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.

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.

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.

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.

Determination of the minimum inhibitory concentration of Cryptococcus neoformans and Cryptococcus gattii against fluconazole by flow cytometry.

Recent studies have used flow cytometry (FCM) as an important alternative method to determine the antifungal susceptibility of yeasts compared to the broth microdilution Clinical and Laboratory Standards Institute (CLSI) reference procedure. We present a comparative study of the broth microdilution method and flow cytometry to assess the in vitro antifungal susceptibility of Cryptococcus neoformans (n = 16) and C. gattii (n = 24) to fluconazole. The minimum inhibitory concentration (MIC) assays by flow cytometry were defined as the lowest drug concentration that showed ∼50% of the count of acridine orange negative cells compared to that of the growth control. Categorical classification showed all C. neoformans isolates were susceptible to fluconazole. Three isolates of C. gattii were susceptible dose-dependent and the remaining 21 isolates were classified as susceptible. MICs comparison of both methodologies demonstrated 100% categorical agreement of the results obtained for C. neoformans and C. gattii. The MICs obtained with the CLSI-approved method and flow cytometry were compared by the Spearman correlation test and a significant Pv = 0.001. The flow cytometric method has the advantage of analyzing a large and constant number of cells in less time, i.e., 9 h incubation for fluconazole using acridine orange versus 72 h for broth microdilution method. In conclusion, the two methods were comparable and flow cytometry method can expedite and improve the results of in vitro susceptibility tests of C. neoformans and C. gattii against fluconazole and also allows comparative studies in vitro/in vivo more rapidly, which along with clinical data, could assist in selecting the most appropriate treatment choice.

Cryptococcus gattii VGII in a Plathymenia reticulata hollow in Cuiabá, Mato Grosso, Brazil.

Little is known about the ecology of agents of cryptococcosis in Mato Grosso, without any data regarding to the sources of both agents in the environment. This study aimed to investigate Cryptococcus gattii and Cryptococcus neoformans associated with decay in tree hollows within the urban area of three different cities of Mato Grosso. Seventy-two environmental samples collected from 72 living trees in the cities of Cuiabá, Várzea Grande and Chapada dos Guimarães were sampled and analysed. One tree (Plathymenia reticulata, Leguminosae) in the city of Cuiabá yielded 19 colonies identified as C. gattii molecular type VGII. The isolation of C. gattii VGII in the downtown city of Cuiabá is important because it fits in the Northern Macroregion, suggesting expanding and urbanisation of this genotype in different Brazilian cities.

Fatal Cryptococcus gattii genotype AFLP6/VGII infection in a HIV-negative patient: case report and a literature review.

Cryptococcus gattii, a species belonging to the Cryptococcus complex which occurs endemically in tropical and subtropical regions, has been reported as a causative agent of cryptococcosis in healthy individuals. We report a case of meningitis in HIV-negative patient from Cuiaba, MT, in the Midwestern region of Brazil. Cryptococcus gattii AFLP6/VGII was isolated from cerebrospinal fluid and molecular typing was performed by URA5-RFLP. The in vitro susceptibility profile was determined using the standard method according to the document M27A3, CLSI 2008. C. gattii AFLP6/VGII was shown to be susceptible to the antifungals tested. Treatment with 0.8 mg/kg of amphotericin B was initiated; however, the patient died 2 days after the onset of therapy.

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.

Techniques for the detection of pathogenic Cryptococcus species in wood decay substrata and the evaluation of viability in stored samples.

In this study, we evaluated several techniques for the detection of the yeast form of Cryptococcus in decaying wood and measured the viability of these fungi in environmental samples stored in the laboratory. Samples were collected from a tree known to be positive for Cryptococcus and were each inoculated on 10 Niger seed agar (NSA) plates. The conventional technique (CT) yielded a greater number of positive samples and indicated a higher fungal density [in colony forming units per gram of wood (CFU x g(-1))] compared to the humid swab technique (ST). However, the difference in positive and false negative results between the CT-ST was not significant. The threshold of detection for the CT was 0.05.10³ CFU x g(-1), while the threshold for the ST was greater than 0.1.10³ CFU(-1). No colonies were recovered using the dry swab technique. We also determined the viability of Cryptococcus in wood samples stored for 45 days at 25ºC using the CT and ST and found that samples not only continued to yield a positive response, but also exhibited an increase in CFU x g(-1), suggesting that Cryptococcus is able to grow in stored environmental samples. The ST.1, in which samples collected with swabs were immediately plated on NSA medium, was more efficient and less laborious than either the CT or ST and required approximately 10 min to perform; however, additional studies are needed to validate this technique.

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.

Cryptococcus neoformans carried by Odontomachus bauri ants.

Cryptococcus neoformans is the most common causative agent of cryptococcosis worldwide. Although this fungus has been isolated from a variety of organic substrates, several studies suggest that hollow trees constitute an important natural niche for C. neoformans. A previously surveyed hollow of a living pink shower tree (Cassia grandis) positive for C. neoformans in the city of Rio de Janeiro, Brazil, was chosen for further investigation. Odontomachus bauri ants (trap-jaw ants) found inside the hollow were collected for evaluation as possible carriers of Cryptococcus spp. Two out of 10 ants were found to carry phenoloxidase-positive colonies identified as C. neoformans molecular types VNI and VNII. The ants may have acted as a mechanical vector of C. neoformans and possibly contributed to the dispersal of the fungi from one substrate to another. To the best of our knowledge, this is the first report on the association of C. neoformans with ants of the genus Odontomachus.

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.

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.

Genotypes of Cryptococcus neoformans and Cryptococcus gattii as agents of endemic cryptococcosis in Teresina, Piauí (northeastern Brazil).

Throughout Brazil, Cryptococcus neoformans is the cause of cryptococcosis, whereas Cryptococcus gattii is endemic to the northern and northeastern states. In this study, the molecular types of 63 cryptococcal isolates recovered from the cerebrospinal fluid of meningitis patients diagnosed between 2008-2010 in Teresina, Piauí, Brazil, were analysed. Out of the 63 patients, 37 (58.7%) were human immunodeficiency virus (HIV)-positive and 26 (41.3%) were HIV-negative. URA5-restriction fragment length polymorphism analysis identified 37/63 (58.7%) isolates as the C. neoformans VNI genotype, predominantly in HIV-positive patients (32/37, 86.5%), and 24/63 (38.1%) as the C. gattii VGII genotype, mostly in HIV-negative patients (21/26, 80.8%). The occurrence of C. gattii VGII in six apparently healthy children and in seven adolescents/young adults in this region reaffirms the endemic occurrence of C. gattii VGII-induced primary cryptococcosis and early cryptococcal infection. Lethality occurred in 18/37 (48.6%) of the HIV-positive subjects and in 13/26 (50%) of the HIV-negative patients. Our results provide new information on the molecular epidemiology of C. neoformans and C. gattii in Brazilian endemic areas.

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.

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.

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.

Regional pattern of the molecular types of Cryptococcus neoformans and Cryptococcus gattii in Brazil.

The molecular types of 443 Brazilian isolates of Cryptococcus neoformans and Cryptococcus gattii were analyzed to determine their geographic distribution within Brazil and their underlying host conditions. The following data, imported from previous epidemiological studies as well as two culture collections, were analyzed for: place of isolation, source (clinical or environmental), host risk factors, species, serotype, mating type, and molecular type. Molecular typing by PCR-fingerprinting using primers for the minisatellite-specific core sequence of the wild-type phage M13 or microsatellites [(GACA)4, (GTG)5], restriction fragment length polymorphism of URA5 gene analysis, and/or amplified fragment length polymorphism (AFLP) identified eight major genotypes: VNI/AFLP1, VNII/AFLP1A, VNIII/AFLP2, and VNIV/AFLP3 for C. neoformans, and VGI/AFLP4, VGII/AFLP6, VGIII/AFLP5, and VGIV/AFLP7 for C. gattii. The most common molecular type found in Brazil was VNI (64%), followed by VGII (21%), VNII (5%), VGIII (4%), VGI and VNIV (3% each), and VNIII (< 1%). Primary cryptococcosis caused by the molecular type VGII (serotype B, MAT alpha) prevails in immunocompetent hosts in the North and Northeast regions, disclosing an endemic regional pattern for this specific molecular type in the Northern Brazil.

Primary endemic Cryptococcosis gattii by molecular type VGII in the state of Pará, Brazil.

In order to study the infectious agents causing human disseminated cryptococcosis in the state of Pará, North Brazil, 56 isolates of Cryptococcusspp. (54 isolated from cerebral spinal fluid and two from blood cultures) from 43 cases diagnosed between 2003-2007 were analysed. The species were determined through morphological and physiological tests and genotypes were determined by URA5-RFLP and PCR-fingerprinting (wild-type phage M13). The following species and genotypes were identified: Cryptococcus neoformans VNI (28/56, 50%), Cryptococcus gattii VGII (25/56, 44.64%) and C. gattii VGI (3/56, 5.26%). The genotype VNI occurred in 12 out of 14 HIV-positive adults, whereas the genotype VGII occurred in 11 out of 21 HIV-negative adults (p < 0.02, OR = 6.6 IC95% 0.98-56.0). All patients less than 12 years old were HIV negative and six cases were caused by the VGII genotype, one by the VGI and one by VNI. Therefore, endemic primary mycosis in HIV-negative individuals, including an unexpectedly high number of children, caused by the VGII genotype deserves further study and suggests the need for surveillance on cryptococcal infection in the state of Pará, Eastern Amazon.