Shifting epidemiology and microbiological profiles of cryptococcosis in a Brazilian tertiary center: A descriptive study of 94 patients

Abstract

Background

The epidemiology of cryptococcosis is shifting, with an increasing number of cases occurring among non-HIV immunosuppressed individuals. In this context of a new epidemiological presentation of the disease, further studies are needed to better describe the microbiological profiles associated with different immune status.

Aims

To characterize cryptococcosis cases according to immune status, focusing on species distribution and time to culture positivity.

Methods

We conducted a retrospective study of patients with positive Cryptococcus spp. cultures from various biological materials between 2017 and 2022 at a Brazilian tertiary hospital. Clinical and laboratory data were extracted, including immune status, biological sample type, Cryptococcus species, and time to positivity in aerobic and fungal cultures. Patients were classified into HIV-positive, non-HIV immunosuppressed, or non-immunosuppressed/non-HIV. Descriptive analyses were performed.

Results

Among the 94 patients analyzed, 29 (30.9%) were HIV-positive, 39 (41.5%) were non-HIV immunosuppressed, and 19 (20.2%) were non-immunosuppressed/non-HIV. A total of 288 isolates were identified, predominantly Cryptococcus neoformans (72/94, 76.6%), but Cryptococcus gattii (17/94, 18%) infection was non-neglectable. The mean time to culture positivity was 5 days (range 2–24 days) for aerobic cultures and 10 days (range 0–63 days) for fungal cultures.

Conclusions

This work reinforces the need for future studies to better represent non-HIV immunosuppressed patients, to address infections caused by C. gattii, and to consider the wide variability in culture growth time as a diagnostic limitation.

Resumen

Antecedentes

La epidemiología de la criptococosis está cambiando, con un número creciente de casos en individuos inmunosuprimidos, pero no infectados por el VIH. En este contexto de una nueva presentación epidemiológica de la enfermedad se necesitan más estudios para describir mejor los perfiles microbiológicos asociados con los diferentes estados inmunológicos.

Objetivos

Caracterizar los casos de criptococosis según el estado inmunológico de los pacientes, con atención en la distribución de las especies y el tiempo transcurrido hasta la positividad de los cultivos.

Métodos

Realizamos un estudio retrospectivo de pacientes con cultivos positivos para Cryptococcus spp. obtenidos de diversos materiales biológicos entre 2017 y 2022 en un hospital terciario brasileño. Se extrajeron datos clínicos y de laboratorio, incluyendo el estado inmunológico, el tipo de muestra biológica, las especies de Cryptococcus aisladas y el tiempo hasta la positividad en cultivos aeróbicos y fúngicos. Los pacientes fueron clasificados en los grupos VIH positivo, inmunosuprimidos no infectados por VIH, y no-inmunosuprimidos/no-VIH. Se realizaron análisis descriptivos.

Resultados

De un total de 94 pacientes analizados, 29 (30,9%) eran VIH positivos, 39 (41,5%) eran inmunosuprimidos no-VIH y 19 (20,2%) eran no-inmunosuprimidos/no-VIH. Se identificaron 288 aislamientos, con el predominio de Cryptococcusneoformans (72/94, 76,6%), aunque la infección por Cryptococcusgattii (17/94, 18%) no fue desdeñable. El tiempo medio hasta la positividad fue de 5 días (rango 2-24 días) para los cultivos aeróbicos y de 10 días (rango 0-63 días) para los cultivos fúngicos.

Conclusiones

Este trabajo refuerza la necesidad de futuros estudios que representen mejor a los pacientes inmunosuprimidos no-VIH, que aborden las infecciones causadas por C. gattii y que consideren la amplia variabilidad en el tiempo de crecimiento de los cultivos como una limitación diagnóstica.

Cryptococcosis is a systemic mycosis caused by species of the Cryptococcus genus, with Cryptococcus neoformans and Cryptococcus gattii complexes¹⁰ being the primary pathogenic agents in humans. Infection occurs through the inhalation of yeasts or basidiospores from the environment, potentially resulting in primary pulmonary infection and hematogenous dissemination, particularly to the central nervous system (CNS).^4,9,10

Cryptococcosis has a high mortality rate, especially in immunosuppressed individuals, including those with HIV/AIDS, transplant recipients, and patients on immunosuppressive therapy. The World Health Organization has classified C. neoformans species complex as a critical-priority pathogen¹⁸ due to its high associated mortality, ranging from 41% to 61% in immunocompromised patients.⁸ Cryptococcal meningitis significantly contributes to the disease burden, particularly among patients living with HIV/AIDS, estimated to globally account for 15% of AIDS-related deaths.¹³

Cryptococcosis remains a public health concern in Brazil, with 5755 related deaths recorded from 2000 to 2012, among which cryptococcosis was the primary cause (mortality rate: 6.09/million inhabitants) in 19.5% of the patients.² Cryptococcosis typically presents a slow and insidious clinical course, often leading to delayed diagnoses despite the availability of highly effective diagnostic methods, ultimately contributing to increased mortality.¹⁶

The clinical laboratory plays a crucial role in achieving a diagnosis through microbiological methods such as culture, cerebrospinal fluid (CSF) examination, and cryptococcal antigen (CrAg) detection.^5,6 Culture results can take 7–14 days; however, earlier fungal growth enables prompt starting of antifungal therapy.⁵ Furthermore, studies have shown that CSF sterility after antifungal induction therapy is associated with better prognosis, reinforcing the importance of laboratory monitoring.^6,15

Given the increasing recognition of cryptococcosis among non-HIV immunosuppressed individuals and the limited data describing their microbiological profiles, we aimed to characterize cryptococcosis cases according to immune status. Specifically, we sought to describe species distribution, clinical sample types, and the variability in time to culture positivity, with emphasis on its implications for timely diagnosis and therapeutic decision-making.

Methods

This was a retrospective, observational, cross-sectional study of routinely processed clinical samples submitted to the microbiology laboratory between 2017 and 2022, at the Clinical Microbiology Laboratory of the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo.

We included patients with positive cultures for Cryptococcus spp. from any clinical sample. Patients were classified into three groups based on medical records: HIV-positive (documented infection), non-HIV immunosuppressed (e.g., transplant recipients, patients on corticosteroids or other immunosuppressants), and non-immunosuppressed/non-HIV individuals. Patients with incomplete medical records or unclear documentation regarding HIV status or other immunosuppressive conditions were classified as ‘Undetermined’ and analyzed as a separate group.

After culture identification, the clinical sample type and distribution over time were analyzed. For further analysis, only the first sample per patient was selected. If both fungal and aerobic cultures from the same specimen were requested, fungal culture was prioritized.

Clinical samples such as CSF, respiratory specimens (e.g., sputum, bronchoalveolar lavage), urine, and tissue biopsies were processed for fungal cultures by inoculating them onto Sabouraud agar, being incubated at 37 °C up to 30 days. Yeast growth was assessed daily during the first week, every other day in the second week, and twice weekly thereafter. When yeast-like colonies were observed, India ink staining was performed and colonies were subcultured onto CHROMagar Candida (bioMérieux) for preliminary differentiation. Final species identification was performed using MALDI-TOF mass spectrometry (VITEK® MS, bioMérieux).

In the case of aerobic cultures, blood samples mainly were inoculated into BD BACTEC™ bottles and incubated under automated aerobic conditions; other sterile fluids (e.g., ascitic, pleural, or peritoneal fluid) were inoculated occasionally. Once flagged positive by the system, subcultures were performed by plating onto blood agar and incubating at 37 °C up to 48 h. Yeast suspected colonies were identified using MALDI-TOF MS.

For urine specimens, samples were plated on chromID™ CPS® medium and incubated at 37 °C up to 48 h. Respiratory and biopsy specimens were also cultured on blood agar under the same conditions. Yeast colonies identification was performed with MALDI-TOF MS.

Data on patients’ age, gender, Cryptococcus species isolated, clinical sample, referring healthcare unit, date of medical request, latex agglutination test results, CSF physical and biochemical parameters, clinical diagnosis, and subsequent negative cultures were retrieved from the hospital's electronic medical records. Reference values for CSF cytochemical analysis were based on established normal ranges: cell count <5 cells/μL, glucose >40 mg/dL, and protein <45 mg/dL.¹⁷

Statistical analysis

Data analysis was descriptive. Categorical variables were presented as absolute numbers and percentages. Continuous variables were reported as medians and range. Analyses were conducted using IBM SPSS Statistics for Windows, Version 30.0.0 (IBM Corp., Armonk, NY, USA). All charts and tabular data were compiled using Microsoft Excel for Microsoft 365 (Microsoft Corp., Redmond, WA, USA).

Results

Ninety four patients with culture-confirmed cryptococcosis were included. Table 1 summarizes the characteristics of the patients included in the study. Twenty nine patients (30.9%) were HIV-positive, 39 (41.5%) were non-HIV immunosuppressed, 19 (20.2%) were non-immunosuppressed/non-HIV, and 7 (7.4%) had undetermined immunological status. The overall median age was 49 years (range, 17–92), with HIV-positive patients being younger (median 33 years, range 19–51), in contrast to non-HIV immunosuppressed and non-immunosuppressed individuals (median ages 55 and 58 years, respectively). Males predominated across all groups, comprising 67% of the cohort (63/94), with the highest proportion among HIV-positive patients (82.8%). Over the years, we observed an increased percentage of HIV negative patients with cryptococcosis (Fig. 1).

Table 1.

Clinical and microbiological characteristics of patients with cryptococcosis, stratified by immunological status.

Variables	HIV positive	Immunossuppresssed	Non-HIV/Non-immunossuppressed	Undetermined	Total
	n (%)	n (%)	n (%)	n (%)	n (%)
Male	24 (82.76)	25 (64.1)	10 (52.63)	4 (57.1%)	63 (67.02)
Female	5 (17.24)	14 (35.9)	9 (47.37)	3 (42.9%)	31 (32.98)
Age (Median)	33 (19–51)	55 (23–92)	58 (33–86)		49 (17–92)
Cryptococcus species
C. neoformans	27 (93.1%)	30 (76.9%)	12 (63.1%)	3 (42.9%)	72 (76.6%)
C. gattii	2 (6.9%)	8 (20.5%)	5 (26.3%)	2 (28.6%)	17 (18%)
C. albidus	0	1 (2.6%)	0	1 (14.3%)	2 (2.1%)
C. laurentii	0	0	1 (5.3%)	0	1 (1.1%)
C. iniguttulatus	0	0	1 (5.3%)	0	1 (1.1%)
Cryptococcus sp.	0	0	0	1 (14.3%)	1 (1.1%)
Aerobic culture TTP
Mean (days)	5	6	6	4	5
Median (days)	5	5	5.5	4	5
Min–max (days)	(2–24)	(2–24)	(2–16)	(3–5)	(2–24)
Fungal culture TTP
Mean (days)	8	11	12	17	10
Median (days)	6	8	7	12.5	7
Min–max (days)	(0–31)	(3–47)	(2–63)	(7–34)	(0–63)
CrAg latex agglutination in CSF
>1/512	19 (73%)	11 (42.3%)	1 (12.5%)	1 (33.3%)	31 (50%)
<1/512	3 (11.5%)	6 (23.1%)	5 (62.5%)	2(66.6%)	16 (25.8%)
Non-reactive	4 (15.4%)	9 (34.6%)	2 (25%)	0	15 (24.28%)
Not performed	3	13	11	4	31

SD: standard deviation; CSF: cerebrospinal fluid, reported as dilution factors; TTP: time to positivity.

Fig. 1.

Distribution of the cryptococcosis cases among the different clinical groups over the five years of the study.

The mean time for growth in aerobic culture was 5 days, ranging from 2 to 24 days, with no significant differences among clinical groups. The median time was 5 days, except in the non-HIV/non-immunosuppressed group, where it was slightly longer (5.5 days, range: 2–16 days). For fungal culture, the mean growth time was 10 days, ranging from 0 to 63 days (Table 1).

Regarding the biological materials analyzed, CSF was the most prevalent, accounting for approximately 62.5% of isolates, followed by blood with 18.4%, biopsies with 13.19%, urine with approximately 3%, and respiratory samples with 2.78%. Among the most frequently isolated species, C. neoformans species complex was the most prevalent, but the frequency of C. gattii with 17 isolates (18%) was non-negligible.

Regarding CSF cellularity, 52% of HIV-positive patients exhibited abnormalities, whereas 48% had normal values (Table 2). Among non-HIV immunosuppressed patients, the majority (71.4%) showed cellularity alterations. In the non-immunosuppressed/non-HIV group, cellularity was normal in 50% of the cases and abnormal in the other half. In terms of glycorrhachia, 52% of HIV-positive patients exhibited reduced CSF glucose concentrations. In contrast, 42.9% of non-HIV immunosuppressed patients had abnormal glycorrhachia values, with a higher proportion (57.1%) having normal concentrations. The non-immunosuppressed/non-HIV group showed the lowest frequency of glycorrhachia alterations, with 75% of patients presenting normal values. Regarding CSF protein levels, protein abnormalities were highly prevalent among all groups, with the highest frequency in non-HIV immunosuppressed patients (89.3%), followed by HIV-positive (76%), and non-immunosuppressed/non-HIV (75%).

Table 2.

Cryptococcosis patients by clinical group and cytochemical parameters values in in cerebrospinal fluid.

	HIV positive		Immunossuppressed		Non-HIV/non-immunossuppressed		Undertermined
	Normal	Abnormal	Normal	Abnormal	Normal	Abnormal	Normal	Abnormal
Cellularity	12 (48%)	13 (52%)	8 (28.6%)	20 (71.4%)	4 (50%)	4 (50%)	0	3 (100%)
Glycorrhachia	12 (48%)	13 (52%)	16 (57.1%)	12 (42.9%)	6 (75%)	2 (25%)	1 (33.3%)	2 (66.7%)
Proteins	6 (24%)	19 (76%)	3 (10.7%)	25 (89.3%)	2 (25%)	6 (75%)	1 (33.3%)	2 (66.7%)

CrAg testing in CSF using latex agglutination was performed in 63 patients, of whom 31 (59.3%) had high titers (>1:512), 16 (25.8%) had titers <1:512, and 15 (24.2%) were non-reactive (Table 1). In 31 patients (33%), the CrAg test was not performed or data were unavailable.

Discussion

In this study we provide a descriptive overview of microbiological profiles among patients with cryptococcosis diagnosed at a Brazilian tertiary center over a five-year period. We observed a high frequency of non-HIV immunosuppressed patients, a non-neglectable frequency of C. gattii, and a highly variable time to positive cultures.

In this analysis of 94 patients and 288 Cryptococcus spp. isolates over a five-year period we observed a notable epidemiological shift in cryptococcosis. While initially cases were more frequent among HIV-positive individuals, they increasingly involved non-HIV immunosuppressed patients, particularly from 2019 onwards. This trend may reflect multiple, non-mutually exclusive factors, including expanded access to antiretroviral therapy, reducing the incidence of advanced HIV disease, a growing population of transplant recipients and individuals receiving immunosuppressive or biologic therapies, and broader implementation of CrAg testing for early detection in high-risk groups. Additionally, an increased referral of complex immunosuppressed patients to our tertiary care center may have contributed. Although these hypotheses remain speculative, they underscore the need to include non-HIV-infected populations in future research on cryptococcosis.

C. neoformans species complex is the most frequently isolated species, particularly among HIV-positive patients, due to their profound cellular immunosuppression, as highlighted in 2022 by Rajasingham et al. in their global burden study on cryptococcal meningitis in people living with HIV/AIDS.¹³ In contrast, C. gattii species complex is more commonly associated with non-HIV immunosuppressed individuals, such as solid organ transplant recipients³ or those receiving prolonged corticosteroid therapy.^7,11 In our study, we observed a non-neglectable frequency of C. gattii and uncommon species such as Cryptococcus albidus, Cryptococcus laurentii, and Cryptococcus uniguttulatus. This is important because most studies provide data on C. neoformans, which might not apply to other species.

Culture from body sites such as blood, CSF, sputum, urine or bronchoalveolar lavage fluid is considered the gold standard for the diagnosis of cryptococcosis, although it may take 1–10 days to yield fungal growth.^1,6 Among the 288 isolates analyzed, 133 were obtained from aerobic culture, while 155 were recovered in fungal-specific cultures. The distribution of biological materials analyzed in this study reflects established patterns of cryptococcal infection, with CSF being the most commonly analyzed specimen, accounting for approximately 62.5% of all isolates. Blood cultures accounted for 18.4% of isolates, reflecting the systemic dissemination of Cryptococcus species, which is particularly common in advanced HIV infection. The data also emphasize the importance of performing blood culture in these patients, particularly in cases with sepsis.^12,14

The mean time to culture positivity was notably shorter for aerobic cultures (5 days) compared to fungal cultures (10 days). This duration may appear long when considering the anticipated time frame, as most samples are kept in an incubator up to 48 h before the microorganism is identified, typically totaling 3 days. The increase in average time is attributed to hemocultures, which account for approximately 20% of the samples. In these cases, incubation in hemoculture tubes takes up to 5 days, followed by culture in a specific medium. This finding is consistent with previous research demonstrating that fungal pathogens, particularly species of Cryptococcus, typically require 1–10 days for growing in culture, and extended incubation periods, due to their slower growth rates, are necessary to yield positive results.^1,6 However, we found a wide range of time to positive culture, which underscores this limitation of fungal cultures in the management of cryptococcal infection.

Our study has some limitations: as a retrospective observational study, our analysis is subject to inherent biases, including incomplete data collection. This might have had an impact on how the patients were classified within the groups. However, it seems improbable that the fact that the majority of patients were non-HIV positive was impacted. The study was conducted at a single tertiary care hospital in Brazil, limiting the generalizability of the findings to other regions. The tertiary nature might have increased the number of non-HIV immunosuppressed patients (e.g. transplants or autoimmune diseases). The limited sample size, especially within specific subgroups, constrained conducting formal statistical comparisons and limits the strength of the conclusions that can be drawn from the data. Although cultures were performed, antifungal susceptibility testing was not done systematically, which limits assessing potential drug resistance patterns, an important factor in treatment failures and a topic for future studies.

Conclusion

This work highlights the need for future investigations to more comprehensively include non-HIV immunosuppressed patients, who represent a growing proportion of cryptococcosis cases. A greater focus on C. gattii species complex infections is warranted given it represented almost one fifth of the cases. Additionally, it should be accepted that the broad variability in culture growth time is a key limitation in the diagnosis of this disease.

Authors’ contributions

S.M.R.: conceptualization, visualization, study design, writing original draft; V.F. de O.: visualization, writing original draft; A.P.C.: visualization, writing original draft; H.R.G.: data collection; M.T.: visualization; A.S.G.K.M.; visualization and study design; G.D.S.: conceptualization; A.R.S.J.: data collection; A.L.M.: conceptualization, data collection; W.L.L.K: writing review; E.M.P.A.A.: writing review and editing; M.M.C.M.: conceptualization, writing review and editing, supervision. All authors have read and agreed to the published version of the manuscript.

Institutional review board statement

The study was conducted in accordance with the Declaration of Helsinki. The ethical approval of the study was obtained from the local ethics committee (Protocol No. 72638123.1.0000.0068).

Consent for publication

All authors have read and agreed to the published version of the manuscript.

Funding

The authors did not receive specific funding for this study.

Conflicts of interest

M.M.C.M has received support for attending educational meetings from Knight and Mundipharma. The remaining authors declare having no conflict of interest.

Availability of data and material

Not applicable.