Cryptococcal antigen prevalence in HIV-infected Tanzanians: a cross-sectional study and evaluation of a point-of-care lateral flow assay

Transcription

1 Tropical Medicine and International Health doi: /tmi volume 18 no 9 pp september 2013 Short Communication Cryptococcal antigen prevalence in HIV-infected Tanzanians: a cross-sectional study and evaluation of a point-of-care lateral flow assay Joan Rugemalila 1, Venance P. Maro 1, Gibson Kapanda 2, Arnold J. Ndaro 3 and Joseph N. Jarvis 4 1 Department of Medicine, Kilimanjaro Christian Medical University College, Moshi, Tanzania 2 Department of Epidemiology and Biostatistics, Kilimanjaro Christian Medical University College, Moshi, Tanzania 3 Kilimanjaro Clinical Research Institute, Biotechnology Laboratory, Moshi, Tanzania 4 Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine,UK Abstract objectives Cryptococcal antigen (CRAG) screening at antiretroviral therapy (ART) initiation and pre-emptive antifungal treatment for those testing positive could prevent many cases of cryptococcal meningitis (CM). To investigate whether CRAG screening would be feasible in Tanzania, we conducted a cross-sectional study measuring CRAG prevalence in ART clinic patients and comparing the novel lateral flow assay (LFA) with the cryptococcal latex agglutination (LA) test. methods Consecutive HIV-infected outpatients with CD4 counts <200 cells/µl, who were ART naive or had been on ART for <6 months, were screened for CRAG using the LA and LFA kits. For further assay validation, HIV-infected inpatients with suspected cryptococcal disease were also tested using the LA and LFA kits. results Cryptococcal antigen was detected in seven of 218 ART clinic attendees (3%). Six patients (5%) with CD4 cell counts 100 cells/µl (n = 124) were CRAG-positive. Agreement between the LA and LFA test in the 218 outpatients was 100%. Another 101 inpatients were tested for CRAG, of whom 56 (55%) were CRAG-positive on both the LA and LFA tests. One patient was positive using the LFA test but negative on the LA test. The overall agreement between the two assays was 99.7%, kappa coefficient 0.99 (standard error 0.06, P < 0.001). conclusions Five percentage of ART clinic patients with CD4 cell counts 100 cells/µl in northern Tanzania had asymptomatic cryptococcal antigenaemia, suggesting that CRAG screening would be worthwhile in the Tanzanian ART programme. The LFA is a reliable, cheap and practical alternative to LA for detection of CRAG. keywords HIV, cryptococcal antigen, screening, lateral flow assay, Tanzania Introduction Cryptococcal meningitis (CM) is estimated to cause over deaths annually in sub-saharan Africa (Park et al. 2009) and is a leading cause of mortality in HIVinfected patients initiating antiretroviral therapy (ART) in Africa (Jarvis & Harrison 2007; Lawn et al. 2008; Walker et al. 2012). Recent data suggest that most CM cases that develop after ART initiation could be prevented by screening patients for subclinical cryptococcal infection at ART programme entry, using cryptococcal antigen (CRAG) detection assays, and pre-emptively treating those who test positive with high-dose fluconazole (Jarvis et al. 2009, 2010, 2012; Meya et al. 2010). Highly sensitive and specific CRAG detection assays, usually latex agglutination (LA) kits, have been available for many years, but such assays require laboratory infrastructure and capacity often lacking in low-resource settings. The development of a new simple to use point-of-care lateral flow dipstick assay (LFA) has the potential to markedly facilitate both CRAG screening of patients initiating ART and earlier diagnosis of patients presenting with symptomatic cryptococcal disease in much of Africa (Jarvis et al. 2011; Lindsley et al. 2011) John Wiley & Sons Ltd 1075

2 The prevalence of cryptococcal antigenaemia in patients entering ART programmes in Tanzania is unknown. This is important to quantify as the cost-effectiveness of CRAG screening for patients initiating ART depends upon the CRAG prevalence in the patient population screened (Meya et al. 2010; Jarvis et al. 2013). We performed a cross-sectional study to determine the prevalence of cryptococcal antigenaemia in patients initiating or who had recently initiated ART at a clinic in northern Tanzania. In parallel, we evaluated the performance of the novel cryptococcal LFA compared with the conventional LA test, both in the ART clinic attendees and in symptomatic inpatients with suspected cryptococcal disease. Methods Patients were recruited and samples collected between 1 September 2011 and 29 February 2012 at Kilimanjaro Christian Medical Centre (KCMC), Moshi, the tertiary referral hospital serving northern Tanzania. The study was approved by the Kilimanjaro Christian Medical University College Research Ethics Committee. All study participants provided written informed consent. For unconscious patients in the hospital, primary relatives and/or spouses provided written informed consent. Consecutive HIV-infected outpatients aged 13 years and older with CD4 cell counts <200 cells/ll (FACS Count, Becton Dickinson, Mountain View, CA, USA) who were either ART naive or had been on ART for <6 months were recruited at the care and treatment clinic (CTC). For further validation of the lateral flow assay, HIV-infected inpatients aged 13 years and older on the medical wards at KCMC with suspected CM presenting during the study period were also recruited. Patients taking fluconazole or treated for CM in the 6 months prior to study enrolment were excluded. Demographic, clinical and laboratory data, including a full symptom questionnaire, were recorded at study enrolment, and 3 ml of whole blood was collected from each participant. Serum samples were obtained by centrifugation and tested for CRAG using the latex agglutination (LA) and lateral flow assay (LFA) kits (Immuno-Mycologics Inc, Norman, OK, USA) as per manufacturer s instructions. Positive results were reported to the responsible clinicians for further investigation and treatment at their discretion. Data were analysed using Stata, version 12.0 (StataCorp, College Station, Texas, USA). CRAG prevalence with 95% confidence intervals (95% CIs) was calculated in the outpatient cohort. Baseline variables were compared across CRAG-positive and CRAG-negative groups with the Mann Whitney U test, the Chi-squared test or Fisher s exact test as appropriate. Associations between ART status, CD4 cell count and CRAG positivity were examined using cross tabulations and calculation of odds ratios, and a simple logistic regression model with likelihood ratio testing. Agreement between LA and LFA CRAG tests was assessed in both the outpatient cohort, and the whole study population (inpatients and outpatients) using Cohen s kappa coefficient. Statistical significance was defined as P Results The study included 218 outpatients. 43% (n = 94) were male, the median age was 39 (IQR 31 48) years, 44% (n = 96) were on ART, and the median CD4 cell count was 96 (IQR ) cells/µl. Seven patients (3%, 95%CI 1 7%) were CRAG-positive, all on both the LA and LFA test. The median LA CRAG titre was 1:128 (IQR 1:32 1:512). None of the patients had symptoms suggestive of CM on direct questioning (headache, neck stiffness, photophobia or vomiting). CRAG-positive patients had significantly lower median CD4 cell counts than CRAG-negative patients: 19 cells/µl (IQR 9 58 cells/µl) vs. 97 cells/µl (IQR cells/µl), P < (Table 1). Patients with a CD4 cell >50 cells/µl had markedly lower odds of being CRAG positive than those with CD4 cell counts 50 cells/µl after adjusting for ART status (adjusted odds ratio 0.03, 95% confidence interval ). ART-naive patients had higher odds of being CRAG-positive than patients on ART in unadjusted analysis; however, this association did not remain significant after adjustment for CD4 cell count (Table 2). When analysis was restricted to just those patients with a CD4 cell count 100 cells/µl (n = 124), six patients (5%, 95% CI 2 10%) were CRAG-positive (Table 1). Agreement between the LA and LFA test in the 218 asymptomatic patients was 100%. An additional 101 inpatients with clinically suspected cryptococcal disease were tested for CRAG using both the LA and LFA kits (Table 1), of whom 56 (55%) were CRAG-positive on both the LA and LFA tests. One patient was positive using the LFA test but negative on the LA test. This patient had a CD4 cell count of 129 cells/µl, was on ART, and was symptomatic with cough and fever, but no headache, neck stiffness or photophobia. Thus, in the total cohort of 319 in- and outpatients, 64 patients were LFA positive, 63 of whom were also LA positive. No patients were LA positive but LFA negative (Table 2). The overall agreement between the two assays was 99.7%, giving a kappa coefficient of 0.99 (standard error 0.06, P < 0.001) John Wiley & Sons Ltd

3 Table 1 Baseline demographic, clinical and laboratory data All patients CRAG ve* CRAG +ve P-value Outpatients n = (97%) 7 (3%) Sex (% male) 43% (94) 44% (92) 29% (2) 0.7 Age (years) 39 (31 48) 39 (31 48) 38 (30 55) 0.6 ART (% on ART) 44% (96) 45% (95) 14% (1) 0.1 CD4 count (cells/ll) 96 (69 115) 97 (72 116) 19 (9 58) <0.001 Outpatients CD4 100 cells/ll n = (95%) 6 (5%) Sex (% male) 44% (54) 44% (52) 33% (2) 0.5 Age (years) 38 (30 48) 37 (30 47) 43 (30 55) 0.4 ART (% on ART) 39% (48) 40% (47) 17% (1) 0.4 CD4 count (cells/ll) 74 (54 89) 75 (59 90) 17 (9 36) <0.001 Inpatients n = (44%) 57 (56%) Sex (% male) 53% (54) 57% (25) 51% (29) 0.6 Age (years) 38 (30 48) 40 (32 51) 38 (30 47) 0.5 ART (% on ART) 27% (27) 36% (16) 19% (11) 0.06 CD4 count (cells/ll) 33 (18 78) 82 (48 104) 25 (12 32) <0.001 Headache 67 (66%) 20 (45%) 47 (82%) <0.001 Neck stiffness 18 (18%) 0 (0%) 18 (32%) <0.001 Photophobia 1 (1%) 0 (0%) 1 (2%) 1.0 Coma 13 (13%) 1 (2%) 12 (21%) Vomiting 22 (22%) 7 (16%) 15 (26%) 0.2 Fever 53 (52%) 15 (34%) 38 (67%) Cough 63 (62%) 28 (64%) 35 (61%) 0.8 *Lateral flow assay (LFA) results. Data shown are % (n) or median (IQR) unless otherwise stated. P-values from Mann Whitney U test, the Chi-squared test or Fisher s exact test as appropriate. Table 2 Association between cryptococcal antigen detection (by LFA), ART status and CD4 cell count in ART outpatient clinic attendees (n = 218) LFA positive% (n) OR P aor* P ART status ART (n = 96) 1% (1) 1 < No ART (n = 122) 5% (6) 3.3 ( ) 1.7 ( ) CD4 cell count 0 50 cells/ll (n = 29) 17% (5) 1 < < cells/ll (n = 95) 1% (1) 0.02 ( ) 0.03 ( ) >100 cells/ll (n = 94) 1% (1) 0.02 ( ) 0.02 ( ) *aor = adjusted odds ratio with 95% confidence intervals. Adjusted for CD4 cell count category and ART status. P-values calculated using likelihood ratio testing. Discussion Cryptococcal antigenaemia was detectable in 5% of patients with CD4 cell counts 100 cells/µl attending an ART clinic in northern Tanzania. These are the first CRAG prevalence data from ART clinic attendees in Tanzania and are consistent with other East African data showing a high prevalence of subclinical cryptococcal infection in asymptomatic HIV-infected individuals with advanced immune suppression(lara-peredo et al. 2000; Tassie et al. 2003; Liechty et al. 2007; Meya et al. 2010; Meyer et al. 2013). Liechty et al. (2007) found a 5.8% prevalence of cryptococcal antigenaemia in a cohort of 377 patients with CD4 cell counts 100 cells/µl and no prior history of CM at ART initiation in rural Uganda. A second study in Kampala, Uganda, detected a higher CRAG prevalence of 8.8% in a similar cohort of 295 patients (Meya et al. 2010), and a recent Kenyan study reported a CRAG prevalence of 11% in a cohort of 514 ART-naive patients with CD4 cell counts 100 cells/µl (Meyer et al. 2013). These findings are also in keeping with data from Southern African ART programmes, where CRAG prevalence in 2013 John Wiley & Sons Ltd 1077

4 Table 3 Agreement between cryptococcal lateral flow assay (LFA) and latex agglutination (LA) tests in 319 Tanzanian patients Lateral flow assay (LFA) Positive Negative Total Latex agglutination (LA) Positive Negative Total % agreement. Cohen s kappa coefficient patients screened prior to treatment initiation ranges from four to seven per cent (Jarvis et al. 2009; NHLS 2013). The reason for the slightly lower CRAG prevalence found in our study than the recent studies from Kampala (Meya et al. 2010) and western Kenya (Meyer et al. 2013) may relate to our inclusion of patients on ART. This was carried out for operational reasons to maximise sample size within the recruitment time frame. The duration of ART permitted prior to patient recruitment was limited to 6 months, and the available data suggest that cryptococcal antigenaemia persists for over 1 year in most patients following treatment for CM (Jarvis et al. 2011). A small number of patients with asymptomatic low-level antigenaemia may have cleared their infection within the first 6 months of ART (Jarvis et al. 2009; Kwan et al. 2012), leading us to underestimate the true CRAG prevalence. More importantly, as CM usually develops very early after ART initiation (Jarvis et al. 2010) and has a high casefatality rate, a number of these patients may have been missed as they would either be dead, or excluded from the study due to their previous CM, again leading to an underestimation of the CRAG prevalence in the section of the cohort on ART. Although the cross-sectional method of our study means that we have no information about what subsequently happened to patients with subclinical cryptococcal infection, evidence from several independent studies demonstrates that asymptomatic cryptococcal antigenaemia is a strong risk factor for CM and mortality in patients initiating ART (Liechty et al. 2007; Jarvis et al. 2009; Meya et al. 2010). The CRAG prevalence of 5% found in this cohort of ART clinic attendees with CD4 cell counts 100 cells/µl suggests that CRAG screening and pre-emptive treatment for CRAG-positive patients would be a worthwhile and almost certainly cost-effective (Meya et al. 2010; Jarvis et al. 2013) intervention in the Tanzanian ART programme. Our results also highlight the good agreement between the conventional LA CRAG detection assays and the dipstick format LFA, further adding to the accumulating data demonstrating close agreement between the two tests (Jarvis et al. 2011; Lindsley et al. 2011; Binnicker et al. 2012; McMullan et al. 2012; Hansen et al. 2013). The only discordance between the LA and LFA tests in our study was in a single patient with pulmonary symptoms who was positive using the LFA but had a negative LA. Rather than a false positive LFA result, this may have been a false negative LA result in a patient with low-level cryptococcal antigenaemia, reflecting the ability of the LFA to detect lower levels of antigen than the conventional LA assays (Gates- Hollingsworth & Kozel 2013). These data both emphasise the high burden that HIVassociated cryptococcal disease places on in-patient hospital services in Tanzania (Kisenge et al. 2007; Wajanga et al. 2011), with 57 patients diagnosed with cryptococcal infection during a six-month period, and provide further justification for targeted CRAG testing of HIVinfected inpatients and improved resources for the treatment of CM in Tanzania (Wajanga et al. 2011). In conclusion, 5% of patients with CD4 cell counts 100 cells/µl attending an ART clinic in northern Tanzania had detectable asymptomatic cryptococcal infection, suggesting that CRAG screening and targeted pre-emptive antifungal treatment in this patient group may reduce morbidity and mortality due to CM. The LFA is a reliable, cheap and practical alternative to LA for detection of CRAG in regions such as Tanzania. Acknowledgements Immuno-Mycologics Inc (IMMY) donated the latex agglutination and lateral flow assay kits. The ministry of health and social welfare is acknowledged for sponsoring the postgraduate training. We thank the nurses at the CTC who helped with data collection and the patients involved in the study. Funding was obtained from the Ministry of Health and Social Welfare, government of the United Republic of Tanzania. The sponsors were not involved in study design, data analysis or preparation of the manuscript. References Binnicker MJ, Jespersen DJ, Bestrom JE & Rollins LO (2012) Comparison of four assays for the detection of cryptococcal antigen. Clinical and Vaccine Immunology 19, Gates-Hollingsworth MA & Kozel TR (2013) Serotype sensitivity of a lateral flow immunoassay for cryptococcal antigen (CrAg). Clinical and Vaccine Immunology 20, John Wiley & Sons Ltd

5 Hansen J, Slechta ES, Gates-Hollingsworth MA et al. (2013) Large-scale evaluation of the immuno-mycologics lateral flow and enzyme-linked immunoassays for detection of cryptococcal antigen in serum and cerebrospinal fluid. Clinical and Vaccine Immunology 20, Jarvis JN & Harrison TS (2007) HIV-associated cryptococcal meningitis. AIDS 21, Jarvis JN, Lawn SD, Vogt M, Bangani N, Wood R & Harrison TS (2009) Screening for cryptococcal antigenemia in patients accessing an antiretroviral treatment program in South Africa. Clinical Infectious Diseases 48, Jarvis JN, Lawn SD, Wood R & Harrison TS (2010) Cryptococcal antigen screening for patients initiating antiretroviral therapy: time for action. Clinical Infectious Diseases 51, Jarvis JN, Percival A, Bauman S et al. (2011) Evaluation of a novel point-of-care cryptococcal antigen test on serum, plasma, and urine from patients with HIV-associated cryptococcal meningitis. Clinical Infectious Diseases 53, Jarvis JN, Govender N, Chiller T et al. (2012) Cryptococcal antigen screening and preemptive therapy in patients initiating antiretroviral therapy in resource-limited settings: a proposed algorithm for clinical implementation. Journal of the International Association of Physicians in AIDS Care 11, Jarvis JN, Harrison TS, Lawn SD, Meintjes G, Wood R & Cleary S (2013) Cost effectiveness of cryptococcal antigen screening as a strategy to prevent hiv-associated cryptococcal meningitis in South Africa. PLoS ONE 8, e Kisenge PR, Hawkins AT, Maro VP et al. (2007) Low CD4 count plus coma predicts cryptococcal meningitis in Tanzania. BMC Infectious Diseases 14, 39. Kwan CK, Leelawiwat W, Intalapaporn P et al. (2012) Utility of cryptococcal antigen screening and evolution of asymptomatic cryptococcal antigenaemia among HIV-infected women starting antiretroviral therapy in Thailand. 19th Conference on Retroviruses and Opportunistic Infections (CROI). Seattle, WA. Lara-Peredo O, Cuevas LE, French N, Bailey JW & Smith DH (2000) Cryptococcal infection in an HIV-positive Ugandan population. Journal of Infection 41, 195. Lawn SD, Harries AD, Anglaret X, Myer L & Wood R (2008) Early mortality among adults accessing antiretroviral treatment programmes in sub-saharan Africa. AIDS 22, Liechty CA, Solberg P, Were W et al. (2007) Asymptomatic serum cryptococcal antigenemia and early mortality during antiretroviral therapy in rural Uganda. Tropical Medicine & International Health: TM & IH 12, Lindsley MD, Mekha N, Baggett HC et al. (2011) Evaluation of a newly developed lateral flow immunoassay for the diagnosis of cryptococcosis. Clinical Infectious Diseases 53, McMullan BJ, Halliday C, Sorrell TC et al. (2012) Clinical utility of the cryptococcal antigen lateral flow assay in a diagnostic mycology laboratory. PLoS ONE 7, e Meya DB, Manabe YC, Castelnuovo B et al. (2010) 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 resourcelimited settings. Clinical Infectious Diseases 51, Meyer AC, Kendi CK, Penner JA et al. (2013) The impact of routine cryptococcal antigen screening on survival among HIV-infected individuals with advanced immunosuppression in Kenya. Tropical Medicine & International Health: TM & IH 18, NHLS (2013) Laboratory-based screening for cryptococcal disease. Report for the period: 3 September - 21 December National Institute for Communicable Diseases; (accessed 27 Feb 2013). Park BJ, Wannemuehler KA, Marston BJ, Govender N, Pappas PG & Chiller TM (2009) Estimation of the current global burden of cryptococcal meningitis among persons living with HIV/AIDS. AIDS 23, Tassie JM, Pepper L, Fogg C et al. (2003) Systematic screening of cryptococcal antigenemia in HIV-positive adults in Uganda. Journal of Acquired Immune Deficiency Syndromes 33, Wajanga BM, Kalluvya S, Downs JA, Johnson WD, Fitzgerald DW & Peck RN (2011) Universal screening of Tanzanian HIV-infected adult inpatients with the serum cryptococcal antigen to improve diagnosis and reduce mortality: an operational study. Journal of the International AIDS Society 11, 48. Walker AS, Prendergast AJ, Mugyenyi P et al. (2012) Mortality in the year following antiretroviral therapy initiation in HIV-infected adults and children in Uganda and Zimbabwe. Clinical Infectious Diseases 55, Corresponding Author Joseph N. Jarvis, Department of Clinical Research, Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, Keppel Street, London, WC1E 7HT, UK. joejarvis@doctors.net.uk 2013 John Wiley & Sons Ltd 1079