individuals, in which definitive diagnosis was based on documented HSV-2 seroconversion, 155 true seroconversions were identified of which only 60 Chapter 1: Introduction

20 7 were correctly identified clinically; another 14 patients were incorrectly clinically diagnosed. Thus, the sensitivity was 39% and specificity was 99%, while positive and negative predictive values in that population were 81% and 96% respectively.[33] Simple Giemsa or Wright stains of tissue scrapings can be a helpful adjunct in providing a rapid presumptive diagnosis, as infected cells may fuse to form multinucleated giant cells that are a histologic hallmark of herpesvirus infection. However, this technique cannot distinguish between HSV-1, HSV-2 and varicella zoster virus (VZV), requires experience in the identification of giant cells, and is seldom performed in busy outpatient settings. Microbiologic confirmation of the symptomatic outbreaks is thus preferred, and provides the added advantage of type identification which may be clinically useful. Specimens are obtained by swabbing the base of a lesion, and can be tested using methods such as tissue culture or PCR. Tissue culture diagnosis involves identification of characteristic cytopathic effects on susceptible cell cultures hours after inoculation, followed by viral typing using antibody staining. Spin-amplification can accelerate this process to under 24 hours. The sensitivity of tissue culture depends on both the stage of disease, with greater yields observed with primary versus recurrent episodes due to greater viral loads, as well as the stage of the lesion; roughly 95%, 70% and 30% of vesicular, ulcerative and crusted lesions will produce a positive result, respectively.[48] Specimens should be transported to the laboratory as soon as possible, preferably on ice, to maximize yield. Compared with culture, PCR testing offers the advantages of a 3-4 times increase in sensitivity and decreased dependence on specimen transport conditions, although costs are higher.[41] Because the above methods require the presence of clinically apparent symptoms, and because the majority of HSV reactivations are subclinical, serologic testing provides an important diagnostic tool. Published guidelines identify three clinical scenarios in which this testing may be useful: culture-negative recurrent symptoms, a clinical history consistent with herpes that has not been microbiologically confirmed, and sexual partners of patients with documented genital herpes.[49] These tests have also been invaluable as research tools. Chapter 1: Introduction

21 8 The University of Washington Western Blot (WB) has long been considered the gold standard type-specific serology test, and involves separation of denatured viral proteins by molecular weight on a nitrocellulose strip, followed by reaction with clinical serum samples.[50] However, it is impractical for widespread use because of expense, labour requirements, and the lack of a readily available antigen source.[51] Commercially available type-specific HSV antibody tests have thus been developed, although marketed products vary in their accuracy. The recommended assays detect human IgG antibodies to the glycoprotein gg-1 (in HSV-1) or gg-2 (in HSV-2), the major viral envelope proteins which evoke type-specific responses. The HerpeSelect 1 and 2 enzyme-linked immunosorbent assay (ELISA) IgG (Focus Technologies) are paired Food and Drug Administration (FDA)-approved, commonly used assays that have shown sensitivities of % for HSV-1 and % for HSV-2, and specificities of % for HSV-1 and 93-97% for HSV-2, in cohorts of pregnant women and sexually active adults.[52-54] Additional typespecific gg-based assays include those manufactured by Kalon, Diagnology (POCkit-HSV-2) and Gull; key operating characteristics of these assays are summarized in Table 1.1. Another methodology, targeting gg2 and the infected cell protein-35 (ICP-35) complex, is the HerpeSelect 1 and 2 Immunoblot assay, with reported sensitivities of % for HSV-1 and % for HSV-2, and specificities of 93-95% for HSV-1 and 94-98% for HSV-2 compared to Western blot.[55, 56] Other commercially available assays, such as those manufactured by Diasorin, Zeus, are based on whole virus or crude antigen mixtures that cannot adequately distinguish HSV-1 mono-infection, HSV-2 mono-infection, or co-infection with both types, despite marketing claims to the contrary.[57] Other limitations of serologic testing warrant further discussion. First, it is now recognized that the operating characteristics of available assays may vary depending on the population studied. In the case of the HerpeSelect ELISA, for instance, the manufacturer s recommended laboratory threshold for defining HSV-2 seropositivity is an index value of 1.1, but literature suggests that increasing this threshold to may improve specificity depending on the geographic setting, and particularly in HIV-infected populations.[58-60] Second, all serologic tests may be negative Chapter 1: Introduction

22 9 in the setting of recently acquired infection. In one study using the HerpeSelect HSV-2 ELISA, 93% of primary and 73% of non-primary HSV-2 infections tested positive within 90 days; the median times to seropositivity were 21 and 23 days for primary and non-primary infections respectively.[61] Using the HerpeSelect HSV-1 ELISA, 73% of primary infections tested positive within 90 days, with median time to seropositivity 25 days.[61] Management of HSV Infection HSV infections are readily treated with the antiviral drug acyclovir and related compounds such as valacyclovir and famciclovir. Acyclovir is an acyclic guanosine analogue that must be phosphorylated by virally encoded thymidine kinases to the monophosphate form. Cellular thymidine kinases further phosphorylate acyclovir monophosphate into the active triphosphate form, which inhibits viral replication through uptake by viral DNA polymerase into the growing nucleic acid and causing chain termination. Valacyclovir, the valyl ester of acyclovir, is a prodrug that achieves higher bioavailability (54.5% versus 15-30%), permitting a lower pill burden for similar therapeutic effect.[62] Famciclovir is a pro-drug of the related compound penciclovir, which is similarly phosphorylated to an active triphosphate form that competitively inhibits the viral DNA polymerase. Additional agents with activity against HSV include the nucleoside analogues cidofovir, ganciclovir (and pro-drug valganciclovir), as well as the pyrophosphate analogue foscarnet, all of which also target the viral DNA polymerase, although these agents are not generally used for HSV therapy due to their increased toxicity. Clinical trials support the use of acyclovir and related drugs in two therapeutic strategies. First, episodic treatment involves short courses of medication for the management of both first episodes and symptomatic recurrences of HSV. In immunocompetent hosts, commonly used regimens for first episode genital herpes include acyclovir 200 mg five times daily, acyclovir 400 mg three times daily, valacyclovir 1000 mg twice daily, and famciclovir 250 mg three times daily, each for 7-10 days or longer if needed; these regimens reduce the duration of symptoms, accelerate the healing of lesions, and shorten the duration of viral shedding by 2-7 days.[48, 63, 64] In recurrent episodes of genital herpes, regimens such as acyclovir 200 mg five times daily, acyclovir 800 mg twice daily, valacyclovir 500 mg twice daily, and famciclovir 125 mg twice daily, each for 5 days, are associated with 1-2 day decreases in these same outcomes; evidence Chapter 1: Introduction

23 10 also supports simplifying the valacyclovir regimen to 1000 mg once daily or shortening the 500 mg duration to 3 days.[48, 63, 64] Second, for individuals with frequent HSV recurrences, chronic suppressive therapy with either acyclovir 400 mg twice daily, valacyclovir mg once daily, or famciclovir 250 mg twice daily is associated with 70-80% reductions in recurrences after 4 months.[48, 63, 64] Because the natural history of genital herpes infection is characterized by a gradual reduction in the number of recurrences over time, the need for ongoing therapy should be reassessed on a periodic basis. Epidemiology of HIV, HSV-2 Co-Infection Risk factors for genital herpes include an increased number of lifetime partners, previous history of other sexually transmitted infections (STIs) and earlier age of first sexual intercourse, as for other STIs.[46] Similar risk factors apply to HIV, and in particular, the sexual transmission of HIV is enhanced by the presence of both ulcerative and non-ulcerative STIs in either the HIVinfected and uninfected partner. Meta-analyses on this topic have generally shown effect sizes in the range of 2-5 for the impact of various STIs on both HIV acquisition and transmission.[65-67] Because HIV and HSV-2 share a common mode of transmission through sexual activity, there is considerable overlap in their epidemiology. Studies consistently show the prevalence of HSV-2 to be higher in the HIV-infected than in the general population, with Canadian estimates of 54.5% compared to %, respectively.[68-70] The incidence of HSV-2 is likely also higher in HIV-infected populations, and was estimated at 1.8 (95%CI=0.8,2.8) and 7.4 per 100 personyears (95%CI=3.5,12.6) in two prospective HIV cohorts respectively,[71, 72] compared with a modeled estimate of 0.84 (95%CI=0.77,0.91) in the United States general population.[73] This overlap between the HIV and HSV-2 epidemics is of considerable public health and clinical significance due to at least three forms of negative synergy between these two common pathogens, as described below. Chapter 1: Introduction

24 11 Negative Synergy 1: HSV-2 may be fuelling the HIV epidemic As mentioned above, it has long been recognized that classic STIs such as gonorrhoea, chlamydia and syphilis may be important cofactors in the propagation of HIV infection, and several randomized controlled trials of various STI management strategies for preventing HIV were conducted over the last two decades.[74-77] These studies met with mixed success, and a number of plausible explanations have been advanced to explain the differences in their results, most notably, the important epidemiologic differences between the stage of the HIV epidemics in the different trial settings.[78, 79] Importantly, however, these trials focused on bacterial STIs, and the high burden of untreated HSV-2 infection in the study cohorts may have been another important reason that most trials failed to impact on HIV transmission rates. Since that time, a large number of studies have shown that HSV-2 seropositivity increases the risk of HIV acquisition, with one systematic review showing a relative risk of 2.1 (95%CI= ).[80] Similar results were obtained in a subsequent meta-analysis, in which the relative risk of HIV associated with HSV-2 infection was considered separately for men (summary adjusted relative risk, RR=2.7, 95%CI=1.9,3.9), women (RR=3.1, 95%CI=1.7,5.6), and among men who have sex with men (MSM; RR=1.7, 95%CI=1.2,2.4).[81] HSV-2 is similarly believed to increase the likelihood of onward HIV transmission; a study in Uganda showed that genital ulcer disease, the bulk of which was caused by HSV-2, increased the risk of transmitting HIV to sexual partners by roughly four-fold.[82] Several biologic mechanisms may underlie this negative synergy (Figure 1.1). Micro- or macroscopic physical disruptions of the epithelial barrier of genital mucosal surfaces by HSV-2 may provide portals of entry and exit for HIV during sexual contact. The near constant rate at which HSV virions are likely released into the genital tract alluded to above suggests that such miniscule disruptions are common in infected persons. HSV-2 may further increase local HIV replication through mechanisms discussed further below, and indeed HIV is detectable in genital lesions of patients with STIs.[83, 84] In addition, the host immune response to HSV-2 reactivations involves an influx of CD4-positive T-cells co-expressing the co-receptor CCR5, thereby increasing the pool of locally susceptible target cells in persons sexually exposed to HIV.[85-87] Chapter 1: Introduction

25 12 Notably, three randomized trials of daily acyclovir 400 mg twice daily for HSV-2 suppression, including two international trials (HPTN 039 and the Partners in Prevention trial) and a third trial conducted among heterosexual women in Tanzania, did not show a benefit on either decreasing HIV acquisition [88, 89] or transmission.[90] There are several possible reasons for this lack of effect. Poor adherence with study medications may be one factor, with 90% adherence observed over only 51-52% of person-years of follow-up in the Tanzanian trial,[89] although estimated adherence was high at 94% and 96% in the other trials. A high burden of other co-infections in the study populations such as tuberculosis, malaria, geohelminths and other parasitic infections may also have been a factor; meta-analyses have suggested that treatment of such infections can lower HIV viral load, implying potential confounding effects on HIV transmission rates.[91, 92] Inadequate dosing or duration of acyclovir may also have contributed to the negative results. Importantly, it has further been found that HSV-2-specific immune responses, including the influx of HIV target cells described above, may persist in genital skin up to 20 weeks after resolution of clinical symptoms, and that this occurs despite twice daily use of acyclovir 400 mg.[93] Clearly, the local immune modulating effects of HSV-2 infection are long-lasting, and prolonged doses of high potency drugs may be required to fully suppress the effects of HSV-2 infection, if this is achievable at all. Recent findings that significant HSV shedding persists even during high dose use of valacyclovir (1g three times daily) suggests that currently available medications may be insufficient for this task.[45] Negative synergy 2: HIV exacerbates HSV-2 infection HIV exacerbates HSV-2 infection in several ways (Figure 1.2). Mucocutaneous shedding of HSV is greater in frequency and quantity in the context of HIV. A study among 176 women showed the prevalence of asymptomatic genital HSV-2 culture positivity to be roughly four times higher in HIV-infected than in uninfected participants (13.2% vs. 3.6%, p=0.04; OR =4.1, 95%CI=1.0,27.4),[40] while a study among 81 MSM showed more than a three-fold increase in the proportion of days with asymptomatic anogenital HSV-2 culture positivity (9.7% vs 3.1% of days).[25] Reactivations also occur more frequently and with increasing levels of both HIVassociated immunosuppression and plasma HIV RNA.[94] Chapter 1: Introduction

26 13 Symptomatic reactivations of HSV-2 are also more frequent and severe with increasing HIVrelated immunosuppression,[95] and can cause extensive lesions featuring deep ulceration and necrosis; chronic mucocutaneous HSV infection is considered an AIDS defining illness.[96, 97] Further, HSV-2 can cause serious systemic illnesses in HIV patients including esophagitis, retinitis, meningoencephalitis, pneumonitis and hepatitis, although such manifestations are distinctly uncommon. As in other immunocompromised populations, HSV-2 can also produce atypical hypertrophic, verrucous, and nodular lesions in HIV-infected hosts regardless of CD4 count.[98, 99] Such lesions may be mistaken for neoplasms, but pathology features mixed inflammatory infiltrates sometimes featuring characteristic giant cells, and culture or PCR can be used to confirm the diagnosis. This exacerbation and perturbation of the usual clinical manifestations of HSV-2 in the setting of HIV is felt to be attributable to HIV-related impairment of T-cell immunity against the virus. Both the breadth and the magnitude of T-cell responses to HSV-2 are downregulated in HIV/HSV-2 co-infected individuals compared to HSV-2 monoinfected individuals.[100, 101] HSV-2 can thus be considered an opportunistic pathogen in HIV/AIDS, and the generally recommended doses of antivirals used for both episodic and chronic suppressive therapy are higher than those used for immunocompetent patients. Reasonable regimens for episodic treatment include acyclovir mg 3-5 times daily, valacyclovir mg 2-3 times daily, and famciclovir mg 2-3 times daily; chronic suppressive doses may involve acyclovir mg 2-3 times daily, valacyclovir 500 mg twice daily, or famciclovir 500 mg twice daily.[102] Complicating the management of HIV, HSV-2 co-infection is the risk of acyclovir resistance which may necessitate second-line therapies. Acyclovir resistance in HSV can arise through a variety of distinct mechanisms, resulting in mutant strains that either are thymidine kinase (TK) deficient, produce decreased levels of TK, produce TK with altered substrate specificity, or, less often, produce an altered DNA polymerase.[103] Although the prevalence of such resistant strains is increased in immunocompromised compared to immunocompetent populations at roughly 4-7% versus <1%, these rates have remained stable across numerous geographic settings despite widespread use of these compounds for over 2 decades,[ ] perhaps because Chapter 1: Introduction

27 14 altered TK imposes a fitness cost on the virus.[107] As such, resistant strains are generally confined to the subset of patients with chronic HSV ulcerations and advanced AIDS and are an infrequent clinical problem. Negative synergy 3: HSV-2 may exacerbate HIV disease Because of the important ways in which HIV exacerbates HSV-2 disease, guidelines for HSV and HIV-co-infected individuals have primarily focused on strategies for controlling HSV-2 using acyclovir and related drugs. However, HSV-2 may exacerbate HIV disease in a reciprocal fashion (Figure 1.2). Numerous molecular mechanisms underlying this interaction have been described. First, numerous studies have demonstrated that co-infection of various human cell lines with HSV (typically using HSV-1) and HIV results in increased HIV replication,[108, 109] and specific HSV gene products have been shown to be responsible. This effect has been best demonstrated for the immediate-early gene products ICP-0, ICP-4 and ICP-27, which transactivate proviral HIV by inducing binding of cellular activators nuclear factor kappa B (NFκB) and Sp1 to the HIV long terminal repeat.[ ] These effects may depend on the status of the HIV infection (latent versus productive infection) and the cell line used, but appear independent of productive HSV replication. In addition, HSV-1 co-infection has been shown to augment HIV transcription by cellular proteins such as leader binding protein-2 (LBP-2).[116] Effects are further observed at the post-transcriptional level; the HSV-1 gene product U S 11 has been shown to chaperone HIV transcripts from the nucleus to the cytoplasm, thereby enhancing HIV protein synthesis in a fashion similar to that of the HIV Rev protein.[117] That HSV-2 and HIV can co-infect the same CD4 cell suggests that these transactivating effects likely have clinical relevance.[118] Second, paracrine effects of cytokines released by HSV-infected cells may also increase HIV replication. Co-incubation of peripheral blood mononuclear cells from HIV-infected individuals with HSV-1 and HSV-2, or with monocyte-derived macrophages previously exposed to HSV, results in increased HIV replication, and is thought to occur through the release of soluble factors.[119, 120] Chapter 1: Introduction

28 15 Third, HSV co-infection can expand the cell tropism of HIV through the formation of viral pseudotypes virions containing the HIV genome but bearing surface glycoproteins from HSV. Such phenotypic mixing abrogates the need for cells to bear the usual HIV receptor, the CD4 molecule, in order to become infected, and permits increased HIV replication. HSV-1 / HIV pseudotypes have been described, and infection of keratinocytes, which normally cannot be infected by HIV, has been observed in clinical samples.[121, 122] Additional mechanisms may also be important, and have been demonstrated for other, related herpesviruses. For instance, human herpesvirus 6 can upregulate CD4 expression on CD8- positive T cells, rendering these cells permissive to HIV infection.[123] The cytomegalovirus (CMV) U S 28 gene product can act as a co-receptor for HIV infection on CD4 cells, thus facilitating HIV entry into susceptible cells.[124] Further, presentation of CMV and Epstein- Barr virus (EBV) antigens to chronically HIV-infected cells stimulates cytokine production that secondarily results in increased HIV expression.[125] Thus there are numerous ways in which HSV-2 co-infection may upregulate HIV replication, and potentially exacerbate HIV pathogenesis. Complementing these mechanistic studies have been in vivo reports of increased plasma HIV viral load in the setting of HSV-2 co-infection (detailed further in Chapter 2), and as described already, plasma HIV viral load is a key driver of HIV disease progression. Importantly, this third negative synergy between HSV-2 and HIV may represent an opportunity for clinical intervention, if control of symptomatic and asymptomatic HSV-2 replication could have clinical benefits for co-infected individuals. HSV-2 Co-infection in HIV: An Opportunity for Intervention? Because acyclovir and its pro-drugs are safe, well-tolerated, and widely available, their use in this setting is a realistic option if benefit can be shown. However, they add to pill burden and cost, so careful evaluation regarding which patient populations may be most amenable to such studies, and of the potential magnitude of benefit in each, is warranted. Chapter 1: Introduction

29 16 Recently, a small number of clinical trials have begun to address these issues and have shown encouraging results in HAART-untreated populations (detailed further in Chapter 2).[126, 127] However, important unanswered questions remain. For instance, in HAART-untreated coinfected individuals at early stages of HIV disease, the adverse impact of HSV-2 co-infection has not been systematically quantified. This knowledge is important because it could theoretically determine the maximum magnitude of benefit that should be expected from HSV-2 suppressive therapy and have implications for the optimal dosing of anti-hsv medications and for better understanding the mechanisms by which these drugs exert their beneficial effects. Further, the role of HSV-1 co-infection has seldom been addressed, even though there are several reasons to suspect that it may be clinically relevant (detailed further in Chapter 3). Finally, it remains unclear whether HSV-2 co-infection represents a similarly important opportunity for clinical intervention in HAART-treated individuals, since modern antiretroviral therapy is already extremely effective at controlling HIV replication. This thesis explores these issues through three related research projects, detailed in the following three chapters. Chapter 2 contains a systematic review of the impact of HSV-2 co-infection on HAART-untreated HIV disease progression. Chapter 3 contains a retrospective cohort study that examines the same question but attempts to address some of the methodologic shortcomings of identified literature; the potential significance of HSV-1 co-infection is explored here as well. Ultimately, this work seeks to determine whether the adverse impact of HSV-2 infection on HIV disease can be exploited for clinical benefit among patients at early stages of HIV disease who do not yet require antiretroviral therapy. As described above, however, guidelines increasingly recommend earlier initiation of HIV treatment, such that HAART-untreated individuals will comprise a shrinking proportion of HIV patients in the future, and the potential significance of HSV-2 co-infection in HAART-treated individuals is of increasing interest. This issue is explored in Chapter 4, which outlines a prospective cohort study of the impact of HAART on HSV-2 shedding. Finally, Chapter 5 concludes by exploring the key implications of these projects and proposing directions for future work. Chapter 1: Introduction

30 17 Table 1.1 Operating characteristics of gg-based HSV serology assays a Assay HSV-1 HSV-2 References Sensitivity Specificity Median days to seroconversion Sensitivity Specificity Median days to seroconversion Diagnology N/A N/A N/A [51] Focus [51-54] Gull [128, 129] Kalon N/A N/A N/A [54] a Sensitivity and specificity compared to Western Blot Chapter 1: Introduction

31 18 Figure 1.1 Enhanced sexual acquisition of HIV in HSV-2 infected persons Figure 1.2 Negative synergies between HIV-1 and HSV-2 in co-infected persons Chapter 1: Introduction