© 2015 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association
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DOI: 10.1111/hiv.12263 HIV Medicine (2015), 16, 640--644

Prevalence of cryptococcal antigenuria at initial HIV diagnosis in KwaZulu-Natal

PK Drain,1 JM Kleene,2 SM Coleman,3 E Losina,1,4 JN Katz,5 J Giddy,6 D Ross,7 KA Freedberg1,4 and IV Bassett1 1Massachusetts General Hospital, Boston, MA, USA, 2Stony Brook School of Medicine, Stony Brook, NY, USA, 3Boston University School of Public Health, Boston, MA, USA, 4Harvard Medical School, Boston, MA, USA, 5Brigham and Women’s Hospital, Boston, MA, USA, 6McCord Hospital, Durban, South Africa and 7St Mary’s Hospital, Durban, South Africa

Objectives The World Health Organization (WHO) recommends screening HIV-infected people for cryptococcal antigens to identify cryptococcosis, a major cause of AIDS-related deaths. As the burden of cryptococcosis is unknown in South Africa’s KwaZulu-Natal province, we assessed the cryptococcal antigenuria prevalence among newly diagnosed HIV-infected adults there.
Methods We conducted a cross-sectional study of newly diagnosed HIV-infected adults who received voluntary HIV testing in an out-patient clinic. Participants provided a urine specimen in a sterile container, and we performed testing with a WHO-endorsed rapid cryptococcal antigen lateral ﬂow assay (Immy Inc., Norman, OK, USA) per the manufacturer’s speciﬁcations. We assessed cryptococcal antigenuria prevalence among participants with CD4 counts < 200 cells/μL, and stratiﬁed results by CD4 count categories.
Results Among 432 participants, the mean (± standard deviation) age was 36.1 ± 9.9 years and 172 (40%) were female. The overall estimated prevalence of cryptococcal antigenuria was 9.0% [95% conﬁdence interval (CI) 6.5–12.1%]. CD4 counts were available for 319 participants (74%); the median CD4 count was 75 cells/μL [interquartile range (IQR) 34–129 cells/μL]. Participants with a negative cryptococcal antigenuria screening test had a median CD4 count of 79 cells/μL (IQR 36–129 cells/μL), while participants with a positive cryptococcal test had a median CD4 count of 41 cells/μL (IQR 10–112 cells/μL). The estimated prevalence of cryptococcal antigenuria among participants with CD4 counts < 50 cells/μL was 12.5% (95% CI 7.0–20.1%), which was signiﬁcantly higher than that among participants with CD4 counts of 50–200 cells/μL (4.8%; 95% CI 2.3–8.7%).
Conclusions Nearly 1 in 10 newly diagnosed HIV-infected adults with CD4 counts < 200 cells/μL in KwaZulu-Natal had evidence of cryptococcal antigenuria. Point-of-care CD4 count testing and cryptococcal antigen screening may rapidly identify cryptococcosis at the time of HIV diagnosis.
Keywords: CD4 count, cryptococcal urine antigen, cryptococcosis, cryptococcus, diagnosis, HIV/AIDS, South Africa
Accepted 8 February 2015

Correspondence: Dr Paul K Drain, Medical Practice Evaluation Center, Massachusetts General Hospital, 50 Staniford St, 9th ﬂoor, Boston, MA 02114, USA. Tel: 617 726 9339; fax: 617 726 4120; e-mail: pdrain@partners.org
The copyright line for this article was changed on 20 September 2016 after original online publication.
This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modiﬁcations or adaptations are made.

Introduction
Cryptococcosis, a fungal infection that leads to cryptococcal meningitis, is a leading cause of AIDS-related mortality world-wide [1]. The World Health Organization (WHO) recommends screening all HIV-infected people with CD4 counts ≤ 100 cells/μL for cryptococcal antigens [2], as levels of circulating cryptococcal antigens predict the development of cryptococcal meningitis and mortality [3–5]. Two

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studies have shown a new, rapid lateral ﬂow assay to have excellent sensitivity (combined sensitivity of 98%) when performed on urine specimens and compared with latex agglutination testing of serum specimens [3,6].
In South Africa, cryptococcosis accounts for approximately 63% of meningitis cases, which is attributable in part to the high burden of HIV/AIDS [7]. The KwaZuluNatal province has the highest HIV prevalence in South Africa [8], yet there have been no studies on cryptococcosis prevalence in this region. We assessed the prevalence of cryptococcal antigenuria, which is strongly correlated with circulating serum and plasma cryptococcal antigens [3], among newly diagnosed HIV-infected adults attending several out-patient clinics in KwaZulu-Natal. We chose to assess urine specimens as urine has been reported to contain cryptococcal antigen and has additional appeal as a noninvasive biological specimen. The acceptability and validation of an inexpensive rapid test could lead to widespread adoption of cryptococcosis screening in HIV care and treatment programmes.
Methods
Study sites and participants
We conducted a cross-sectional study on adults presenting for voluntary HIV counselling and testing from October 2011 to January 2013. The parent study has been described elsewhere [9]. In brief, participants were recruited from four out-patient clinical areas offering HIV testing services: McCord Hospital, St Mary’s Hospital, and two municipal health clinics in urban and peri-urban areas of Durban. All patients were approached sequentially as they presented for HIV testing. Throughout the study, all four clinical sites offered free HIV counselling and rapid testing on week days. Eligible participants were ≥ 18 years old, not receiving antiretroviral therapy, and seeking out-patient care for various reasons. We excluded those known to be HIV-infected, pregnant, or unwilling to disclose HIV test results to the research team. All participants were informed of the study’s objectives, enrolled in a private space, and provided written informed consent to participate in the study prior to HIV and cryptococcal antigen testing. Participant data were maintained in a secure database and participant conﬁdentiality was preserved throughout the study. The ethics committees of McCord Hospital (IRB00005803) and St Mary’s Hospital in Durban, and Partners HealthCare in Boston (Protocol #: 2006-P-001379/40) approved the study.

Study procedures
In this study, a urine sample was requested from those who were HIV-infected. Urine samples were collected in a sterile container. Urine specimens were refrigerated within 6 h of collection before transfer to a −20°C frost-free freezer (actual temperature ranged from −18° to −22°C) for testing at the completion of cohort enrolment. All participants were offered CD4 count testing as part of their routine care. All HIV testing, care, and treatment were provided in accordance with current South African Department of Health guidelines.
We performed cryptococcal antigen testing using a rapid lateral ﬂow assay (Immy Inc., Norman, OK, USA) on urine samples of participants with CD4 counts < 200 cells/μL. The rapid cryptococcal antigen assay has been approved by the United States Food and Drug Administration and endorsed by the WHO [2]. The rapid lateral ﬂow assay has an estimated sensitivity of 98% when used on urine specimens [3,6]. According to the manufacturer, the rapid lateral ﬂow assay performs as well on thawed urine samples. Test kits were maintained in a sealed pouch at room temperature (15–26°C) before usage. All urine samples were allowed to thaw completely before testing. For each test, one drop of diluent buffer solution, provided by the test manufacturer, was added to a small vial. We pipetted 40 μL of urine into the vial and placed a rapid test into the vial. After 10 min, two researchers (PKD and JMK), blinded to participants’ demographics and CD4 count, independently interpreted the test results using a reference card provided by the manufacturer. Participants were considered urine cryptococcal antigen positive by the appearance of a colour band of any intensity, as deﬁned by the manufacturer. If the ‘control’ band was not present or the two readers were not in agreement, the test process was repeated using the same urine sample until the two readers reached agreement.
Statistical analyses
In primary analyses, we measured the overall prevalence of cryptococcal antigenuria among the cohort. In secondary analyses, we stratiﬁed the prevalence of cryptococcal antigenuria by CD4 count stratum and compared the prevalence in participants with CD4 counts < 50 cells/μL with that in participants with CD4 counts of 50–200 cells/μL. As this is a descriptive study, we calculated exact 95% conﬁdence intervals (CIs) for the overall and stratiﬁed estimates of prevalence. The P-values were generated to determine the signiﬁcance of differences between people who were cryptococcal antigenuria positive and those who were cryptococcal antigenuria negative, and to compare estimated cryptococcal antigenuria prevalences among

© 2015 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association

HIV Medicine (2015), 16, 640--644

642 PK Drain et al.

Table 1 Baseline characteristics

Total (n = 432)

CrAg negative (n = 393)

CrAg positive (n = 39)

Age (years) (mean ± SD) Gender [n (%)]
Female Male CD4 cell count Result available [n (%)] CD4 count (cells/μL) [median (IQR)]

36.1 ± 9.9
172 (39.8) 260 (60.2)
319 (73.8) 75 (34, 129)

36.2 ± 9.9
157 (39.9) 236 (60.1)
295 (75.1) 79 (36, 129)

35.3 ± 10.1
15 (38.5) 24 (61.5)
24 (61.5) 41 (10, 112)

We used Fisher’s exact test to compare percentages, t-tests to compare means, and the Mann−Whitney test to compare medians. CrAg, cryptococcal antigenuria; IQR, interquartile range; SD, standard deviation.

P-value
0.59
1.00 1.00
0.08 0.28

categories of CD4 cell count. We used Fisher’s exact test to compare categorical data, t-tests to compare continuous data, and the Mann−Whitney test to compare medians. The sample size was generated to provide an exact 95% binomial CI of ± 3%, assuming 10% CrAg prevalence in this cohort. All reported P-values were two-tailed, and a P-value < 0.05 was considered statistically signiﬁcant. We collected data using Microsoft Excel and conducted statistical analyses using SAS version 9.4 (SAS Institute, Cary, NC, USA).
Results
We tested urine specimens from 432 newly diagnosed HIVinfected adults. Mean age was 36.1 years (standard deviation ± 9.9 years) and 40% were female (Table 1). CD4 counts were available for 319 participants (74%). Among the entire cohort, the median CD4 count was 75 cells/μL [interquartile range (IQR) 34–129 cells/μL]. There was no signiﬁcant difference in cryptococcal antigenuria at initial HIV diagnosis by age or gender.
Median CD4 count was 41 cells/μL (IQR 10–112 cells/μL) among participants with a positive cryptococcal antigenuria result, and 79 cells/μL (IQR 36–129 cells/μL) among participants with a negative cryptococcal antigenuria result. Fewer participants with cryptococcal antigenuria obtained a CD4 count result, but this was not statistically signiﬁcant. Participants who had evidence of cryptococcal antigenuria had lower CD4 counts compared with participants who tested cryptococcal antigenuria negative, but this difference was not statistically signiﬁcant.
Thirty-nine participants were urine cryptococcal antigen positive, giving an overall estimated cryptococcal antigenuria prevalence in the cohort of 9.0% (95% CI 6.5– 12.1%) (Table 2). When stratiﬁed by CD4 count, the estimated cryptococcal antigenuria prevalence was 7.5% (95% CI 4.9–11.0%) among participants with CD4 counts < 200 cells/μL, and 8.8% (95% CI 5.2–18.8%) among participants with CD4 counts < 100 cells/μL. Among participants with CD4 counts < 50 cells/μL, the estimated cryptococcal

Table 2 Cryptococcal antigenuria prevalence among HIV-infected adults stratiﬁed by CD4 count category

Number positive/ number tested

Prevalence (%) (95% CI)

Total Stratiﬁed by CD4 count category
<200 cells/μL <100 cells/μL <50 cells/μL Missing CD4 cell count

39/432
24/319 17/192 14/112 15/113

9.0 (6.5–12.1)
7.5 (4.9–11.0) 8.8 (5.2–18.8) 12.5 (7.0–20.1) 13.3 (7.6–21.0)

CI, conﬁdence interval.

antigenuria prevalence was 12.5% (95% CI 7.0–20.1%). Among the 113 participants who did not obtain a CD4 count, the estimated prevalence of cryptococcal antigenuria was 13.3% (95% CI 7.6–21.0%).
Overall, cryptococcal antigenuria prevalence was greater among participants with lower CD4 counts. There was no difference in estimated cryptococcal antigenuria prevalence between those with CD4 counts of 100–200 cells/μL (5.5%; 95% CI 2.2–11.0%) and those with CD4 counts < 100 cells/μL (P = 0.29). However, the estimated prevalence of cryptococcal antigenuria among participants with CD4 counts < 50 cells/μL was signiﬁcantly higher than that in participants with CD4 counts of 50–200 cells/μL (4.8%; 95% CI 2.3–8.7%) (P = 0.01).
Discussion
In a cohort of newly diagnosed HIV-infected adults with CD4 counts < 200 cells/μL in KwaZulu-Natal, the estimated prevalence of cryptococcal antigenuria at HIV diagnosis was nearly 1 in 10 patients. As expected, we found the prevalence of cryptococcal antigenuria to be higher among participants with lower CD4 counts. We also found a high rate of cryptococcal antigenuria prevalence among participants who did not obtain a CD4 count result, which is a group at risk for being lost to follow-up. As death from

© 2015 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association

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Cryptococcal antigenuria at HIV diagnosis 643

cryptococcosis has been observed in one African study with a CD4 count as high as 365 cells/μL [10], current WHO screening recommendations (CD4 count ≤ 100 cells/μL) may miss some HIV-infected people with potentially lifethreatening cryptococcosis.
To our knowledge, this is the ﬁrst prevalence study of cryptococcal antigenuria in KwaZulu-Natal province, which has a very high burden of both HIV infection and tuberculosis (TB). Our prevalence estimates are consistent with those of most other studies of HIV-infected adults in sub-Saharan Africa [11–14], which have been as high as 21% among adults with CD4 counts < 150 cells/μL in Ethiopia [15]. A study in Cape Town, South Africa reported an estimated overall cryptococcal antigenaemia prevalence of 7% at the time of HIV diagnosis [4]. Few studies have examined the prevalence of cryptococcal antigens among HIV-infected adults with CD4 counts > 200 cells/μL. A recent study in Ethiopia reported an estimated serum cryptococcal antigenaemia prevalence of 5.8% (three of 52) among HIV-infected adults with CD4 counts of 200– 350 cells/μL [15]. Our observed prevalence of cryptococcal antigenuria among newly diagnosed HIV-infected adults is probably attributable to a high burden of cryptococcosis in the study region, but others have suggested greater sensitivity of the rapid lateral ﬂow assay as compared with standard latex agglutination assays [6].
Our study had several strengths and limitations. First, we performed testing on frozen, not fresh, urine samples. Cryptococcal antigens circulate at lower levels in urine than in plasma or serum [3], and may be less detectable in frozen samples. Thus, our methodology may have led to some false negative results, thereby underestimating the true burden of infection. Secondly, as this was a pilot study to assess the burden of disease, we did not conduct a laboratory reference standard test for cryptococcosis or assess patient outcomes. Two studies, however, have demonstrated that the rapid lateral ﬂow assay has 98% sensitivity when performed on urine specimens and compared with latex agglutination testing of serum specimens [3,6], and there have been no studies of test speciﬁcity. Finally, we evaluated the rapid cryptococcal antigen test among HIV-infected adults in a TB-endemic region, so these results may not be generalizable to other populations.
In conclusion, we found a prevalence of cryptococcal antigenuria of around 10% among newly diagnosed HIVinfected adults with CD4 counts < 200 cells/μL in KwaZuluNatal. Because the rapid lateral ﬂow assay is a simple, inexpensive and rapid test to detect cryptococcal antigenuria, screening HIV-infected people at the clinical point of care may be appropriate [12]. As the WHO recommends routine cryptococcosis screening for HIV-infected patients with CD4 counts ≤ 100 cells/μL and the relationship

between cryptococcal antigenuria and patient outcomes is uncertain, test validation studies for urine should include HIV-infected people with CD4 counts > 100 cells/μL and assess clinical outcomes.
Acknowledgements
We would like to acknowledge the excellent work and valuable contributions of our research staff and nurses. We thank each of the clinical sites for sharing their enthusiasm and space, as well as the men and women who participated in this study.
Conﬂicts of interest: We declare that we have no conﬂicts of interest.
Financial support: Research reported in this publication was supported by the Harvard Global Health Institute, the Fogarty International Clinical Research Scholars and Fellows Program at Vanderbilt University (R24 TW007988), The Program in AIDS Clinical Research Training [grant T32 AI007433 (PKD)], the National Institute of Mental Health [R01 MH090326 (IVB) and R01 MH073445 (RPW)], the National Institute of Allergy and Infectious Disease [K23 AI108293 (PKD) and R01 AI058736 (KAF)], the Harvard University Center for AIDS Research (P30 AI060354), the National Institute of Arthritis and Musculoskeletal and Skin Diseases [K24 AR057827 (EL)], and the National Center for Research Resources (the Harvard Catalyst UL1 RR 025758). The content is solely the responsibility of the authors and does not represent the ofﬁcial views of the National Institutes of Health or other funding agencies.
References
1 Park BJ, Wannamueler KA, Marston BJ. Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 2009; 23: 525–530.
2 World Health Organization. Rapid Advice: Diagnosis, Prevention and Management of Cryptococcal Disease in HIV-Infected Adults, Adolescents and Children. Geneva, WHO, 2011.
3 Jarvis JN, Percival A, Bauman S et al. Evaluation of a novel point-of-care cryptococcal antigen test on serum, plasma, and urine from patients with HIV-associated cryptococcal meningitis. Clin Infect Dis 2011; 53: 1019–1023.
4 Jarvis JN, Lawn SD, Vogt M, Bangani N, Wood R, Harrison TS. Screening for cryptococcal antigenemia in patients accessing an antiretroviral treatment clinic in South Africa. Clin Infect Dis 2009; 48: 856–862.
5 Liechty CA, Solberg P, Were W et al. Asymptomatic serum cryptococcal antigenemia and early mortality during antiretroviral therapy in rural Uganda. Trop Med Int Health 2007; 12: 929–935.

© 2015 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association

HIV Medicine (2015), 16, 640--644

644 PK Drain et al.

6 McMullan BJ, Halliday C, Sorrell TC et al. Clinical utility of the cryptococcal antigen lateral ﬂow assay in a diagnostic mycology laboratory. PLoS ONE 2012; 7: e49541.
7 Jarvis JN, Meintjes G, Williams A, Brown Y, Crede T, Harrison TS. Adult meningitis in a setting of high HIV and TB prevalence: ﬁndings from 4961 suspected cases. BMC Infect Dis 2010; 10: 67.
8 Department of Health. The 2010 National Antenatal Sentinel HIV and Syphilis Prevalence Survey in South Africa. Pretoria, Republic of South Africa, 2011.
9 Bassett IV, Giddy J, Chaisson CE et al. A randomized trial to optimize HIV/TB care in South Africa: design of the Sizanani trial. BMC Infect Dis 2013; 13: 390.
10 French N, Gray K, Watera C et al. Cryptococcal infection in a cohort of HIV-1-infected Ugandan adults. AIDS 2002; 16: 1031–1038.
11 Meya DB, Manabe YC, Castelnuovo B et al. Cost-effectiveness of serum cryptococcal antigen screening to prevent deaths among HIV-infected persons with a CD4+ cell count < or = 100 cells/microL who start HIV

therapy in resource-limited settings. Clin Infect Dis 2010; 51: 448–455. 12 Rugemalila J, Maro VP, Kapanda G, Ndaro AJ, Jarvis JN. Cryptococcal antigen prevalence in HIV-infected Tanzanians: a cross sectional study and evaluation of a point-of-care lateral ﬂow assay. Trop Med Int Health 2013; 18: 1075–1079. 13 Oyella J, Meya D, Bajunirwe F, Kamya MR. Prevalence and factors associated with cryptococcal antigenemia among severely immunosuppressed HIV-infected adults in Uganda: a cross-sectional study. J Int AIDS Soc 2012; 15: 15. 14 Alemu AS, Kempker RR, Tenna A et al. High prevalence of cryptococcal antigenemia among HIV-infected patients receiving antiretroviral therapy in Ethiopia. PLoS ONE 2013; 8: e58377. 15 Beyene T, Woldeamanuel Y, Asrat D, Ayana G, Boulware DR. Comparison of cryptococcal antigenemia between antiretroviral naïve and antiretroviral experienced HIV positive patients at two hospitals in Ethiopia. PLoS ONE 2013; 8: e75585.

© 2015 The Authors. HIV Medicine published by John Wiley & Sons Ltd on behalf of British HIV Association

HIV Medicine (2015), 16, 640--644