How To Determine The Risk Of Kidney Disease

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1 Treating HIV and African ethnicity; towards chronic care Annelot Schoffelen

2 ISBN: Cover design: L. Schellekens & R. Jongeneel Layout: R. Sanders Printed by: Gildeprint Drukkerijen, Enschede Copyright Annelot F. Schoffelen, the Netherlands, All rights reserved. No part of this thesis may be reproduced, stored or transmitted, in any form or by any means, without prior admission of the author. The copyright of the articles that have been accepted for publication or that have been published are transferred to the respective journals. Publication of this thesis was financially supported by: Boehringer Ingelheim bv, Janssen-Cilag B.V., Divisie Interne Geneeskunde & Dermatologie UMC Utrecht, Cluster Interne Geneeskunde & Infectieziekten UMC Utrecht

3 Treating HIV and African ethnicity; towards chronic care Behandeling van HIV bij patiënten van Afrikaanse afkomst; op weg naar betere zorg (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 7 juli 2015 des ochtends te uur door Annelot Fennigje Schoffelen geboren op 12 juni 1980 te Heerlen

4 Promotor: Copromotor: Prof. dr. A.I.M. Hoepelman Dr. R.E. Barth

5 Voor de mensen in Elandsdoorn

6 Beoordelingscommissie: Prof. dr. D.E. Grobbee Prof. dr. R.A. Coutinho Prof. dr. M.C. Verhaar Prof. dr. F.L.J. Visseren Prof. dr. P. Reiss Paranimfen: Dr. I. Mutsaers Dr. J.M. Schoffelen

7 Contents Chapter 1. Introduction 9 Part 1: African ethnicity and HIV infection in the Netherlands Chapter 2. Lower incidence of Pneumocystis jirovecii pneumonia among Africans in the Netherlands; host or environmental factors? 23 Chapter 3. Diminished impact of ethnicity as a risk factor for chronic kidney disease in the current HIV treatment era 37 Part 2: Treating HIV in rural South Africa Chapter 4. Sustained virological response on second-line antiretroviral therapy following virological failure in HIV-infected patients in rural South Africa 63 Chapter 5. Albuminuria is associated with traditional cardiovascular risk factors and viral load in HIV-infected patients in rural South Africa 83 Chapter 6. Carotid intima media thickness in HIV-infected subjects in rural South Africa is related to traditional cardiovascular risk factors but not to HIV-related factors 105 Chapter 7. Summary, general discussion and future perspectives 129 Chapter 8. Summary in Dutch 153 Chapter 9. Acknowledgements 163 Chapter 10. Curriculum vitae 171

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9 1 st Introduction Annelot F. Schoffelen

10 Chapter 1 HIV infection: from acute illness to chronic disease The global burden of human immunodeficiency virus (HIV) is immense. In 2013, an estimated number of 35 million adults and children were living with HIV worldwide [1]. Since the rise of HIV infection in the early 1980s, extensive research has provided much knowledge on the virus and its effects and has led to advances in treatment and prevention in the past three decades. The development of combination antiretroviral therapy (ART) resulted in the ability to suppress HIV-viral load and partially restore immune function, thereby preventing AIDS-related complications such as opportunistic infections. Its implementation improved health and life expectancy and reduced the risk of HIV transmission. AIDS-related illnesses are no longer the primary threat to people living with chronic HIV infection. New complications of HIV and its therapy have emerged instead [2-4]. In the current treatment era, HIV-infected people are at increased risk of developing various co-morbidities, such as cardiovascular disease and malignancies, the so-called non- AIDS events [5-8]. Several factors are thought to explain the excess risk for non-aids morbidity. First, the prevalence of traditional risk factors, such as tobacco use, alcohol, and unhealthy diet, is relatively high in HIV-infected populations. Second, multiple toxic effects of ART have been described, for example tubular dysfunction associated with tenofovir use [9, 10]. The use of ART is associated with several metabolic risk factors, including insulin resistance and dyslipidemia, which again can predispose people to developing cardiovascular events [11-13]. Lastly, chronic HIV infection is assumed to have on-going negative health effects in spite of successful virological treatment. Growing evidence suggests that HIV-infected people age more rapidly than HIV-negative subjects [2, 3]. Chronic systemic inflammation, immune dysregulation and the increased hypercoagulability as seen in HIV-infected patients may interfere with endothelial function and may lead to accelerated atherosclerosis [12, 14, 15]. The cause of these inflammatory processes is thought to be multifactorial, including continuous low-level HIV replication despite adequate viral suppression [16-18], common viral co-infections (e.g. cytomegalovirus and hepatitis C) [19, 20], and chronic translocation of gut microbial products in the systemic circulation as a result of breakdown of the integrity of the gut mucosa [21]. Treating HIV infection and African ethnicity 10 The shift from HIV infection characterized by acute AIDS-related diseases towards emerging long-term complications has been thoroughly studied in Western populations. Most research has been performed in Europe and the United States. Much less is known on chronic HIV infection and its complications in patients from sub-saharan Africa (SSA).

11 Introduction It is important to learn more on long-term effects of HIV and African ethnicity, regarding the growing numbers of sub-saharan African people living with HIV. The vast majority (>70%) of the worldwide HIV-infected population resides in SSA, covering an estimated number of 24.7 million in 2013 [1]. In some countries the burden is very high, such as South Africa, where approximately 19% of adults between 15 and 49 years of age are HIV-positive. The massive roll-out of ART in resource-limited settings (RLS) started about a decade ago, almost 10 years later than in Western countries, and has positively resulted in increased survival for patients in these areas as well. The number of AIDS-related deaths has diminished and the incidence of new HIV infections has declined significantly. As in high-income countries, HIV infection has become a chronic disease in SSA, with more and more people on lifelong treatment. Also in a Western country such as the Netherlands, a substantial proportion (14%) of HIV-infected patients is of African ancestry [22]. Treating chronic HIV infection in the sub-saharan African population might be different from the well-studied Western patients for several reasons. First of all, socio-economic and cultural factors have a large impact on HIV care. Politics influence the organization of health care systems in a country. Financial constraints in RLS limit the availability of resources, such as medical personnel, diagnostics and follow-up testing, medication, etc. The restricted obtainability of the number of antiretroviral drugs in a country can potentially lead to diminished efficacy, or toxicity-related problems. Furthermore, cultural characteristics could potentially play a role in the utilization of health care services, disease presentation, or the occurrence of psychosocial health problems. The prevalence of traditional risk factors, such as tobacco use, alcohol and other substance use, might vary for the SSA population compared to Western subjects. Second, demographic characteristics influence long-term HIV-related complications. The majority of SSA HIV-positives are females (approximately 60%) in contrast to 80% being males in Western populations [1, 22]. The median age of HIV-infected people in SSA is lower compared to Western countries, and HIV transmission occurs predominantly through heterosexual contact in contrast to MSM (men-having-sex-with-men) contact. This demographic profile can be seen in the HIV-infected population in SSA areas, but also among patients from African ethnicity in Western countries. Late presentation of HIV infection is still common in SSA [23, 24]. In the Netherlands as well, late presenters (defined as having a CD4 cell count below 350 cells/µl or AIDS-defining disease) are more prevalent among heterosexual patients originating from SSA in comparison to Dutch heterosexual patients (65% versus 53%) 11

12 Chapter 1 [22]. More progressive disease at the moment of first HIV diagnosis or presentation at a health care facility obviously increases the risk for contracting opportunistic infections or other AIDS-defining illnesses, but potentially also for non-aids events [25, 26]. In addition, the increased risk for tuberculosis among SSA patients should be taken into account when treating HIV infection. It remains a highly prevalent comorbidity in this population, even in spite of adequate HIV treatment, and is strongly associated with increased mortality risk [27, 28]. Viral subtypes of the HIV virus circulating in sub-saharan African populations differ from those extensively studied within Western patient groups. HIV-2 is primarily found in Western Africa, and its infection is known to have a lower transmissibility and disease progression rate than HIV-1 infection [29]. HIV-2 is naturally resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). HIV-1 subtypes N and O are almost exclusively detected in patients originating from Western-Central Africa and are less sensitive to NNRTIs as well. HIV-1 subtype C is the major subtype circulating in Southern Africa, in contrast to subtype B, which predominates in Western countries [30]. Genetic variety and polymorphism of subtype C might play a role in selection and transmission of resistance mutations. The prevalence of NNRTI resistance is on a rise in Southern Africa since the roll-out of ART [31]. Sub-Saharan African residents and most African patients (over 80%) living in the Netherlands have contracted HIV in their country of origin and as a result are infected with different and potentially more resistant viral subtypes compared to patients infected in Europe [22]. Finally, the genetic makeup in African ethnicity varies from that of Western patients and could influence the occurrence of long-term complications of HIV and its treatment. Potentially, host variability of the immune system could have an impact on progression of HIV disease and its response to treatment, or on the susceptibility for opportunistic infections. Furthermore, specific HIV-associated complications have been associated with African ancestry in previous studies, such as the occurrence of HIV-associated nephropathy (HIVAN), which may be related to genetic variability [32, 33]. Outline of this thesis 12 The aim of this thesis was to have a look into long-term complications of (treating) HIV infection in sub-saharan Africans, regarding the above-mentioned differences of this growing population compared to the well-studied Western patients. The following subjects were addressed.

13 Introduction The first part of this thesis discusses the role of African ethnicity in the occurrence of HIV-related complications in Western settings. Two studies were performed among patients enrolled in the observational AIDS Therapy Evaluation in the Netherlands (ATHENA) cohort, which follows HIV-positive patients registered for care in one of the 27 designated treatment centers in the Netherlands [22]. Chapter 2 describes the results of a study analyzing the risk of contracting Pneumocystis jirovecii pneumonia for patients of sub-saharan African origin compared to Western patients. Early literature suggests that racial differences in the incidence of this fungal opportunistic infection may exist, but results are conflicting [34-37]. We hypothesized that genetic susceptibility plays a role in the development of PJP, and could be one of the factors attributing to the observed racial differences. Therefore, a retrospective observational cohort study was performed, to investigate whether there are differences in PJP incidence among patients originating from SSA compared with patients from other regions of origin. The role of ethnicity as a risk factor for chronic kidney disease (CKD) in the current HIV treatment era is evaluated in Chapter 3. In the early years of the HIV epidemic, when HIVAN accounted for a substantial part of renal impairment, CKD was predominantly observed in individuals of African ancestry. However, this may be different in the most recent years, since the spectrum of HIV-related CKD has changed and new kidney-related conditions have emerged instead after the introduction of ART. For the second part of the thesis, treatment of HIV infection and some associated long-term complications were studied in rural SSA, where the majority of people living in RLS reside. The setting of the research was Elandsdoorn, a township situated in a poor, rural area in Limpopo, a province in the northeast of the Republic of South Africa. HIV prevalence among antenatal clinic attendees in 2012 was 22.3% in this province [38]. Ndlovu Medical Centre (NMC) is a health care center in Elandsdoorn ( that provides paid for service primary health care, as well as prevention-, tuberculosis- and HIV/AIDS programs. A fully funded ART program is available since 2003, including HIV treatment and testing. It serves a population of approximately people and around 3600 HIVinfected patients are in care at the clinic. In Chapter 4 we describe the virological, immunological and clinical efficacy of second-line ART in patients visiting NMC. In the past, good initial, on-treatment responses to first-line therapy were observed in this setting. Still, a substantial proportion of people experienced virological failure later during follow-up, which 13

14 Chapter 1 might result in the selection of virological resistance mutations [39-41]. Such mutations could negatively influence the efficacy of second-line or consecutive ARTregimens. At the moment, no further ART options are available in RLS following a failing protease-inhibitor based second-line regimen. Regarding the growing number of people requiring treatment, it is essential to evaluate the outcome of this therapy. Non-communicable diseases such as cardiovascular disease are rising in RLS. Currently, the vast majority (>75%) of the 17.5 million people worldwide who die annually of cardiovascular disease and diabetes live in RLS [42-44]. In addition, observations in Western populations show that cardiovascular and kidney disease risks are more prevalent among HIV-infected persons. Therefore, we set up a cross-sectional observational study, looking at cardiovascular and renal disease risk factors and the presence of atherosclerosis in HIV-positives from this South-African rural population. Chapter 5 and Chapter 6 describe the results of investigations in more than 900 subjects included in this research. The prevalence of albuminuria was determined, and its association with cardiovascular risk factors and HIV-related factors was analyzed in Chapter 5. In addition, the presence of atherosclerosis was evaluated by ultrasound measurements of carotid intima media thickness, which will be further explained in Chapter 6. Finally, the results of this thesis are summarized in Chapter 7 and a general overview and perspectives for the future are discussed. 14

15 Introduction References 1. UNAIDS. UNAIDS GAP Report - HIV estimates with uncertainty bounds on Jan 28, Guaraldi G, Orlando G, Zona S, Menozzi M, Carli F, Garlassi E, et al. Premature age-related comorbidities among HIV-infected persons compared with the general population. Clin Infect Dis. 2011;53(11): Hasse B, Ledergerber B, Furrer H, Battegay M, Hirschel B, Cavassini M, et al. Morbidity and aging in HIV-infected persons: the Swiss HIV cohort study. Clin Infect Dis. 2011;53(11): Deeks SG, Lewin SR, Havlir DV. The end of AIDS: HIV infection as a chronic disease. Lancet. 2013;382(9903): Bedimo RJ, McGinnis KA, Dunlap M, Rodriguez-Barradas MC, Justice AC. Incidence of non- AIDS-defining malignancies in HIV-infected versus noninfected patients in the HAART era: impact of immunosuppression. J Acquir Immune Defic Syndr. 2009;52(2): Freiberg MS, Chang CC, Kuller LH, Skanderson M, Lowy E, Kraemer KL, et al. HIV infection and the risk of acute myocardial infarction. JAMA Intern Med. 2013;173(8): Neuhaus J, Angus B, Kowalska JD, La Rosa A, Sampson J, Wentworth D, et al. Risk of all-cause mortality associated with nonfatal AIDS and serious non-aids events among adults infected with HIV. AIDS. 2010;24(5): Palella FJ, Jr., Baker RK, Buchacz K, Chmiel JS, Tedaldi EM, Novak RM, et al. Increased mortality among publicly insured participants in the HIV Outpatient Study despite HAART treatment. AIDS. 2011;25(15): Scherzer R, Estrella M, Li Y, Choi AI, Deeks SG, Grunfeld C, et al. Association of tenofovir exposure with kidney disease risk in HIV infection. AIDS. 2012;26(7): Mocroft A, Kirk O, Reiss P, De Wit S, Sedlacek D, Beniowski M, et al. Estimated glomerular filtration rate, chronic kidney disease and antiretroviral drug use in HIV-positive patients. AIDS. 2010;24(11): Lorenz MW, Stephan C, Harmjanz A, Staszewski S, Buehler A, Bickel M, et al. Both long-term HIV infection and highly active antiretroviral therapy are independent risk factors for early carotid atherosclerosis. Atherosclerosis. 2008;196(2): Palella FJ, Jr., Phair JP. Cardiovascular disease in HIV infection. Current opinion in HIV and AIDS. 2011;6(4): van Wijk JP, Cabezas MC. Hypertriglyceridemia, Metabolic Syndrome, and Cardiovascular Disease in HIV-Infected Patients: Effects of Antiretroviral Therapy and Adipose Tissue Distribution. International journal of vascular medicine. 2012;2012: Deeks SG, Phillips AN. HIV infection, antiretroviral treatment, ageing, and non-aids related morbidity. BMJ. 2009;338:a

16 Chapter Neuhaus J, Jacobs DR, Jr., Baker JV, Calmy A, Duprez D, La Rosa A, et al. Markers of inflammation, coagulation, and renal function are elevated in adults with HIV infection. J Infect Dis. 2010;201(12): Ostrowski SR, Katzenstein TL, Pedersen BK, Gerstoft J, Ullum H. Residual viraemia in HIV-1- infected patients with plasma viral load <or=20 copies/ml is associated with increased blood levels of soluble immune activation markers. Scand J Immunol. 2008;68(6): Mavigner M, Delobel P, Cazabat M, Dubois M, L Faqihi-Olive FE, Raymond S, et al. HIV-1 residual viremia correlates with persistent T-cell activation in poor immunological responders to combination antiretroviral therapy. PLoS One. 2009;4(10):e Buzon MJ, Massanella M, Llibre JM, Esteve A, Dahl V, Puertas MC, et al. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nat Med. 2010;16(4): Kovacs A, Al-Harthi L, Christensen S, Mack W, Cohen M, Landay A. CD8(+) T cell activation in women coinfected with human immunodeficiency virus type 1 and hepatitis C virus. J Infect Dis. 2008;197(10): Hunt PW, Martin JN, Sinclair E, Epling L, Teague J, Jacobson MA, et al. Valganciclovir reduces T cell activation in HIV-infected individuals with incomplete CD4+ T cell recovery on antiretroviral therapy. J Infect Dis. 2011;203(10): Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, et al. Microbial translocation is a cause of systemic immune activation in chronic HIV infection. Nat Med. 2006;12(12): van Sighem A, Gras L, Smit C, Stolte I, Reiss P 2014;Pageshttp:// files/8914/1527/1076/shm_monitoring_report_2014.pdf on January 27, Kigozi IM, Dobkin LM, Martin JN, Geng EH, Muyindike W, Emenyonu NI, et al. Late-disease stage at presentation to an HIV clinic in the era of free antiretroviral therapy in Sub-Saharan Africa. J Acquir Immune Defic Syndr. 2009;52(2): Drain PK, Losina E, Parker G, Giddy J, Ross D, Katz JN, et al. Risk factors for late-stage HIV disease presentation at initial HIV diagnosis in Durban, South Africa. PLoS One. 2013;8(1):e Zhang S, van Sighem A, Kesselring A, Gras L, Prins J, Hassink E, et al. Risk of non-aids-defining events among HIV-infected patients not yet on antiretroviral therapy. HIV Med Schouten J, Wit FW, Stolte IG, Kootstra NA, van der Valk M, Geerlings SE, et al. Cross-sectional comparison of the prevalence of age-associated comorbidities and their risk factors between HIVinfected and uninfected individuals: the AGEhIV cohort study. Clin Infect Dis. 2014;59(12): Gupta A, Wood R, Kaplan R, Bekker LG, Lawn SD. Tuberculosis incidence rates during 8 years of follow-up of an antiretroviral treatment cohort in South Africa: comparison with rates in the community. PLoS One. 2012;7(3):e

17 Introduction 28. Gupta A, Wood R, Kaplan R, Bekker LG, Lawn SD. Prevalent and incident tuberculosis are independent risk factors for mortality among patients accessing antiretroviral therapy in South Africa. PLoS One. 2013;8(2):e Nyamweya S, Hegedus A, Jaye A, Rowland-Jones S, Flanagan KL, Macallan DC. Comparing HIV-1 and HIV-2 infection: Lessons for viral immunopathogenesis. Rev Med Virol. 2013;23(4): McCutchan FE. Global epidemiology of HIV. Journal of medical virology. 2006;78 Suppl 1:S7-S Hamers RL, Sigaloff KC, Kityo C, Mugyenyi P, de Wit TF. Emerging HIV-1 drug resistance after roll-out of antiretroviral therapy in sub-saharan Africa. Curr Opin HIV AIDS. 2013;8(1): Choi AI, Rodriguez RA, Bacchetti P, Bertenthal D, Volberding PA, O Hare AM. Racial differences in end-stage renal disease rates in HIV infection versus diabetes. Journal of the American Society of Nephrology : JASN. 2007;18(11): Lucas GM, Lau B, Atta MG, Fine DM, Keruly J, Moore RD. Chronic kidney disease incidence, and progression to end-stage renal disease, in HIV-infected individuals: a tale of two races. The Journal of infectious diseases. 2008;197(11): Hu DJ, Fleming PL, Castro KG, Jones JL, Bush TJ, Hanson D, et al. How important is race/ethnicity as an indicator of risk for specific AIDS-defining conditions? J Acquir Immune Defic Syndr Hum Retrovirol. 1995;10(3): Del Amo J, Petruckevitch A, Phillips AN, Johnson AM, Stephenson JM, Desmond N, et al. Spectrum of disease in Africans with AIDS in London. AIDS. 1996;10(13): Stansell JD, Osmond DH, Charlebois E, LaVange L, Wallace JM, Alexander BV, et al. Predictors of Pneumocystis carinii pneumonia in HIV-infected persons. Pulmonary Complications of HIV Infection Study Group. Am J Respir Crit Care Med. 1997;155(1): Kaplan JE, Hanson DL, Navin TR, Jones JL. Risk factors for primary Pneumocystis carinii pneumonia in human immunodeficiency virus-infected adolescents and adults in the United States: reassessment of indications for chemoprophylaxis. J Infect Dis. 1998;178(4): The 2012 National Antenatal Sentinel HIV and Herpes Simplex type-2 prevalence Survey, South Africa, National Department of Health on Jan 28, Barth RE, van der Meer JT, Hoepelman AI, Schrooders PA, van de Vijver DA, Geelen SP, et al. Effectiveness of highly active antiretroviral therapy administered by general practitioners in rural South Africa. Eur J Clin Microbiol Infect Dis. 2008;27(10): Barth RE, Tempelman HA, Moraba R, Hoepelman AI. Long-Term Outcome of an HIV- Treatment Programme in Rural Africa: Viral Suppression despite Early Mortality. AIDS Res Treat. 2011;2011: Barth RE, Aitken SC, Tempelman H, Geelen SP, van Bussel EM, Hoepelman AI, et al. Accumulation of drug resistance and loss of therapeutic options precede commonly used criteria for treatment failure in HIV-1 subtype-c-infected patients. Antivir Ther. 2012;17(2):

18 Chapter Mayosi BM, Flisher AJ, Lalloo UG, Sitas F, Tollman SM, Bradshaw D. The burden of noncommunicable diseases in South Africa. Lancet. 2009;374(9693): Ikem I, Sumpio BE. Cardiovascular disease: the new epidemic in sub-saharan Africa. Vascular. 2011;19(6): WHO. Global status report on noncommunicable diseases bitstream/10665/148114/1/ _eng.pdf?ua=1 on Jan 28,

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21 Part one African ethnicity and HIV infection in the Netherlands

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23 2 nd Lower incidence of Pneumocystis jirovecii pneumonia among Africans in the Netherlands; host or environmental factors? Annelot F. Schoffelen 1, Steven F.L. van Lelyveld 1, Roos E. Barth 1, Luuk Gras 2, Frank de Wolf 2, Mihai G. Netea 3, Andy I.M. Hoepelman 1, on behalf of the ATHENA national observational HIV cohort 4 1 Department of Internal Medicine and Infectious Diseases, University Medical Centre Utrecht, Utrecht, the Netherlands 2 Stichting HIV Monitoring, Amsterdam, the Netherlands 3 Department of Internal Medicine and Nijmegen Institute for Infection, Inflammation and Immunity, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands 4 Study group members are listed at the end of the text AIDS 2013; 27:

24 Chapter 2 Abstract Background and objective HIV-associated Pneumocystis jirovecii pneumonia (PJP) remains one of the commonest opportunistic infections in Western countries. Although it has been suggested that racial differences in PJP incidence exist, early studies report conflicting results. This study aimed to investigate differences in PJP incidence in a developed country among patients originating from sub-saharan Africa compared with other regions of origin. Design and methods A retrospective observational cohort study was performed among HIVinfected patients from the Dutch ATHENA cohort. The main outcome measure was occurrence of PJP. Results A total number of 1055 PJP infections were diagnosed. Patients originating from sub-saharan Africa had a significantly lower risk for having PJP at the time of HIV diagnosis after adjustment of confounders compared with patients from Western origin (Western Europe, Australia and New Zealand; adjusted odds ratio (aor) 0.21 (95% confidence interval (CI), )). Other factors associated with higher PJP risk were increasing age (aor 1.01 per year (95% CI, )), a low CD4 count at HIV diagnosis (CD4 <50 versus >350 cells/μl aor (95% CI, )) and a high plasma HIV-RNA (> copies/ml) at HIV diagnosis (aor 1.41 (95% CI, )). Moreover, a clearly lower risk for PJP acquisition later during follow-up was observed among sub-saharan Africans versus Western patients (adjusted hazard ratio 0.60 (95% CI, )). Conclusion Among HIV-infected patients living in the Netherlands, PJP occurrence is substantially lower in patients originating from sub-saharan Africa, as compared to Western patients. Differences in genetic susceptibility may partially explain the lower PJP incidence in these patients. 24

25 Lower incidence of Pneumocystis jirovecii pneumonia among Africans in the Netherlands Introduction Pneumocystis jirovecii pneumonia (PJP) remains one of the commonest HIV-related opportunistic infections in Western countries, despite the combination antiretroviral therapy (cart)-associated decline in HIV-related morbidity and improved diagnostic and treatment strategies for P. jirovecii infection [1-3]. Early studies show that during the first decade of the HIV pandemic, prevalence of PJP in HIV-infected adults living in sub-saharan Africa was low [4]. More recent reports, however, present more PJP cases among HIV-infected people in these regions [4-6]. A possible explanation for this increase is an improvement in diagnostics in resource-limited settings (RLS), and could therefore reflect an underestimation of the actual PJP prevalence in the earlier years. However, racial differences in the occurrence of PJP have been observed in Western countries as well, with a lower incidence in African patients [7, 8]. In contrast, some early publications did not support these results, finding no racial differences in PJP occurrence [9, 10]. We hypothesized that genetic susceptibility is important in the development of PJP, and could be one of the factors attributing to the observed racial differences in PJP incidence. Therefore, we performed an observational, retrospective cohort study in the Netherlands to investigate whether there are differences in the incidence of PJP among patients originating from sub-saharan Africa compared with patients from other regions of origin. Methods The observational AIDS Therapy Evaluation in the Netherlands (ATHENA) cohort follows HIV-positive patients, who are registered in one of the 26 designated treatment centers in the Netherlands [11]. The ATHENA database includes information on patient demographics, immunological and virological parameters, detailed treatment data, data on adverse events, and AIDS-defining and selected non-aids-defining clinical events during follow-up. All HIV-infected patients diagnosed between June 1996 and January 2011 were enrolled in the current study. Follow-up was started at the date of HIV or PJP diagnosis, whichever occurred first. End of follow-up was defined as the date of PJP occurrence or the last visit date if no PJP occurred. For further analyses, patients were categorized into one of the following groups: patients from Western origin (Western Europe including the Netherlands, and Australia / New Zealand), sub-saharan Africa, Asia and the Pacific, Latin America and the Caribbean, Central and Eastern Europe, North America, and North Africa and the Middle East. 25

26 Chapter 2 Statistical analysis The primary endpoint was a PJP diagnosis, as registered in the ATHENA database according to the 1993 Centers for Disease Control (CDC) AIDS case definition [12]. Baseline continuous data were compared between regions of origin with the Student s t-test or the Mann-Whitney U-test. The distribution of categorical variables was compared with the chi-squared test. Two different multivariable models were used to determine the association between region of origin and PJP occurrence, either at baseline (before or within one month after HIV diagnosis) or more than one month after HIV diagnosis. The patients who had been diagnosed with PJP at baseline were analyzed by using a logistic regression model, whereas a Cox proportional hazards model was used to analyze the association between region of origin and incidence of PJP more than one month after HIV diagnosis. Patients with missing data regarding HIV-RNA values or CD4 cell counts were excluded from analyses. Adjusted odds ratios (aors) and adjusted hazard ratios (ahrs) were calculated by correcting for multiple independent variables, which included sex, age, mode of HIV transmission, year of HIV diagnosis, and HIV-RNA and CD4 cell count at baseline. For these analyses, determinants that were associated with the outcome with a P-value lower than 0.10 in univariate analysis were included. A P-value less than 0.05 was considered statistically significant. Statistical analyses were done using SPSS version Data were analyzed and reported according to the STROBE statement checklist for observational studies [13]. Results 26 Thirteen thousand, eight hundred and forty-four HIV-infected patients were included, and median duration of follow-up was 4.6 years [IQR years] with a total of patient-years. Most patients (8379/13 844, 60.5%) were from Western origin; sub- Saharan Africa was the second-most frequent region of origin (2608/ patients, 18.8%). Median CD4 cell count at time of HIV diagnosis was 326 cells/μl [IQR cells/μl]. This was significantly lower in patients originating from sub-saharan Africa (230 cells/μl [IQR cells/μl]) compared with Western patients (370 cells/μl [IQR cells/μl]) (P<0.001). Baseline characteristics are summarized in Table 1. During the total follow-up period, PJP was diagnosed in 1055/ (7.6%) patients. Among sub-saharan Africans, only 3.7% (96/2608) of patients were diagnosed with PJP, which was significantly lower than the number of PJP events among Western patients (715/8379 (8.5%); P<0.001).

27 Lower incidence of Pneumocystis jirovecii pneumonia among Africans in the Netherlands Table 1. Baseline characteristics North America North Africa, Middle East Central and Eastern Europe Latin America, the Caribbean South Asia, East Asia, Pacific Sub-Saharan Africa Total group Western Europe, Australia, New Zealand N (100) 8379 (60.5) 2608 (18.8) 537 (3.9) 1609 (11.6) 379 (2.7) 134 (1.0) 198 (1.4) Men (78.5) 7606 (90.8) 1128 (43.3) 349 (65.0) 1182 (73.5) 312 (82.3) 130 (97.0) 165 (83.3) Age (years) 36.8 [ ] 39.4 [ ] 31.4 [ ] 34.0 [ ] 34.4 [ ] 33.1 [ ] 37.1 [ ] 36.1 [ ] 73 (36.9) 88 (44.4) 37 (18.7) 122 (91.0) 6 (4.5) 6 (4.5) 189 (49.9) 114 (30.1) 76 (20.1) 787 (48.9) 724 (45.0) 98 (6.1) 260 (48.4) 218 (40.6) 59 (11.0) 117 (4.5) 2202 (84.4) 289 (11.1) 6154 (73.4) 1570 (18.7) 655 (7.8) 7702 (55.6) 4922 (35.6) 1220 (8.8) Mode of HIV transmission Homosexual Heterosexual Rest CD4 at baseline (cells/µl) 326 [ ] 370 [ ] 230 [ ] 250 [70-420] 300 [ ] 370 [ ] 350 [ ] 290 [ ] 4.72 [ ] 4.81 [ ] 4.51 [ ] 4.70 [ ] 4.51 [ ] 4.63 [ ] 4.78 [ ] 4.71 [ ] VL at baseline (log 10 copies/ml) Follow-up (years) 4.6 [ ] 4.4 [ ] 5.4 [ ] 4.6 [ ] 5.0 [ ] 3.8 [ ] 4.8 [ ] 4.3 [ ] Person-years (8.6) 13 (6.6) 12 (9.0) 8 (6.0) 22 (5.8) 14 (3.7) 129 (8.0) 95 (5.9) 64 (11.9) 49 (9.1) 96 (3.7) 60 (2.3) 715 (8.5) 568 (6.8) 1055 (7.6) 807 (5.8) 4 (2.0) 4 (3.0) 8 (2.1) 34 (2.1) 15 (2.8) 36 (1.4) 147 (1.8) 248 (1.8) PJP Before or <1 month after HIV diagnosis > 1 month after HIV diagnosis 45 [20-110] 42.5 [20-120] 50 [10-120] 35 [15-70] 45 [20-90] 50 [25-180] 55 [28-305] 65 [23-133] CD4 at PJP diagnosis (cells/µl) 5.18 [ ] 5.23 [ ] 5.11 [ ] 5.15 [ ] 5.00 [ ] 5.17 [ ] 5.19 [ ] 5.00 [ ] VL at PJP diagnosis (log 10 copies/ml) Data are given as number (%) or median [IQR]. N: number of patients, IQR: interquartile range, VL: viral load, PJP: Pneumocystis jirovecii pneumonia. Mode of HIV transmission Rest: intravenous drug use, blood or blood products, vertical transmission, unknown / other. 27

28 Chapter 2 Of the 1055 PJP cases, 807 (76.5%) were diagnosed at baseline and were thus included in a logistic regression model. Of the group of patients (807 with and without PJP), 211 (1.6%) were excluded because of missing values. Analyses were therefore performed on a total of patients, 788 of whom (5.9%) were diagnosed with a PJP infection. After adjustment for all included variables, originating from sub-saharan Africa remained associated with a significantly lower risk of having PJP at baseline (aor 0.21; 95% CI, ), as compared to Western patients. In patients originating from Latin America and the Caribbean the risk of having PJP was lower as well (aor 0.67; 95% CI, ). However, such differences were not observed between patients from other regions of origin and Western patients. Other factors that were predictive for PJP were older age, lower CD4 cell count and high plasma HIV-RNA (> copies/ml) at the time of HIV diagnosis (Table 2). Twenty-four per cent of all PJP cases (248/1055) occurred more than one month after HIV diagnosis, resulting in an incidence rate of 3.4 events per 1000 person-years in the total group, with a similar rate among Western patients versus 2.5 events per 1000 person-years among sub-saharan Africans. These cases were included in a Cox proportional hazards analysis, which was performed on patients (total group minus the 807 baseline PJP cases and 194 (1.5%) patients with missing data). Again, a clearly lower risk for PJP acquisition among sub-saharan Africans compared with Western patients (ahr 0.60 (95% CI, )) was observed. In this analysis, a lower CD4 cell count at baseline and not having homosexual or heterosexual contact as mode of HIV transmission were also associated with contracting PJP (ahr of 4.72 (95% CI, ) for CD4 <50 compared with >350 cells/μl and ahr 2.04 (95% CI, ), respectively). Moreover, results suggested an association between a PJP infection and the period in which HIV was diagnosed ( ahr 0.69 (95% CI, ) and ahr 0.53 (95% CI, ) as compared to ). Twelve percent (125/1055) of the PJP cases died during follow-up. The risk of death was independent of the region of origin, as was the median survival time between PJP diagnosis and time of death (data not shown). 28

29 Lower incidence of Pneumocystis jirovecii pneumonia among Africans in the Netherlands Table 2. Univariate and multivariable analysis PJP at baseline Variable Univariate analysis OR (95% CI) P-value Multivariable analysis aor (95% CI) P-value Sex (women) 0.69 ( ) < ( ) 0.68 Age at HIV diagnosis 1.04 ( ) < ( ) Mode of HIV transmission Homosexual 1.00 (Reference) (Reference) - Heterosexual 1.02 ( ) ( ) 0.80 Rest 1.80 ( ) < ( ) 0.47 Year HIV diagnosis (Reference) (Reference) ( ) ( ) ( ) < ( ) ( ) < ( ) 0.57 HIV-RNA at baseline (log 10 copies/ml) Log HIV-RNA at baseline (Reference) (Reference) - Log HIV-RNA at baseline > ( ) < ( ) <0.001 CD4 at baseline (cells/µl) CD4 < ( ) < ( ) <0.001 CD ( ) < ( ) <0.001 CD ( ) < ( ) <0.001 CD ( ) < ( ) <0.001 CD4 > (Reference) (Reference) - Region of origin Western Europe, Australia, New Zealand 1.00 (Reference) (Reference) - Sub-Saharan Africa 0.32 ( ) < ( ) <0.001 South Asia, East Asia, Pacific 1.40 ( ) ( ) 0.56 Latin America, Caribbean 0.87 ( ) ( ) Central and Eastern Europe 0.53 ( ) ( ) 0.11 North America 0.89 ( ) ( ) 0.58 North Africa, Middle East 0.97 ( ) ( ) 0.45 Included cases: , number of diagnoses of PJP: 788. PJP: Pneumocystis jirovecii pneumonia, OR: Odds Ratio, aor: adjusted Odds Ratio. Mode of HIV transmission Rest: intravenous drug use, blood or blood products, vertical transmission, unknown / other. 29

30 Chapter 2 Discussion 30 Among HIV-infected patients living in the Netherlands, patients originating from sub-saharan Africa have a lower risk of contracting PJP, as compared to patients from Western origin. This observation strengthens the hypothesis that host factors may play a role in susceptibility to this opportunistic pulmonary infection. To our knowledge, this cohort contains one of the largest groups of PJP cases that have been studied to date. Previously, only a few studies have been published on this subject with conflicting results. In a British study, comparing African and non- African HIV-infected patients, PJP was approximately half as frequent in African participants as in non-africans [7]. Another study, performed in the United States, showed similar results, reporting that African-Americans had less than half the risk of developing PJP, as compared to European-American participants [8]. These findings are in line with the results in our study, which was performed in an even larger cohort including more PJP cases. In contrast, other American studies did not find ethnicity to be a risk factor for PJP incidence in HIV-infected patients [9, 10]. It is hypothesized that geographical, and thus racial, variations in reported incidence of PJP result from geographical P. jirovecii strain variations [14]. Currently, it is not yet clear whether PJP is the result of reactivation of long-standing latent infection with P. jirovecii when the host s immune function declines, or whether recent de novo exposure is the common mechanism for acquisition of Pneumocystis infection [15]. However, both animal and human studies suggest that the latter is the most likely explanation [15, 16]. If recent exposure before PJP is indeed frequent, most patients in our cohort would have been infected with Pneumocystis in the Dutch climate, and thus harbor similar strains. This suggests that host susceptibility, rather than P. jirovecii strain, may be responsible for the observed difference in PJP incidence between the various regions of origin. In some reports, genetic variability has been associated with the risk of developing PJP in HIV-infected patients, such as a deletion in the CCR5 gene (CCR5-Δ32), the CCRL2 F167Y mutation and a histidine-histidine polymorphism of one of the Fc segment receptors of IgG (FcγRIIa), but exact mechanisms remain to be explored [17-19]. To our knowledge, no studies on a potential relation between HLA gene polymorphisms and PJP susceptibility have been published to date. Toll-like receptors (TLRs) and Dectin-1 are known to play an important role in controlling fungal infections [20]. It has been shown that Dectin-1 knockout mice are more susceptible to Pneumocystis infection than wild-type mice [21]. As polymorphisms in TLRs are associated with susceptibility for various infectious diseases [22], we hypothesize that genetic variability in TLRs and/or Dectin-1 may explain differences in the

31 Lower incidence of Pneumocystis jirovecii pneumonia among Africans in the Netherlands susceptibility to Pneumocystis infections. Obviously, further research on this subject needs to be performed to clarify the role of genetic susceptibility in the incidence of PJP. Strengths of this study include the use of a well-defined cohort of HIV-infected patients followed prospectively, whose data are recorded in a standardized manner, and the large number of patients included. However, there are some limitations. Information regarding the length of stay in the Netherlands prior to the HIV diagnosis is lacking. Furthermore, we used region of origin as a surrogate for race or ethnicity. We expect these factors to greatly overlap, but small inconsistencies, causing variety in host PJP susceptibility within groups, cannot be ruled out. Lastly, although a clinical PJP diagnosis is common and widely accepted, we are not informed whether all PJP diagnoses have been confirmed by cytological or histological tests and how many were presumptive. It is to be expected that this would not influence the outcome of our analyses, as patients from various regions of origin are most likely to have undergone similar diagnostic procedures in specialized centers in the Netherlands. In summary, this Dutch observational cohort study shows that the occurrence of PJP in HIV-infected patients originating from sub-saharan Africa is significantly lower compared with patients from Western origin. These observed differences may partially be explained by a variation in genetic susceptibility of the hosts. This theory needs to be addressed in future translational studies. Acknowledgments The ATHENA national observational cohort has been made possible through the collaborative efforts of the following physicians (* site coordinating physicians): Academisch Medisch Centrum bij de Universiteit van Amsterdam, Amsterdam: Prof. dr. J.M. Prins*, Prof. dr. T.W. Kuijpers, Dr. H.J. Scherpbier, Dr. J.T.M. van der Meer, Dr. F.W.M.N. Wit, Dr. M.H. Godfried, Prof. dr. P. Reiss, Prof. dr. T. van der Poll, Dr. F.J.B. Nellen, Prof. dr. J.M.A. Lange, Dr. S.E. Geerlings, Dr. M. van Vugt, Drs. D. Pajkrt, Drs. J.C. Bos, Drs. M. van der Valk, Drs. M.L. Grijsen, Dr. W.J. Wiersinga. Academisch Ziekenhuis Maastricht, Maastricht: Dr. S. Lowe*, Dr. G. Schreij, Dr. A. Oude Lashof, Dr. D. Posthouwer. Catharina-ziekenhuis, Eindhoven: Drs. M.J.H. Pronk*, Dr. B. Bravenboer. Erasmus Medisch Centrum, Rotterdam: Dr. M.E. van der Ende*, Dr. T.E.M.S. de Vries-Sluijs, Dr. C.A.M. Schurink, Dr. J.L. Nouwen, Dr. M.H. Nispen tot Pannerden, Dr. A. Verbon, Drs. B.J.A. Rijnders, Dr. E.C.M. van Gorp, Dr. R.J. Hassing, Drs. A.W.M. Smeulders. Erasmus Medisch Centrum Sophia, Rotterdam: Dr. N.G. Hartwig, Dr. G.J.A. Driessen. Flevoziekenhuis. Almere: Dr. J. Branger*. HagaZiekenhuis, Den Haag: Dr. E.F. Schippers*, Dr. C. van Nieuwkoop, 31

32 Chapter 2 Drs. E.P. van Elzakker. Isala Klinieken, Zwolle: Dr. P.H.P. Groeneveld*, Dr. M.A. Alleman, Drs. J.W. Bouwhuis. Kennemer Gasthuis: Dr. R. Soetekouw*, Prof. dr. R.W. ten Kate. Leids Universitair Medisch Centrum, Leiden: Dr. F.P. Kroon*, Prof. dr. J.T. van Dissel, Dr. S.M. Arend, Dr. M.G.J. de Boer, Drs. H. Jolink, Dr. H.J.M. ter Vollaard, Drs. M.P. Bauer. Maasstadziekenhuis, Rotterdam: Dr. J.G. den Hollander*, Dr. K. Pogany. Medisch Centrum Alkmaar, Alkmaar: Drs. G. van Twillert*, Drs. W. Kortmann*. Medisch Centrum Haaglanden, Den Haag: Dr. E.M.S. Leyten*, Dr. L.B.S. Gelinck. Medisch Spectrum Twente, Enschede: Drs. G.J. Kootstra*, Drs. C.E. Delsing. Onze Lieve Vrouwe Gasthuis, Amsterdam: Prof. dr. K. Brinkman*, Dr. W.L. Blok, Dr. P.H.J. Frissen, Drs. W.E.M. Schouten, Drs. G.E.L. van den Berk. Sint Elisabeth Ziekenhuis, Tilburg: Dr. J.R. Juttmann*, Dr. M.E.E. van Kasteren. Sint Lucas Andreas Ziekenhuis, Amsterdam: Dr. J. Veenstra*, Dr. K.D. Lettinga. Slotervaartziekenhuis, Amsterdam: Dr. J.W. Mulder*, Drs. S.M.E. Vrouenraets, Dr. F.N. Lauw. Stichting Medisch Centrum Jan van Goyen, Amsterdam: Drs. A. van Eeden*, Dr. D.W.M. Verhagen. Universitair Medisch Centrum Groningen, Groningen: Drs. H.G. Sprenger*, Drs. R. Doedens, Dr. E.H. Scholvinck, Drs. S. van Assen, Dr. W.F.W. Bierman. Universitair Medisch Centrum Sint Radboud, Nijmegen: Dr. P.P. Koopmans*, Dr. M. Keuter, Dr. A.J.A.M. van der Ven, Dr. H.J.M. ter Hofstede, Dr. A.S.M. Dofferhoff, Dr. A Warris, Dr. R. van Crevel. Universitair Medisch Centrum Utrecht, Utrecht: Prof. dr. A.I.M. Hoepelman*, Dr. T. Mudrikova, Dr. M.M.E. Schneider, Drs. C.A.J.J. Jaspers, Dr. P.M. Ellerbroek, Dr. J.J. Oosterheert, Dr. J.E. Arends, Dr. M.W.M. Wassenberg, Dr. R.E. Barth. Vrije Universiteit Amsterdam, Amsterdam: Dr. M.A. van Agtmael*, Dr. R.M. Perenboom, Drs. F.A.P. Claessen, Dr. M. Bomers, Dr. E.J.G. Peters. Wilhelmina Kinderziekenhuis, Utrecht: Dr. S.P.M. Geelen, Dr. T.F.W. Wolfs, Dr. L.J. Bont. Ziekenhuis Rijnstate, Arnhem: Dr. C. Richter*, Dr. J.P. van der Berg, Dr. E.H. Gisolf. Admiraal De Ruyter Ziekenhuis, Vlissingen: Drs. M. van den Berge*, Drs. A. Stegeman. Medisch Centrum Leeuwarden, Leeuwarden: Dr. M.G.A. van Vonderen*, Drs. D.P.F. van Houte. Medisch Centrum Zuiderzee, Lelystad: Dr. S. Weijer*, Dr. R. el Moussaoui. Sint Elisabeth Hospitaal, Willemstad - Curaçao: Dr. C. Winkel, Drs. F. Muskiet, Drs. Durand, Drs. R. Voigt. Funding The ATHENA national observational cohort is maintained by the HIV Monitoring Foundation, supported by the Dutch Ministry of Health. 32

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