Prevalence of Cryptococcal Antigenemia and Lateral Flow Assay Accuracy in Severely Immunosuppressed AIDS Patients.

This study aimed to estimate the prevalence of cryptococcal antigenemia detected by lateral flow assay (LFA) in AIDS patients and its accuracy in the diagnosis of cryptococcosis. Conducted at a university hospital in Brazil from March 2015 to July 2017, it included AIDS patients over 18 years old with a CD4+ count ≤ 200 cells/mm3. Cryptococcal antigen (CrAg) detection using LFA and latex agglutination (LA), along with blood and urine cultures, were performed. The reference standard was the identification of Cryptococcus spp. in clinical specimens through microbiological or histopathological examination. Among 230 patients, the prevalence of CrAg detected by LFA (CrAg LFA) was 13.0%. Factors associated with cryptococcal antigenemia included fever, vomiting, seizures, and a lack of antiretroviral therapy. The sensitivity and specificity of CrAg LFA were 83.9% and 98.0%, respectively. The positive predictive value (PPV) was 86.7%, the negative predictive value (NPV) was 97.5%, and overall accuracy was 96.1%. Cross-reactions were observed in patients with histoplasmosis and paracoccidioidmycosis, but not with aspergillosis or positive rheumatoid factor. The study concludes that the LFA is a useful tool for detecting cryptococcal antigenemia in severely immunocompromised AIDS patients due to its high NPV, specificity, and PPV.

Antifungal susceptibility and multilocus sequence typing (MLST) of Cryptococcus neoformans complex from HIV-associated cryptococcal meningitis patients in Manaus, Amazonas, Brazil.

Cryptococcus neoformans is primarily responsible for cases of cryptococcal meningitis in individuals with HIV/AIDS. This study evaluated the susceptibility of C. neoformans obtained from individuals with cryptococcal meningitis associated with HIV/AIDS in Manaus, Amazonas, Brazil, against the action of the antifungals amphotericin B, flucytosine, fluconazole, itraconazole and posaconazole and analyzed it using Multilocus Sequence Typing (MLST) in order to identify the Sequence Types (STs). We analyzed 30 isolates of C. neoformans, from 24 HIV/AIDS patients diagnosed with cryptococcosis from 2017 to 2019 in a reference hospital, in addition to 3 environmental isolates: 1 isolate obtained in the home of a patient and 2 isolates obtained from neighboring homes of patients. 86.6% (n = 26/30) of the clinical isolates were identified as C. neoformans VNI ST93, 6.6% (n = 2/30) as C. neoformans VNI ST5, 3.3% (n = 1/30) as C. neoformans VNI ST32 and 3.3% (n = 1/30) as C. neoformans VNB ST232. The environmental isolates were identified as C. neoformans VNI ST93 (n = 3/3). 96.6% (n = 29/30) isolates were sensitive to amphotericin B, though there was variation in the MIC. 60% (n = 18/30) presented a MIC above the proposed epidemiological cutoff values for one or more antifungals. All environmental isolates were sensitive to the tested antifungals. The MLST showed that there is an important relationship between C. neoformans VNI ST93 and individuals with HIV/AIDS, including in the environmental isolates analyzed. C. neoformans VNB ST232 was observed for the first time in Amazonas. Amphotericin B was proven to be the best drug, but fluconazole and posaconazole also showed relevant action.

Histoplasmosis at a Reference Center for Infectious Diseases in Southeast Brazil: Comparison between HIV-Positive and HIV-Negative Individuals.

Objectives: Histoplasmosis is a systemic mycosis, present globally. We aimed to describe cases of histoplasmosis (Hc) and to establish a risk profile associated with Hc in HIV-infected patients (HIV+). Methods: This was a retrospective study of patients with a clinical laboratory diagnosis of Hc. Data were fed into REDCap, and statistical analysis was performed with R. Results: We included 99 records, 65 HIV+ and 34 HIV-. Average age was 39 years. Median time from onset to diagnosis was 8 weeks in HIV- and 22 weeks in HIV+. Disseminated histoplasmosis occurred in 79.4% of HIV+, vs. 36.4% of HIV- patients. Median CD4 count was 70. Co-infection with tuberculosis was present in 20% of HIV+ patients. Blood cultures were positive in 32.3% of HIV+ vs. 11.8% of HIV- (p = 0.025) patients; bone marrow culture was positive in 36.9% vs. 8.8% (p = 0.003). Most HIV+ patients (71.4%) were hospitalized. On univariate analysis, anemia, leukopenia, intensive care, use of vasopressors and mechanical ventilation were associated with death in HIV+ patients. Conclusions: Most of our patients with histoplasmosis were HIV+, presenting advanced AIDS. Diagnosis was late in HIV+ patients, and they frequently presented disseminated Hc, required hospitalization, and died. Early screening for Hc in HIV+ and drug-induced immunosuppressed patients is crucial.

Faster Cryptococcus Melanization Increases Virulence in Experimental and Human Cryptococcosis.

Cryptococcus spp. are human pathogens that cause 181,000 deaths per year. In this work, we systematically investigated the virulence attributes of Cryptococcus spp. clinical isolates and correlated them with patient data to better understand cryptococcosis. We collected 66 C. neoformans and 19 C. gattii clinical isolates and analyzed multiple virulence phenotypes and host-pathogen interaction outcomes. C. neoformans isolates tended to melanize faster and more intensely and produce thinner capsules in comparison with C. gattii. We also observed correlations that match previous studies, such as that between secreted laccase and disease outcome in patients. We measured Cryptococcus colony melanization kinetics, which followed a sigmoidal curve for most isolates, and showed that faster melanization correlated positively with LC3-associated phagocytosis evasion, virulence in Galleria mellonella and worse prognosis in humans. These results suggest that the speed of melanization, more than the total amount of melanin Cryptococcus spp. produces, is crucial for virulence.

Mixed infection by Cryptococcus neoformans and Cryptococcus gattii and coinfection with paracoccidioidomycosis in PLHIV.

We present a rare condition of mixed C. neoformans and C. gattii infection in a person living with HIV with false-negative CrAg LFA in the CSF and co-infection with paracoccidioidomycosis. Signs and symptoms are relative to respiratory tract and skin, confounding with other opportunistic disease. After negatives CrAg LFA and Indian ink staining in CSF, there was isolation of C. gattii in sputum and C. neoformans in CSF, in addition to reagent serology (double immunodiffusion) for PCM with 1/16 titer. The patient was treated with amphotericin B and TMP-SMX with good clinical response and recovery of cellular immunity after initiation of antiretroviral therapy.

Cryptococcosis in HIV/AIDS patients in northern Brazil: Clinical aspects, molecular types and isolation of agents from environmental samples associated with patients.

OBJECTIVES: In the state of Amazonas, northern Brazil, cryptococcosis is endemic, with a predominance of Cryptococcus neoformans in individuals with HIV/AIDS, and Cryptococcus gattii VGII in non-HIV individuals. This study analysed the clinical isolates and clinical-epidemiological characteristics of HIV/AIDS patients diagnosed with cryptococcosis in a tertiary healthcare facility in Manaus, Amazonas and investigated the presence of agents of cryptococcosis in environmental samples. METHODS: A survey was made of data from HIV/AIDS patients diagnosed with cryptococcosis between January 2017 and December 2019, and environmental samples were collected at the patients' and their neighbours' homes. The isolates were submitted to morphophysiological analysis and PCR-RFLP typing to determine the molecular types. RESULTS: Clinical-epidemiological characteristics of 55 patients and 75 clinical isolates were analysed. Neurocriptococcosis was the clinical form observed in 98.2% (n = 54/55) of patients. A total of 38.1% (n = 21/55) of patients died within 100 weeks, of which 21.8% (n = 12/55) died less than a month after the diagnosis of cryptococcosis. C. neoformans VNI (n = 68/75), C. neoformans VNII (n = 1/75), C. gattii VGI (n = 3/75) and C. gattii VGII (n = 3/75) were identified. Mixed infection was observed in two patients, one by C. neoformans VNI and VNII and the other by C. neoformans VNI and C. gattii VGI. Cryptococcus VNI was detected in three (n = 3/51) households, one of a patient (n = 1/17) and two households that neighbour patients' houses (n = 2/34). CONCLUSIONS: This study demonstrated the prevalence of C. neoformans VNI, which is a cause of cryptococcosis in patients with HIV/AIDS in the state of Amazonas, and revealed a greater diversity of molecular types affecting these patients in the region than in previous studies. In the studied group, a high mortality rate was observed, which reflects the importance of early diagnosis, and evidences cryptococcosis as an AIDS-defining disease and an important public health problem in the region. The home environment proved to be a potential source of infection/reinfection by C. neoformans VNI.

Indoor Dust as a Source of Virulent Strains of the Agents of Cryptococcosis in the Rio Negro Micro-Region of the Brazilian Amazon.

Cryptococcosis, a potentially fatal mycosis in humans, is acquired via exposure to exogenous environmental sources. This study aimed to investigate the frequency, genetic diversity, and virulence of cryptococcal strains isolated from indoor dust in the Rio Negro micro-region of the Brazilian Amazon. A total of 8.9% of the studied houses were positive, recovering nine Cryptococcus neoformans VNI and 16 C. gattii VGII isolates, revealing an endemic pattern in domestic microenvironments. The International Society for Human and Animal Mycology (ISHAM) consensus multilocus sequence typing (MLST) scheme for the C. neoformans/C. gattii species complexes identified two sequence types (STs), ST93 and ST5, amongst C. neoformans isolates and six STs amongst C. gattii isolates, including the Vancouver Island Outbreak ST7 (VGIIa) and ST20 (VGIIb), the Australian ST5, and ST264, ST268 and ST445, being unique to the studied region. Virulence studies in the Galleria mellonella model showed that five C. gattii strains and one C. neoformans strain showed a similar pathogenic potential to the highly virulent Vancouver Island outbreak strain CDR265 (VGIIa). The findings of this study indicate that humans can be exposed to the agents of cryptococcosis via house dust, forming the basis for future studies to analyze the impact of early and continuous exposure to indoor dust on the development of subclinical or clinical infections.

Genotypic and Phenotypic Stability of Mixed Primary Isolates of Cryptococcus gattii and Cryptococcus neoformans: A Comparative Analysis of Four Preservation Methods.

The choice of a suitable preservation method is critical for long-term microorganisms' viability. The strains should be preserved for long periods using reliable and reproducible methods that minimize genotypic and phenotypic variations and viability losses. The methodologies are usually designed for a better performance in isolated microorganisms. However, atypical primary isolates of Cryptococcus neoformans or Cryptococcus gattii, such as mixed species or even different species of a species complex, are a challenge for long-term preservation and taxonomic review studies. The aim of this study was to evaluate which of the four preservation methods tested presented better performance in the preservation of simulated coexistence strains of C. neoformans and C. gattii. Two environmental strains, one C. gattii and one C. neoformans, were mixed in vitro to test four different preservation methods (freezing at -20°C, -80°C, -196°C, and freeze-drying). The colony-forming units from each preservation method were evaluated, and colonies were randomly selected and cultivated in canavanine glycine bromothymol blue (CGB) agar to evaluate the amounts of CGB-positive (C. gattii) and CGB-negative (C. neoformans) colonies resulting from each preservation method after 1 week, 15 days, 1 month, 6 months, and 1 year. According to our results, cryopreservation at -20°C demonstrated was preferable for C. neoformans species, and further studies after long-term storage are necessary. Recovery of yeast cells after freeze-dried preservation in skim milk is better for both species. Ultrafreezing methods evaluated (-80°C and -196°C) also showed better results in the maintenance of C. gattii. Freeze-drying should be preferred for the maintenance of multilineage isolates from the C. neoformans and C. gattii species complexes.

Mortality by cryptococcosis in Brazil from 2000 to 2012: A descriptive epidemiological study.

BACKGROUND: Cryptococcosis is a neglected and predominantly opportunistic mycosis that, in Brazil, poses an important public health problem, due to its late diagnosis and high lethality. METHODS: The present study analysed cryptococcosis mortality in Brazil from January 2000 to December 2012, based on secondary data (Mortality Information System/SIM-DATASUS and IBGE). RESULTS: Out of 5,755 recorded deaths in which cryptococcosis was mentioned as one of the morbid states that contributed to death, two distinct groups emerged: 1,121 (19.5%) registered cryptococcosis as the basic cause of death, and 4,634 (80.5%) registered cryptococcosis associated with risk factors, mainly AIDS (75%), followed by other host risks (5.5%). The mortality rate by cryptococcosis as the basic cause was 6.19/million inhabitants, whereas the mortality rate by cryptococcosis as an associated cause was 25.19/million inhabitants. Meningitis was the predominant clinical form (80%), males were the more affected (69%), and 39.5 years old was the mean age. The highest mortality rate due to cryptococcosis as basic cause occurred in the state of Mato Grosso (10.96/million inhabitants). Mortality rates due to cryptococcosis as associated cause were highest in the states of Santa Catarina (70.41/million inhabitants) and Rio Grande do Sul (64.40/million inhabitants), both in the South Region. Southeast, Northeast and South showed significant time trends in mortality rates. CONCLUSIONS: This study is relevant because it shows the magnitude of cryptococcosis mortality linked to AIDS and removes the invisibility of a particular non-AIDS-related disease, accounting for almost 20% of all cryptococcosis deaths. It can also contribute to control and surveillance programs, beyond highlighting the urgent prioritization of early diagnosis and proper treatment to reduce the unacceptable mortality rate of this neglected mycosis in Brazil.

Cryptococcosis due to Cryptococcus gattii VGII in southeast Brazil: The One Health approach revealing a possible role for domestic cats.

Two cats infected by C. gattii, presented lesions on the nasal region and respiratory signs. Strains were typed as molecular type VGII, mating type alpha, MLST subtypes ST442 and ST185. Since Rio de Janeiro is known as an endemic area for C. neoformans VNI, these cases might be a warning for a possible emergence of C. gattii VGII in southeast Brazil.

Comparative antifungal susceptibility analyses of Cryptococcus neoformans VNI and Cryptococcus gattii VGII from the Brazilian Amazon Region by the Etest, Vitek 2, and the Clinical and Laboratory Standards Institute broth microdilution methods.

Early diagnosis, efficient clinical support, and proper antifungal therapy are essential to reduce death and sequels caused by cryptococcosis. The emergence of resistance to the antifungal drugs commonly used for cryptococcosis treatment is an important issue of concern. Thus, the in vitro antifungal susceptibility of clinical strains from northern Brazil, including C. neoformans VNI (n = 62) and C. gattii VGII (n = 37), to amphotericin B (AMB), 5-flucytosine, fluconazole, voriconazole, and itraconazole was evaluated using the Etest and Vitek 2 systems and the standardized broth microdilution (CLSI-BMD) methodology. According to the CLSI-BMD, the most active in vitro azole was voriconazole (C. neoformans VNI modal MIC of 0.06 µg/ml and C. gattii VGII modal MIC of 0.25 µg/ml), and fluconazole was the least active (modal MIC of 4 µg/ml for both fungi). Modal MICs for amphotericin B were 1 µg/ml for both fungi. In general, good essential agreement (EA) values were observed between the methods. However, AMB presented the lowest EA between CLSI-BMD and Etest for C. neoformans VNI and C. gattii VGII (1.6% and 2.56%, respectively, P < .05 for both). Considering the proposed Cryptococcus spp. epidemiological cutoff values, more than 97% of the studied isolates were categorized as wild-type for the azoles. However, the high frequency of C. neoformans VNI isolates in the population described here that displayed non-wild-type susceptibility to AMB is noteworthy. Epidemiological surveillance of the antifungal resistance of cryptococcal strains is relevant due to the potential burden and the high lethality of cryptococcal meningitis in the Amazon region.

Performance of cryptococcal antigen lateral flow assay in serum, cerebrospinal fluid, whole blood, and urine in HIV-infected patients with culture-proven cryptococcal meningitis admitted at a Brazilian referral center.

Cryptococcal meningitis is the most common cause of opportunistic meningitis in HIV-infected patients in Brazil and causes unacceptable high mortality rates. In this study, HIV-infected patients with a first episode of culture-proven cryptococcal meningitis in cerebrospinal fluid (CSF) were prospectively included in order to evaluate sensitivity of cryptococcal antigen (CrAg) lateral flow assay (LFA) in serum, CSF, whole blood (fingerstick), and fresh urine. In addition, HIV-infected patients with other neurological confirmed diseases were included in order to evaluate the specificity of CrAg LFA in serum. Twenty patients with cryptococcal meningitis were included and in 19 of them, CrAg LFA in CSF, serum, and whole blood were positive (95% sensitivity). In 18 patients, India ink test was positive in CSF (90% sensitivity), and in 16 cases, CrAg LFA was positive in urine (80% sensitivity). Thirty-six HIV-infected patients with other neurological diseases had negative results of CrAg LFA in serum (100% specificity). In conclusion, CrAg LFA in serum, CSF, and whole blood showed high sensitivity and specificity. Whole blood CrAg LFA seems to be a good and reliable strategy to improve AIDS-related cryptococcal meningitis diagnosis in Brazil.

Performance of cryptococcal antigen lateral flow assay in serum, cerebrospinal fluid, whole blood, and urine in HIV-infected patients with culture-proven cryptococcal meningitis admitted at a Brazilian referral center

Cryptococcal meningitis is the most common cause of opportunistic meningitis in HIV-infected patients in Brazil and causes unacceptable high mortality rates. In this study, HIV-infected patients with a first episode of culture-proven cryptococcal meningitis in cerebrospinal fluid (CSF) were prospectively included in order to evaluate sensitivity of cryptococcal antigen (CrAg) lateral flow assay (LFA) in serum, CSF, whole blood (fingerstick), and fresh urine. In addition, HIV-infected patients with other neurological confirmed diseases were included in order to evaluate the specificity of CrAg LFA in serum. Twenty patients with cryptococcal meningitis were included and in 19 of them, CrAg LFA in CSF, serum, and whole blood were positive (95% sensitivity). In 18 patients, India ink test was positive in CSF (90% sensitivity), and in 16 cases, CrAg LFA was positive in urine (80% sensitivity). Thirty-six HIV-infected patients with other neurological diseases had negative results of CrAg LFA in serum (100% specificity). In conclusion, CrAg LFA in serum, CSF, and whole blood showed high sensitivity and specificity. Whole blood CrAg LFA seems to be a good and reliable strategy to improve AIDS-related cryptococcal meningitis diagnosis in Brazil

Paracoccidioidomycosis after Highway Construction, Rio de Janeiro, Brazil.

Transmission of Paracoccidioides spp. fungi to humans is usually related to manipulation of soil. Rural workers are the most affected group. We report an outbreak of paracoccidioidomycosis after deforestation and massive earth removal during construction of a highway in Rio de Janeiro, Brazil. Extensive environmental disturbances might be involved in fungal transmission.

Asymptomatic cryptococcal antigen prevalence detected by lateral flow assay in hospitalised HIV-infected patients in São Paulo, Brazil

OBJECTIVE: To determine the prevalence of asymptomatic cryptococcal antigen (CRAG) using lateral flow assay (LFA) in hospitalised HIV-infected patients with CD4 counts <200 cells/ll. METHODS: Hospitalised HIV-infected patients were prospectively recruited at Instituto de Infectologia Emilio Ribas, a tertiary referral hospital to HIV-infected patients serving the S~ao Paulo State, Brazil. All patients were >18 years old without prior cryptococcal meningitis, without clinical suspicion of cryptococcal meningitis, regardless of antiretroviral (ART) status, and with CD4 counts <200 cells/ll. Serum CRAG was tested by LFA in all patients, and whole blood CRAG was tested by LFA in positive cases. RESULTS: We enrolled 163 participants of whom 61% were men. The duration of HIV diagnosis was a median of 8 (range, 1­29) years. 26% were antiretroviral (ART)-na€ive, and 74% were ARTexperienced. The median CD4 cell count was 25 (range, 1­192) cells/ll. Five patients (3.1%; 95%CI, 1.0­7.0%) were asymptomatic CRAG-positive. Positive results cases were cross-verified by performing LFA in whole blood. CONCLUSIONS: 3.1% of HIV-infected inpatients with CD4 <200 cells/ll without symptomatic meningitis had cryptococcal antigenemia in São Paulo, suggesting that routine CRAG screening may be beneficial in similar settings in South America. Our study reveals another targeted population for CRAG screening: hospitalised HIV-infected patients with CD4 <200 cells/ll, regardless of ART status. Whole blood CRAG LFA screening seems to be a simple strategy to prevention of symptomatic meningitis

Nosocomial candidiasis in Rio de Janeiro State: Distribution and fluconazole susceptibility profile.

One hundred and forty-one Candida species isolated from clinical specimens of hospitalized patients in Rio de Janeiro, Brazil, during 2002 to 2007, were analized in order to evaluate the distribution and susceptibility of these species to fluconazole. Candida albicans was the most frequent species (45.4%), followed by C. parapsilosis sensu lato (28.4%), C. tropicalis (14.2%), C. guilliermondii (6.4%), C. famata (2.8%), C. glabrata (1.4%), C. krusei (0.7%) and C. lambica (0.7%). The sources of fungal isolates were blood (47.5%), respiratory tract (17.7%), urinary tract (16.3%), skin and mucous membrane (7.1%), catheter (5.6%), feces (2.1%) and mitral valve tissue (0.7%). The susceptibility test was performed using the methodology of disk-diffusion in agar as recommended in the M44-A2 Document of the Clinical and Laboratory Standards Institute (CLSI). The majority of the clinical isolates (97.2%) was susceptible (S) to fluconazole, although three isolates (2.1%) were susceptible-dose dependent (S-DD) and one of them (0.7%) was resistant (R). The S-DD isolates were C. albicans, C. parapsilosis sensu lato and C. tropicalis. One isolate of C. krusei was resistant to fluconazole. This work documents the high susceptibility to fluconazole by Candida species isolated in Rio de Janeiro, Brazil.

Nosocomial candidiasis in Rio de Janeiro State: Distribution and fluconazole susceptibility profile

One hundred and forty-one Candida species isolated from clinical specimens of hospitalized patients in Rio de Janeiro, Brazil, during 2002 to 2007, were analized in order to evaluate the distribution and susceptibility of these species to fluconazole. Candida albicans was the most frequent species (45.4%), followed by C. parapsilosis sensu lato (28.4%), C. tropicalis (14.2%), C. guilliermondii (6.4%), C. famata (2.8%), C. glabrata (1.4%), C. krusei (0.7%) and C. lambica (0.7%). The sources of fungal isolates were blood (47.5%), respiratory tract (17.7%), urinary tract (16.3%), skin and mucous membrane (7.1%), catheter (5.6%), feces (2.1%) and mitral valve tissue (0.7%). The susceptibility test was performed using the methodology of disk-diffusion in agar as recommended in the M44-A2 Document of the Clinical and Laboratory Standards Institute (CLSI). The majority of the clinical isolates (97.2%) was susceptible (S) to fluconazole, although three isolates (2.1%) were susceptible-dose dependent (S-DD) and one of them (0.7%) was resistant (R). The S-DD isolates were C. albicans, C. parapsilosis sensu lato and C. tropicalis. One isolate of C. krusei was resistant to fluconazole. This work documents the high susceptibility to fluconazole by Candida species isolated in Rio de Janeiro, Brazil.

In vitro susceptibility testing of amphotericin B for Cryptococcus neoformans variety grubii AFLP1/VNI and Cryptococcus gattii AFLP6/VGII by CLSI and flow cytometry.

Cryptococcus neoformans var. grubii AFLP1/VNI is the main causative agent of cryptococcosis associated with AIDS in the world. Cryptococcus gattii AFLP6/VGII causes mainly endemic primary infection in immunocompetent hosts. To determine the minimum inhibitory concentrations (MICs) of C. neoformans var. grubii AFLP1/VNI and C. gattii AFLP6/VGII against amphotericin B (AMB) in a short period of time, flow cytometry (FCM) with FUN-1 fluorochrome was used to compare with broth microdilution method (CLSI M27-A3). The minimum incubation period was evaluated by minimum fungicidal concentration procedure. Seventeen clinical isolates of C. neoformans var. grubii AFLP1/VNI and 18 of C. gattii AFLP6/VGII were analysed. The time for the determination of MICs by FCM was 2 h against 72 h by CLSI M27-A3 and the comparison of MIC showed a positive significant correlation (P = 0.048). It is important to highlight the role of the FCM as an alternative method to determine the MICs for AMB in within a day, with positive cost-benefit.

Environmental isolation of Cryptococcus gattii VGII from indoor dust from typical wooden houses in the deep Amazonas of the Rio Negro basin.

Cryptococcosis is a human fungal infection of significant mortality and morbidity, especially in the meningoencephalitis form. Cryptococcosis is distributed worldwide and its agents, C. neoformans and C. gattii, present eight major molecular types-VNI-VNIV and VGI-VGIV respectively. The primary cryptococcosis caused by molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent patients in the North and Northeast of Brazil, revealing an endemic regional pattern to this molecular type. Since 1999, C. gattii VGII has been involved in an ongoing outbreak in Canada, and is expanding to the Northwest of the United States, two temperate regions. Exposure to propagules dispersed in the environment, related to various organic substrates, mainly decomposing wood in and around dwellings, initiates the infection process. The present study investigated the presence of the agents of cryptococcosis in dust from dwellings in the upper Rio Negro, municipality of Santa Isabel do Rio Negro in Amazonas state. Indoor dust was collected from 51 houses, diluted and plated on bird seed agar. Dark brown colonies were identified phenotypically, and genotypically by URA5 restriction fragment length polymorphism analysis and multilocus sequence typing (MLST). The mating type was identified using pheromone-specific primers. Three of the 51 houses were positive for C. gattii molecular type VGII, MATα and MATa, showing a high prevalence of this agent. MLST studies identified eight subtypes, VGIIb (ST7), VGIIa (ST20), (ST5) and 5 new subtypes unique to the region. For the first time in the state of Amazonas, C. gattii VGII MATα and MATa were isolated from the environment and correlates with endemic cryptococcosis in this state. This is the first description of MLST subtypes on environmental isolates in the Brazilian Amazon, indicating domiciliary dust as a potential source for human infection with different subtypes of C. gattii VGII MATα and MATa.