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.

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.

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.