High prevalence of Cryptococcal antigenemia using a finger-prick lateral flow assay in individuals with advanced HIV disease in Santarém Municipality, Brazilian Amazon Basin

Abstract

There is scarce information about HIV-related cryptococcosis in the Brazilian Amazon basin where laboratory infrastructure is limited. The serum cryptococcal antigen (CrAg) lateral flow assay (LFA) has simplified diagnosis of cryptococcosis and is recommended for screening in advanced HIV disease. We evaluated the prevalence of cryptococcal antigenemia using finger-prick CrAg LFA in the Brazilian Amazon basin. We enrolled a prospective cohort of outpatients and hospitalized individuals with advanced HIV disease at two centers in Santarém Municipality, Northern Brazil. All individuals were > 18 years old with advanced HIV disease, regardless of antiretroviral therapy (ART) status and with no prior or current history of confirmed cryptococcal meningitis. We tested CrAg LFA on finger-prick whole blood using an exact volume transfer pipette. From August 2018 to October 2019, 104 individuals were enrolled (outpatients 62 [60%] and hospitalized 42 [40%]). Median age was 38 years (interquartile range [IQR] 30–46), and 84 (81%) were male. Sixty-five (63%) individuals were ART-naïve. Prevalence of finger-prick CrAg LFA-positive was 10.6%; 95% CI, 5.4 to 18.1%. Prevalence of finger-prick CrAg LFA-positive among individuals without neurological symptoms was 6.0%; 95% CI, 1.7–14.6%. The number needed to test to detect one CrAg-positive individual was 9.4 persons (95% CI, 5.5–18.5). Prevalence of cryptococcal antigenemia using finger-prick whole blood CrAg LFA was high. Point-of-care approach was important for the diagnosis and screening of cryptococcosis in resource-limited settings. Screening and preemptive therapy strategy should be urgently implemented in individuals with advanced HIV disease in the Brazilian Amazon basin.

Lay Summary

This prospective cohort study was carried-out in the Brazilian Amazon basin. We used a cryptococcal rapid test in patients with AIDS. We included 104 participants, and 11 (10.6%) of them had positive results showing a high prevalence of cryptococcal antigenemia.

Introduction

The World Health Organization (WHO) recommends two strategies to reduce AIDS-related cryptococcal meningitis.¹ First, since cryptococcal antigenemia in peripheral blood precedes neurological manifestations by weeks to months, cryptococcal antigen (CrAg) screening and pre-emptive fluconazole therapy for subclinical infection has arisen as an important approach.^1–4 Second, as mortality in the first two weeks after the diagnosis of cryptococcal meningitis reflects the devastating effects of the disease, early diagnosis and timely combined antifungal treatment are of paramount importance.¹ In this scenario and considering the cost-effectiveness of these strategies, their implementation has been successful in some low- or middle-income countries, particularly in Sub-Saharan Africa.^3,5,6

Brazil is one of the most unequal countries in the world in terms of income distribution. These inequalities exist by socioeconomic status, ethnicity, gender, and geographic region.

In Brazil, despite the declining rate of acquired immunodeficiency syndrome (AIDS) detection in the past decade, there are multiple regional epidemics. The incidence of AIDS has increased in the northern region, covering the Amazon basin. This fact is particularly relevant in Pará State, where AIDS detection rate doubled between 2006 and 2016, an effect partially explained by the spread of the epidemic to rural areas.⁷ HIV diagnosis often occurs late with one-third of new diagnoses presenting with CD4 + T lymphocyte counts < 200 cells/mm³.⁸

The Brazilian Ministry of Health recommends the CrAg test for the diagnosis and screening of people living with HIV/AIDS (PLWHA) with CD4 + T lymphocyte counts < 100 cells/mm³ from areas with a high prevalence of cryptococcal antigenaemia.⁹ However, the prevalence of cryptococcal infection is not well-known in the Amazon basin, and CrAg screening has not been implemented in routine clinical practice due to lack of screening tests in the local public health services.

The aim of the present study was to estimate the prevalence of cryptococcal antigenemia in individuals with advanced HIV disease in a municipality of Brazilian Amazon basin.

Methods

In this prospective cohort study, individuals were enrolled from August 2018 to October 2019. Patients were invited to participate during routine clinical visits in the outpatient HIV clinic Centro de Referência do Sidadão or at admission in the medical wards of Hospital Municipal Dr. Alberto Tolentino Sotelo at Santarém Municipality, if they met inclusion criteria. Both centers are the main referral healthcare facilities for infectious diseases of the Brazilian National Health Service for the Lower Amazon Region and the Tapajós Region, in the west-central of the Pará State, northern Brazil, covering a population of approximately 1000 000 inhabitants spread over an area of more than 500 000 km² (Figure 1). These centers provide treatment for an estimated of 3000 PLWHA.

Figure 1.

Map of Brazil highlighting the State of Pará in the Northern of the country (A), showing the lower Amazon region and Tapajós regions (B) and the localization of Santarém Municipality where are located the study sites (C).

We included individuals with confirmed diagnosis of HIV infection, aged ≥ 18 years old, regardless of antiretroviral therapy (ART) status, presenting with advanced HIV disease and no prior or current history of confirmed cryptococcal meningitis. The definition of advanced HIV disease included at least one the following criteria at enrolment: (1) lymphocytes T-CD4 + counts < 200 cells/μl in the last three months; (2) total lymphocyte count < 1200 cells/μl in the last 3 months; or (3) WHO Clinical Stage 3 or 4. We collected demographic, clinical, and laboratory data from patient interviews and/or medical records on a case report form.

Finger-prick testing of whole blood was performed with the CrAg LFA kits (IMMY Inc, Norman, OK, USA), according to manufacturer's instructions (available at: www.immy.com/crag; accessed in October 10, 2020), including the use of an exact volume disposable transfer pipette.¹⁰ A single trained researcher performed all tests. The results of CrAg LFA were reported to the attending physician for further investigation and treatment at their discretion. If a lumbar puncture was performed by the attending physician, CSF CrAg LFA testing was offered. CrAg test, CSF fungal culture, and blood fungal cultures are not available in the routine care where this study was carried-out. In this setting, the only available diagnostic tool for cryptococcosis is the direct examination of CSF with India ink stain.

Asymptomatic cryptococcal meningitis was defined when the patient had no compatible clinical manifestations and showed positive finger-prick CrAg LFA and positive CSF CrAg LFA. Ruled out cryptococcal meningitis was defined when the patient had negative CSF CrAg LFA.

Oropharyngeal candidiasis was clinically diagnosed. Pulmonary tuberculosis was diagnosed with sputum smear microscopy or through the presence of compatible clinical and radiological manifestations associated with the improvement of these parameters after the use of anti-tuberculosis drugs. Pneumocystis jirovecii pneumonia was diagnosed with the presence of compatible clinical and radiological manifestations associated with the improvement of these parameters after the use of trimethoprim-sulfamethoxazole. Cerebral toxoplasmosis was diagnosed with the presence of compatible clinical and neuroradiological manifestations associated with the improvement of these parameters after the use of anti-Toxoplasma therapy.

We present descriptive statistics of participants’ baseline characteristics and estimate the prevalence of cryptococcal antigenemia. We present categorical variables with n (%) and present quantitative variables with median and interquartile range (IQR). We compared proportions with Fisher's exact test or Mantel--Haenszel chi-square test, as appropriate. All statistical tests were considered significant if P < 0.05. We analyzed data using Stata, version 12.0 (Stata-Corp, College Station, TX, USA).

The Research Ethics Committee of the Sao Paulo University School of Medicine approved this study (CAAE 99 408 718.3.0000.0065).

All study individuals provided written informed consent. When necessary, primary relatives and/or those legally responsible provided written informed consent.

Results

In this study, we enrolled 104 individuals of whom 81% (n = 84) were male, and the median age was 38 years (interquartile range [IQR] 30–46). Sixty-five (63%) individuals were ART-naïve. Sixty % (n = 62) of individuals were outpatients, and 40% (n = 42) were hospitalized. Seventy-eight % (n = 81) of individuals had at least one opportunistic infection other than cryptococcosis at baseline and the most frequent diagnoses were: oropharyngeal candidiasis (n = 32), pulmonary tuberculosis (n = 27), Pneumocystis jirovecii pneumonia (n = 15), or cerebral toxoplasmosis (n = 14). Cryptococcal antigenemia was positive in 11 individuals, representing a prevalence of 10.6%; 95% CI, 5.4–18.1%. Details of these individuals are shown in Table 2.

Table 2.

Main characteristics of people living with HIV/AIDS with asymptomatic positive cryptotoccal antigen finger-prick lateral flow assay in Santarém Municipality, Brazilian Amazon Basin.

	Age/sex	Time of HIV diagnosis	ART	Location	Neurological manifestations	Concomitant diseases	CD4 /μL	Total Lymph /μl	HIV VL copies/ml	CSF India Ink/CSF LFA CrAg	Antifungal treatment	Outcome 6 months
1	67/M	15 days	No	Outpatient	Neurocognitive disorder	Pneumocystis pneumonia	NA	312	NA	Negative/NA	AmB	Death day 11
2	48/M	40 days	Yes	Outpatient	None	Chronic diarrhea	382	769	153	Negative/positive	Fluconazole	Alive
3	45/M	16 days	No	Hospitalized	Headache, motor deficit	Pulmonary tuberculosis/Cerebral toxoplasmosis/Pneumocystis pneumonia	73	329	2003	Negative/NA	None	Alive
4	30/F	Same day	No	Outpatient	None	Pneumocystis pneumonia	NA	425	466.963	Negative/negative	Fluconazole	Alive
5	25/M	Same day	No	Hospitalized	Headache	Pneumocystis pneumonia/Chronic diarrhea	NA	332	NA	NA/NA	None	Lost to follow-up
6	52/F	1 day	No	Outpatient	None	Pneumocystis pneumonia/Oroesofageal candidiasis	NA	271	NA	NA/NA	AmB	Death day 12
7	33/M	2 days	No	Outpatient	None	None	119	2148	NA	NA/NA	Fluconazole	Alive
8	63/F	4 years	Yes	Hospitalized	Altered mental status	Pneumocystis pneumonia/Bacterial Pneumonia	230	511	<40	Negative/Negative	Fluconazole	Death day 22
9	57/F	15 days	No	Hospitalized	Headache, motor deficit	Cerebral toxoplasmosis/Oroesophageal candidiasis	74	662	295	NA/NA	None	Alive
10	36/M	2 months	Yes	Hospitalized	Headache	Pulmonary tuberculosis/Oroesophageal candidiasis	NA	70	NA	Negative/Negative	Fluconazole	Lost to follow-up
11	35/M	3 months	No	Hospitalized	Altered mental status	Plasmablastic lymphoma/Oroesophageal candidiasis	NA	582	NA	Negative/Negative	Fluconazole	Death day 22

NA = not available; ART = antiretroviral therapy; CSF = cerebrospinal fluid; Lymph = lymphocyte count; LFA, lateral flow assay; CrAg = cryptococcal antigen; AmB = amphotericin B deoxycholate.

Among individuals without neurological symptoms (n = 67), 4 of them had CrAg LFA-positive results, representing a prevalence of 6.0%; 95% CI, 1.7–14.6%. One case had confirmed asymptomatic cryptococcal meningitis (Patient #2), one case ruled-out cryptococcal meningitis (Patient #4), and two asymptomatic individuals did not receive lumbar punctures (Patients #6 and #7).

Among individuals with neurological symptoms (n = 37), 7 of them had CrAg LFA-positive results, representing a prevalence of 18.9%; 95% CI, 7.6–35.2%. Cryptococcal meningitis was ruled-out in three individuals (Patients #8, #10, and #11). Two symptomatic individuals declined lumbar punctures (Patients #5 and #9), and two individuals (Patients #1 and #3) had negative CSF India Ink results but CSF LFA CrAg was not performed. Thus, cryptococcal meningitis was not excluded in these last four patients.

Prevalence was higher among hospitalized individuals (6/42: 14.3%; 95% CI, 5.4–28.5%), than in outpatients (5/62; 8.1%; 95% CI, 2.7–17.8%) but this difference was not statistically significant with this sample size (P = 0.18). The number needed to test was 9.4 persons (95% CI, 5.5–18.5) to detect one CrAg-positive individual.

Regarding immunosuppression at presentation, all 11 CrAg LFA-positive individuals were in WHO Clinical stage 3 or 4 compared to 83% (77/93) of the CrAg LFA-negative patients (P = 0.42). Of 92 individuals with available white blood cell counts, 91% (10/11) of CrAg LFA-positive had total lymphocyte counts < 1200 cells/μl compared with 74% (60/81) of CrAg LFA-negative individuals (P = 0.331). Only 52 individuals had available CD4 + count and the median (IQR) was 97.5 (53.3--143) cells/mm³. Most individuals included in this study had clinical symptoms (83%, 86/104). General manifestations (fever, weight loss, hyporexia and/or asthenia) were frequent in CrAg LFA-positive participants (100%, 11/11) as well as CrAg LFA-negative individuals (81%, 75/93; P = 0.318). Similarly, neurological manifestations (headache, abnormal mental status, focal neurological deficit and/or seizures) were frequent in CrAg LFA-positive individuals (64%,7/11) when compared with CrAg LFA-negative individuals (32%, 30/93; P = 0.097). Main baseline characteristics of total, CrAg LFA-positive and CrAg LFA-negative individuals are shown in Table 1.

Table 1.

Baseline characteristics of the study population and comparison by cryptococcal antigen lateral flow assay status.

Variable	Total n = 104	CrAg-Positive n = 11	CrAg-Negative n = 93	P-value
Sex				0.266
Male (%)	84 (80.8%)	7 (63.6%)	77 (82.8%)
Female (%)	20 (19.2%)	4 (36.4%)	16 (17.2%)
Age
Median (IQR)	39.5 (29.7–46.3)	45.6 (34.2–55.1)	38.0 (29.4–45.1)	0.750
Local				0.718
Outpatient	62 (59.6%)	6 (54.5%)	56 (60.2%)
Hospitalized	42 (40.4%)	5 (45.5%)	37 (39.8%)
Time of HIV diagnosis				0.140
< 1 year (%)	70 (67.3%)	10 (90.9%)	60 (64.5%)
≥ 1 year (%)	34 (32.7%)	1 (9.1%)	33 (35.5%)
Previous opportunistic infection				0.560
Yes	32 (30.8%)	2 (18.2%)	30 (32.3%)
No	72 (69.2%)	9 (81.8%)	63 (67.7%)
Antiretroviral therapy				0.697
Naïve	65 (62.5%)	8 (72.7%)	57 (61.3%)
Experienced	39 (37.5%)	3 (27.3%)	36 (38.7%)
HIV WHO clinical stage				0.287
1 or 2	16 (15.4%)	0 (0%)	16 (17.2%)
3 or 4	88 (84.6%)	11 (100%)	77 (82.8%)
CD4 count	n = 52	n = 4	n = 48	0.682
< 200 cells/μl	47 (90.4%)	3 (75%)	44 (91.7%)
≥ 200 cells/μl	5 (9.6%)	1 (25%)	4 (8.3%)
Total lymphocyte count	n = 92	n = 11	n = 81	0.405
< 1200 cells/μl	70 (76.1%)	10 (90.9%)	60 (74.1%)
≥ 1200 cells/μl	22 (23.9%)	1 (9.1%)	21 (25.9%)

CrAg = cryptococcal antigen, WHO = World Health Organization, IQR = interquartile rate.

Among 99 individuals with known six-month outcomes, overall mortality rate was higher in the CrAg LFA-positive group (4/9, 44%) when the compared with the CrAg LFA-negative group (9/90, 10%) with a 5.2-fold higher relative risk of 6-month mortality (95% CI, 1.6 to 17.2, P = 0.032). The underlying cause of deaths in 3 cases of the CrAg LFA-positive group was attributed to the following diseases or conditions: Patient #6 died from bacterial sepsis, Patient #8 had end-stage pulmonary fibrosis and was in palliative care and Patient #11 died from digestive bleeding associated with a plasmablastic lymphoma gastric lesion.

Discussion

The present study demonstrates a 10.6% prevalence of cryptococcal antigenemia in individuals with advanced HIV disease in Santarém, Pará State, in the Brazilian Amazon basin. Although data from Latin America are scarce, the prevalence here reported is higher than those reported in previous studies in Brazil and Argentina, which ranged from 3.1 to 8.1%.^11–14 A prior study performed in outpatients PLWHA from Belém, East Region of Pará State, identified a serum CrAg LFA prevalence of 2.6% among 418 samples (95% CI, 1.3–4.7%).¹³ Some arguments may justify the higher prevalence in our cohort when compared to other Latin American studies. First, the design of this study included hospitalized patients and outpatient individuals. As a result, the higher CrAg prevalence of 14.3% among hospitalized persons inflated the overall prevalence, despite of this difference was not statistically significant. Second, the high cryptococcal disease burden in Amazon basin.^13,15,16 The outpatient prevalence was 8.1% in asymptomatic persons, which is among the highest in Latin America. Third, C. neoformans is the most common etiology in PLWHA in this region. However, C. gattii is the main cause of endemic cryptococcosis in immunocompetent or otherwise healthy hosts, but C. gattii can also be commonly observed in PLWHA from Brazilian Amazon basin.^17,18 In this epidemiological scenario, CrAg LFA may have been particularly important as the specific monoclonal antibodies readily detect antigens of both C. neoformans and C. gattii.^19–21

The high prevalence of cryptococcal antigenemia in individuals with advanced HIV disease identified in this study supports the implementation of CrAg screening with preemptive treatment strategy and the use of CrAg LFA in the early diagnosis of cryptococcal disease in the Amazon basin. Despite the absence of economic evaluation in this setting, the use of CrAg LFA was shown to be cost-effective at a prevalence as low as 0.6% in advanced HIV disease.⁵ The current WHO recommendation is to screen all PLWHA with CD4 count < 100 cells/μl using a serum or plasma sample before initiating or reinitiating ART, followed by preemptive antifungal therapy if CrAg LFA-positive, after excluding cryptococcal meningitis. WHO guideline also recommends to consider screening at a CD4 lymphocytes count threshold of < 200 cells/μl.¹ However, screening guidelines also need to be pragmatic for actual implementation in resource-limited settings. Therefore, we used the total lymphocyte count < 1200 cells/μl or WHO HIV Clinical Stage 3/4 as alternatives criteria for screening as CD4 count would not be available in a timely manner for most patients. We selected these criteria from prior studies.^12,22

Cryptococcal antigen screening can be implemented as either a point-of-care test or laboratory-based approach. In this latter option, test can be performed after a clinician order or reflexively during CD4 count analysis using remnant blood from individuals with advanced HIV disease.²³ Reflex CrAg LFA screening has been successfully implemented in some resource-limited settings, such as South Africa,²⁴ but there is no published experience in Latin America. In Brazil, the clinician order is the most commonly used strategy for obtaining the CrAg test and serum latex agglutination is the most available technique. In this context, we evaluated a point-of-care test approach, which does not require laboratory infrastructure and allows a rapid treatment decision, since Brazilian recommendations for the management of PLWHA indicate early ART initiation regardless CD4 count result.⁹ Despite WHO and Brazilian recommendations, CrAg LFA is not available in the public health system of Brazil. In addition, the access to serum samples is limited in our setting and the use of finger-prick blood is a simpler approach. For the screening and preemptive treatment strategy, where low concentrations of CrAg can be present and finger-prick blood may not provide adequate volume, a recent study reported that using a pipette is an optimized point-of-care method when compared with applying the test directly to finger-prick blood.¹⁰ The present study showed that pipetting finger-prick can be performed in routine clinical conditions from a low-resource setting. Point-of-care tools (i.e HIV testing, CD4 count) can be adequately carried-out for health care worker cadre²⁵ or lay counselors.²⁶ The present report and a prior study¹⁰ suggest that CrAg LFA on pipetting finger-prick blood could also be carried out by non-laboratory staff. For the early diagnosis of cryptococcal meningitis, directly finger-prick blood CrAg LFA showed very high agreement with serum, plasma, and CSF CrAg LFA results.^27–29 Thus, finger-prick CrAg LFA is a valuable tool for early diagnosis of cryptococcal meningitis, even when there is no microbiologic evidence of CSF infection.^19,27,30,31 WHO has coined the term ‘ASSURED tests’ (Affordable, Sensitive, Specific, User-friendly, Rapid, Robust, Equipment-free, and Delivered to those who need it) to describe the ideal characteristics of a diagnostic test in resource-limited settings.²⁰ Optimized finger-prick CrAg LFA meets these criteria. Therefore, our study reinforces importance of implementing supply of CrAg LFA as part of a public health approach to the prevention and diagnosis of cryptococcal diseases in advanced HIV disease in Brazil.

In this study, the general manifestations and baseline characteristics of CrAg LFA positive and negative were similar (Table 1). Despite neurological manifestations were numerically more frequent in CrAg LFA-positive individuals when compared with CrAg LFA-negative individuals, only two of eleven patients of the first group had confirmation of other neurological opportunistic diseases.

Eight of eleven CrAg LFA-positive individuals received antifungal therapy, but four of them eventually died (Table 2). Although the observed mortality was high, the underlying cause of death in three patients was not related to cryptococcosis. In addition, details on dosage, course duration and adherence to antifungals were not available. CrAg LFA-positive individuals showed a higher six-month mortality rate when compared with the results of CrAg LFA-negative individuals. This result is compatible with previous reports that showed CrAg LFA-positive as an independent predictor of mortality in PLWHA² and reinforce the need for close monitoring of patients with cryptococcal antigenemia.

This study has several limitations. First, prevalence of cryptococcal antigenemia was underestimated since we do not include patients presenting with confirmed cryptococcal meningitis. In our setting, diagnosis of cryptococcal meningitis is usually performed by a positive CSF India ink stain in other healthcare services and referred to initiate antifungal therapy in our centers. India ink is particularly insensitive in early disease when the fungal burden is < 5000 Cryptococcus yeasts/ml of CSF.³¹ Thus, the inclusion of confirmed cases could highly overestimate the prevalence of antigenemia by referral filter bias. Second, some individuals in the present study had positive finger-prick CrAg LFA, but CSF CrAg LFA was not performed, which limited the final diagnostic classification of the disease. In addition, some of these patients did not receive pre-emptive treatment, showing the local reality. This scenario reflects our routine conditions and highlights the need of systematic evaluation in similar cases. Third, cryptococcal antigenemia prevalence in hospitalized populations is usually higher than in outpatient one and is accepted that asymptomatic outpatient population is ideal for cryptococcal antigenemia screening.³² In this study, prevalence in hospitalized inpatients was higher than in outpatients but both results were high (14.3 and 8.1%, respectively). These results suggest the importance of a diagnostic evaluation for inpatients and screening of outpatients. Fourth, titration of the CrAg LFA tests, other CrAg test as latex agglutination and CSF cultures was not performed due to financial and infrastructure restrictions. Despite that, this scenario allowed us to assess the importance of introducing CrAg LFA under routine clinical conditions. Fifth, missing data of some participants (i.e CD4 count, follow-up information) reflects the laboratory limitations, the nature of the study design and the profile of a subset traveling population included in this study.

In conclusion, we found a high prevalence of cryptococcal antigenemia in individuals with advanced HIV disease in a municipality of Brazilian Amazon basin. In addition, our result highlights the need to implement the point-of-care CrAg LFA test for the diagnosis and strategy of screening and pre-emptive treatment of cryptococcosis in advanced HIV disease in this region.

Declaration of interest

The authors report no conflicts of interest. The authors are responsible for the content and the writing of the paper.