The epidemiological characteristics of the patients with cryptococcosis were evaluated. Males were more commonly infected (70%) than females, and the age group most affected was between 16 and 30 years (53%; median=28.5 years) (Table 1). The majority of patients with cryptococcosis were also infected with HIV (72%) and resided in the east zone of the city of Manaus (25%) (Table 1). Cerebrospinal fluid (CSF) was collected for the isolation of the fungal agents from most patients (93%). Isolates were inoculated on a CGB medium; 9 were positive for the presence of C. gattii, and 31 were positive for C. neoformans.

All of the isolates had high protease production (Pz<0.64) and an average Pz=0.3 for both Cryptococcus species. All but 1 isolate was positive for the production of phospholipase (average Pz=0.53). Twenty-six C. neoformans isolates (83.8%) presented strong positive results (Pz<0.64), and 4 isolates (12.9%) presented positive results (Pz≥0.64 and < 1.0), whereas all 9 isolates of C. gattii were classified as strong-positive producers of phospholipase.

Using the disk diffusion test (CLSI M44-A), 81, 35 and 100% of the isolates of C. neoformans were identified as susceptible to fluconazole, itraconazole and amphotericin B, respectively, and 78%, 56% and 100% of the isolates of C. gattii were susceptible to these antifungal compounds (Table 2). The average MIC for amphotericin B, evaluated by E-test methodology, was 0.26±0.22μg/mL for C. neoformans and 0.58±0.53μg/mL for C. gattii.

DNA was extracted from all clinical isolates and was used to identify the molecular genotypes by URA5-RFLP. All 9 isolates of C. gattii possessed a fingerprint pattern consistent with the VGII molecular genotype, and all 31 isolates of C. neoformans had the profile of the VNI molecular type.

The total number of the inhabitants in the state of Amazonas is approximately 3.5×106 and according to the Mycology Service of the Foundation of Tropical Medicine of Manaus, 20–25 new cases of cryptococcosis occur annually in the region. Manaus, the capital of Amazonas State, is located near the centre of the Amazon Basin on the left bank of the Rio Negro, consisting of Tertiary sediments on sandy-clay land. The region has an average rainfall of 2300mm/year and is thus associated with a hot and humid climate and lush vegetation.31 In this study, the incidence of cryptococcosis was not related to these weather patterns, as the number of isolates was similar throughout the year. The identification of potential environmental sources of C. neoformans and C. gatii that are introduced into the human population requires further investigation. It is hypothesise that the sources of contamination are similar to those described in other countries: human infection with the Cryptoccocus type VNI primarily occurs from contact with bird faeces, and infection with the VGII type usually occurs following contact with decomposing vegetal biomass.

This is one of the first studies to characterise cryptococcosis in the Amazonas State in Brazil. This study determined that the patients most affected by cryptococcosis were male, young (16–30 years old), HIV-positive and inhabitants of the east zone of the city of Manaus.

The number of cases of HIV/AIDS reported annually in the Amazonas State is approximately 700, half of them receive HAART treatment.30 AIDS is the most important risk factor for cryptococcosis, due to the suppression of immune responses by the HIV virus.9,13,26C. neoformans was the species most frequently isolated (n=31; 77.5%), which is consistent with the 82.3% reported in the literature.17,19C. neoformans almost exclusively causes disease in immunocompromised patients, and the majority of the isolates that we obtained came from patients with AIDS.17,19 Among the nine C. gattii isolates, three were obtained from immunocompetent patients, four from AIDS patients and two from patients without documented immune status.

In this study, we obtained three isolates from children aged 0–15 years. C. gattii was the primary causative agent of meningitis in these patients. In a previous study in the Amazonas State from 1988 to 1998, Martins15 reported that the frequency of cryptococcosis in children represented 33% of the total cases. In the Pará state, neighbouring the Amazonas State, 19–24% of cryptococcosis cases were reported in children over the last 10 years.28,29 These studies demonstrate that in the Amazon Rainforest, the prevalence of cryptococcosis in children is high and is also associated with the molecular type VGII. Further epidemiological studies and environmental sampling are necessary to define the importance of the environmental conditions in the incidence of cryptococcosis cases in children.

The production of lipases and proteases is related to the virulence of Cryptococcus pathogenic species.7,8 These enzymes are involved in the destruction of cellular structures to both obtain nutrients for the fungal pathogens and to facilitate spread throughout tissues. Both C. neoformans and C. gattii produced proteases and lipases in similar quantities, and no difference was observed between molecular types.

Antifungal cryptococcosis testing showed 80% and 77% susceptibility to fluconazole in C. neoformans and C. gattii, respectively. This maintenance drug is commonly used in patients with AIDS to prevent opportunistic fungal diseases, and the emergence of resistant strains has been described.6,9C. neoformans and C. gattii had 35% and 55% susceptibility to itraconozole, respectively, and all of the clinical isolates were susceptible to amphotericin B. These results are consistent with previous studies.14,22,27 Amphotericin B is considered to be the gold standard for cryptococcosis treatment.2 In one study, amphotericin B resistance was reported in 10% of clinical isolates,5 but other studies demonstrate low in vitro resistance to this compound.14,27 Although all isolates were considered to be susceptible to amphotericin B (MIC<2μg/mL), the average MIC of the C. gattii isolates (0.56μg/mL) was greater than that of C. neoformans isolates (0.26μg/mL). This result has been previously reported14 and highlights the potential for differential efficacy of therapeutics in the treatment of C. neoformans and C. gattii.

The identification of pathogenic Cryptococcus species and sub-species are important for both clinical guiding and as well as for epidemiology. Recently, false-positive C. gattii results have been reported using the conventional CGB differentiation method due to the existence of C. neoformans isolates that are resistant to canavanine.13 Meanwhile, PCR fingerprinting and PCR-RFLP are being utilized more frequently.9,20,24 In the current study, PCR-RFLP was used to characterize the Cryptococcus isolates as the molecular types VNI and VGII. Furthermore, these results are consistent with previous studies that identified C. neoformans VNI as the primary agent of cryptococcosis.13,16,19 A study that characterized 63 Brazilian isolates described the prevalence of the molecular types VNI (82.3%), VGII (13.6%) and VNII (3.0%),17 and more recent studies in the Pará state reinforce the prevalence of VNI and VGII as being the principal molecular types of C. neoformans and C. gattii, respectively, in the northern region of Brazil.17

Data from this study can assist with current and future management of cryptococcosis in Northern Brazil by identifying the frequency of C. neoformans and C. gattii in patients as well as their susceptibility to commonly used antifungal drugs. These data also contribute to epidemiological studies of the distribution of different molecular types of Cryptococcus species in the Brazilian Amazon.

The authors have no conflict of interest to declare.