Cryptococcal meningitis (CM) is caused by the encapsulated fungi Cryptococcus gattii and Cryptococcus neoformans, which is commonly found in soil enriched with bird guano. Exposure occurs through inhalation of the fungal spores and likely occurs early in life. However, the disease remains dormant because of the suppressive effects of the immune system.1 In people with HIV/AIDS, C. neoformans may reactivate, causing primary pulmonary or disseminated infection, including CM.2 The highest risk for the development of CM is among those with a CD4 count <100 cells per cubic millimeter.3
Worldwide, HIV-associated CM is estimated to affect approximately 1 million persons and cause 600,000 deaths each year, with the largest burden in sub-Saharan Africa.4 In-hospital mortality rates range from 20% to 50% in resource-limited settings,5,6 with higher mortality rates in settings of suboptimal treatment.7 Optimal treatment of CM requires potentially toxic drugs and regular lumbar punctures to manage elevated intracranial pressure. These treatments are expensive, require close monitoring and inpatient hospitalization, and are not widely available in most resource-limited setting.
One method to prevent the development of CM in the first place involves screening patients at high risk for developing CM for early cryptococcal infection.8 This is done by a blood test that detects cryptococcal antigen (CrAg). Research has shown that CrAg is detectable in patients' sera for a median of 3 weeks before the development of symptomatic CM, creating a window for intervention with antifungal treatment to prevent CM.9 In 2011, the World Health Organization (WHO) issued an integrated guideline recommendation for cryptococcal screening among persons with CD4 <100 cells per cubic millimeter who were initiating HIV care.10 Subsequent studies have shown that treatment of early cryptococcal disease can improve outcomes among persons with advanced HIV and is cost-effective down to a CrAg prevalence of 0.6%.11–13
In Nigeria, the high number of new HIV infections combined with late presentations following infection and inadequate treatment coverage results in serious opportunistic infections like CM.4,14 Limited data exist on CM in Nigeria. Osazuwa et al15 evaluated the prevalence of CrAg among ART-naive patients with CD4 <200 cells per cubic millimeter in Benin, a town in southern Nigeria, and found a prevalence of 12.7%. Because of Nigeria's size and previous reports, which suggest that C. neoformans may have a differential environmental distribution in some areas, studies of CrAg prevalence from multiple geographical areas are needed to guide effective and efficient implementation of cryptococcal screening in Nigeria.16,17
Study Setting and Design
This was a retrospective, cross-sectional study to perform CrAg testing on archived whole-blood samples collected from HIV-infected participants at US President's Emergency Plan for AIDS Relief (PEPFAR)-supported sites across Nigeria. Sites in 4 regions, the South East, South West, North West, and North Central, were selected to represent the major geographical and ethnic diversity seen in Nigeria. Because of the low volume of obtainable samples in the North West and North Central regions, samples from these regions were consolidated and their region was considered “North.”
Written informed consent was obtained from all patients on enrollment into care with permission to use archived samples for future studies. The study protocol was approved by the Institutional Review Board and Ethics Committee of University of Nigeria Teaching Hospital, Enugu, Institute of Human Virology Nigeria, and the 68 Nigerian Army Reference Hospital, Yaba, Lagos.
Samples were collected between April 2004 and August 2014 and were tested for CrAg positivity between May 2014 and September 2014. All samples on initial collection and processing were separated into aliquots, placed in cryovials, and stored at −80°C. During the study, samples meeting criteria were identified, retrieved, and kept in a liquid nitrogen freezer until testing. CD4 testing had been performed on samples at the time of initial collection, and resulting CD4 counts were obtained from patients' medical records for use in the analyses of this study.
All stored samples from participants presenting to an outpatient ambulatory setting were selected if they met the inclusion criteria. Samples must have been (1) processed and stored at −80°C, (2) collected from patients with known CD4+ cell count less than 200 cells per cubic millimeter, (3) collected from patients naive to antiretroviral therapy at the time of collection, and (4) collected from patients with written consent to allow performance of further HIV-related testing on archived samples.
All statistical analyses were conducted using STATA version 12. Overall similarities between the 3 regions of the study in demographic and disease characteristics were examined. Demographic characteristics of the populations sampled were compared using Pearson χ2 test. Median CD4 count and age among groups were compared using nonparametric equality-of-medians tests. Differences in proportions of CrAg-positive individuals within and across regions were compared using Fisher exact test. CrAg prevalence by CD4 count stratum, sex, and age group were only compared between the South East and South West regions; the small number of CrAg cases in the North precluded meaningful comparison.
Prevalence ratios for factors related to CrAg screening were calculated using log-binomial regression models.18 For each variable, the prevalence ratio and 95% confidence interval were calculated, adjusting for sample clustering by study site using a generalized linear model. A stratified analysis was also conducted to obtain the prevalence odds ratio for low CD4 count (<100 cells per cubic millimeter) by region.
A total of 2752 stored blood samples from 3 geographic regions were retrospectively screened for CrAg. The numbers of samples screened by region are as follows: 999 in the South East, 1329 in the South West, and 424 in the North. Most of the samples were collected from individuals aged 30–44 years (n = 1585; 57.6%), and 1570 samples (57.1%) were collected from women. Median CD4 counts were 121 in the North, 84 in the South East, and 95 in the South West and were significantly higher among individuals in the North compared with those in the South East and South West (P < 0.001). Mean CD4 count among CrAg-positive specimens was 77 in the North, 46 in the South East, and 41 in the South West without significant differences (P = 0.88).
The overall prevalence of CrAg positivity in specimens with CD4 <200 cells per cubic millimeter was 2.3% (95% confidence interval: 1.8% to 3.0%), although this number varied significantly across the 3 regions (P < 0.001; Table 1). The South East had the highest prevalence at 4.4%. The prevalence of CrAg positivity in specimens with CD4 <100 cells per cubic millimeter was 3.6% and varied significantly across the 3 regions (P < 0.001; Table 1).
Of patients found to be CrAg positive, the median CD4 count was 45 cells per cubic millimeter (range: 7–178 cells per cubic millimeter). Patients with advanced immunosuppression (CD4 < 100 cells per cubic millimeter) were 3.89 times more likely to have been CrAg positive (95% confidence interval: 3.33 to 4.54) than those with CD4 counts between 100 and 200 cells per cubic millimeter (P < 0.001; Table 2), adjusting for sex and age.
Median CD4 counts of CrAg-positive patients were not significantly different overall (P = 0.89). Among patients from the South East and South West, respectively, 82% and 83% who tested positive for CrAg had CD4 <100 cells per cubic millimeter. At a cutoff of CD4 <150 cells per cubic millimeter, the number of CrAg-positive cases detected increased to 91% and 100% in the South East and South West, respectively.
WHO's 2011 Rapid Advice on cryptococcal disease recommends consideration of routine screening for cryptococcal antigen (CrAg) in asymptomatic patients with CD4 count below 100 cells per cubic millimeter in areas with a CrAg prevalence ≥3%.10 In light of these recommendations, this study suggests 2 key findings. First, of the regions studied, cryptococcal disease was found to be most prevalent in Nigeria's South East. With a prevalence of 6.2% among immunocompromised patients (CD4 < 100) screened in this region, cryptococcal disease prevalence in this group is comparable to countries where CrAg screening has been incorporated into national ART guidelines. Even in Nigeria's South West, where this study found prevalence to be low (1.4), screening still would likely be cost-effective, according to recent estimates by Jarvis et al12 citing cost-effectiveness of this intervention down to a prevalence as low as 0.6% in South Africa.
It is worth noting that CrAg estimates from other settings were most often obtained using the WHO recommended screening threshold of CD4 <100 cells per cubic millimeter. This CD4 cutoff used in screening for cryptococcal disease brings up the second key finding of this study: almost one-fifth of patients who tested positive for CrAg in the South West and South East would not have been captured by screening under the WHO guideline. Magambo et al19 also noted a high prevalence of asymptomatic cryptococcal antigenemia (CrAg) in patients with CD4 count between 100 and 200. This calls into question the use of 100 CD4 cells per cubic millimeter as a cutoff. However, very little data exists on the cost-effectiveness of screening patients with CD4 100–200 cells per cubic millimeter, and little is known about differences in development of CM and survival of patients in this range as compared with those with CD4 <100 cells per milliliter.
We found differences in the prevalence of CrAg by region and a lower prevalence when compared to other studies.15Cryptococcus neoformans survives and multiplies in soil at high humidity and relatively low temperatures (4–24°C).20 We saw higher prevalence of CrAg in the Southern regions of Nigeria compared to the North region. The Northern region of Nigeria has hotter temperatures and lower humidity compared to the Southern region, which might decrease the survival of C. neoformans. Additionally, mean CD4 count was higher in the North. We also found lower prevalence of CrAg than has been reported in other studies.15 This might be because of different sample sizes and participant populations (eg, participants recruited from hospitals vs from outpatient clinics).
Although there was not a significant difference between men and women in the total sample, there were differences in the North and Southeast regions. This may be because of a differential risk of expose to fungal spores based on occupation or cultural norms.21
Estimates from this study provide insight into the complexity of Nigeria's cryptococcal disease burden and, on a larger scale, the country's evolving HIV epidemic, although several notable limitations exist. Few samples were analyzed in the North region of the country. Political instability in this region limited the amount of sample collection and testing that could be carried out.
Because of the limitations of the data collected in North, few sound conclusions were able to be drawn about the region. However, other studies do suggest that cryptococcal disease may still pose a public health problem in the northern regions. A hospital-based prevalence study in Shika, a town in the North West region, found that CrAg was prevalent in a slightly larger percentage (2.23%) of HIV patients attending a care center than was seen in this study.22
To our knowledge, this is the largest cryptococcal prevalence study to date in the world. It provides baseline estimates for the prevalence of CrAg in the North, South West, and South East regions of Nigeria, highlighting the regional variation of cryptococcal disease. Additionally, the prevalence found in 2 regions exceeds current estimates for minimum prevalence at which cryptococcal screening is a cost-effective intervention; consideration should be given to implementing cryptococcal screening in these areas. Further research is needed to gain a more complete understanding of the regional complexities of this high-mortality AIDS-related problem facing a country with the second highest HIV burden worldwide.23
This study would not be possible without the staff members of University of Nigeria, IHVN, and 68 Nigeria Army Reference Hospital Yaba-Lagos. The authors are especially indebted to the dedication of the laboratory staff and specific individuals who made this study possible, including Augustine Mpamugo, Isiramen Olajide, Izuchukwu Ibeagha, Julie Mbah, Aaron Hunt, Blessing Oyagha, Faith Toyin Oni, Gimbiya Priscilla Yashim, Dr Owens Wiwa, Dr Michael Obiefune, Dr Alash'le Abimiku, and Dr Patrick Dakum.
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