Cryptococcosis in HIV-AIDS patients from Southern Brazil: Still a major problem.

INTRODUCTION: Cryptococcus neoformans is an opportunistic pathogen that causes ∼15% mortality in AIDS patients. Rio Grande City, Rio Grande do Sul (RS), Brazil, has the highest national rate of HIV/AIDS, considering cities with population more than 100,000 habitants. OBJECTIVE: We aimed to evaluate the clinical and epidemiological profile of cryptococcosis in a reference service for HIV-AIDS patients in the South region of Brazil, over seven years. Material and methods A retrospective study was performed including all cryptococcosis cases diagnosed at the University Hospital, Federal University of Rio Grande (UH-FURG) between January 2010 and December 2016. RESULTS: Seventy cases of cryptococcosis were diagnosis from 2010 to 2016 in the UH-FURG in the seven years of the study. These numbers were responsible for 2.1% to 8.1% of the hospitalizations/year for HIV patients. All were caused by C. neoformans infection (95% C. neoformans var. grubii VNI and 5% C. neoformans var. grubii VNII). Neurocryptococcosis was the major clinical manifestation and cryptococcosis was the HIV- defining condition in 40% of patients. The period of hospitalization was an average of 39.3 days (SD=31.3), and more than half of patients (53%; 37/70) died after a mean of 82 days. DISCUSSION: The present study showed the importance of cryptococcosis as an AIDS-defining disease in HIV-AIDS patients in a tertiary hospital from Southern Brazil. More investment is necessary to reduce the impact of this opportunistic mycosis in HIV-AIDS patients from southern Brazil.

Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for fluconazole, itraconazole, posaconazole, and voriconazole.

Epidemiological cutoff values (ECVs) for the Cryptococcus neoformans-Cryptococcus gattii species complex versus fluconazole, itraconazole, posaconazole, and voriconazole are not available. We established ECVs for these species and agents based on wild-type (WT) MIC distributions. A total of 2,985 to 5,733 CLSI MICs for C. neoformans (including isolates of molecular type VNI [MICs for 759 to 1,137 isolates] and VNII, VNIII, and VNIV [MICs for 24 to 57 isolates]) and 705 to 975 MICs for C. gattii (including 42 to 260 for VGI, VGII, VGIII, and VGIV isolates) were gathered in 15 to 24 laboratories (Europe, United States, Argentina, Australia, Brazil, Canada, Cuba, India, Mexico, and South Africa) and were aggregated for analysis. Additionally, 220 to 359 MICs measured using CLSI yeast nitrogen base (YNB) medium instead of CLSI RPMI medium for C. neoformans were evaluated. CLSI RPMI medium ECVs for distributions originating from at least three laboratories, which included ≥95% of the modeled WT population, were as follows: fluconazole, 8 µg/ml (VNI, C. gattii nontyped, VGI, VGIIa, and VGIII), 16 µg/ml (C. neoformans nontyped, VNIII, and VGIV), and 32 µg/ml (VGII); itraconazole, 0.25 µg/ml (VNI), 0.5 µg/ml (C. neoformans and C. gattii nontyped and VGI to VGIII), and 1 µg/ml (VGIV); posaconazole, 0.25 µg/ml (C. neoformans nontyped and VNI) and 0.5 µg/ml (C. gattii nontyped and VGI); and voriconazole, 0.12 µg/ml (VNIV), 0.25 µg/ml (C. neoformans and C. gattii nontyped, VNI, VNIII, VGII, and VGIIa,), and 0.5 µg/ml (VGI). The number of laboratories contributing data for other molecular types was too low to ascertain that the differences were due to factors other than assay variation. In the absence of clinical breakpoints, our ECVs may aid in the detection of isolates with acquired resistance mechanisms and should be listed in the revised CLSI M27-A3 and CLSI M27-S3 documents.

Cryptococcus neoformans-Cryptococcus gattii species complex: an international study of wild-type susceptibility endpoint distributions and epidemiological cutoff values for amphotericin B and flucytosine.

Clinical breakpoints (CBPs) are not available for the Cryptococcus neoformans-Cryptococcus gattii species complex. MIC distributions were constructed for the wild type (WT) to establish epidemiologic cutoff values (ECVs) for C. neoformans and C. gattii versus amphotericin B and flucytosine. A total of 3,590 amphotericin B and 3,045 flucytosine CLSI MICs for C. neoformans (including 1,002 VNI isolates and 8 to 39 VNII, VNIII, and VNIV isolates) and 985 and 853 MICs for C. gattii, respectively (including 42 to 259 VGI, VGII, VGIII, and VGIV isolates), were gathered in 9 to 16 (amphotericin B) and 8 to 13 (flucytosine) laboratories (Europe, United States, Australia, Brazil, Canada, India, and South Africa) and aggregated for the analyses. Additionally, 442 amphotericin B and 313 flucytosine MICs measured by using CLSI-YNB medium instead of CLSI-RPMI medium and 237 Etest amphotericin B MICs for C. neoformans were evaluated. CLSI-RPMI ECVs for distributions originating in ≥3 laboratories (with the percentages of isolates for which MICs were less than or equal to ECVs given in parentheses) were as follows: for amphotericin B, 0.5 µg/ml for C. neoformans VNI (97.2%) and C. gattii VGI and VGIIa (99.2 and 97.5%, respectively) and 1 µg/ml for C. neoformans (98.5%) and C. gattii nontyped (100%) and VGII (99.2%) isolates; for flucytosine, 4 µg/ml for C. gattii nontyped (96.4%) and VGI (95.7%) isolates, 8 µg/ml for VNI (96.6%) isolates, and 16 µg/ml for C. neoformans nontyped (98.6%) and C. gattii VGII (97.1%) isolates. Other molecular types had apparent variations in MIC distributions, but the number of laboratories contributing data was too low to allow us to ascertain that the differences were due to factors other than assay variation. ECVs may aid in the detection of isolates with acquired resistance mechanisms.

Genetic characterization of environmental isolates of the Cryptococcus neoformans species complex from Brazil.

The genetic affiliation of a large number of isolates of the Cryptococcus neoformans species complex from environmental sources in Brazil has been investigated using amplified fragment length polymorphism (AFLP). The strains of C. neoformans isolated from a single tree, as well as from neighbouring trees, showed high similarity values (> 95%) of their AFLP patterns, thus suggesting considerable genetic homogeneity. The majority of isolates of C. neoformans belonged to AFLP genotype 1, and had serotype A and mating type alpha (= C. neoformans var. grubii). Three isolates belonged to AFLP genotype 2, with serotype D and mating type alpha (= C. neoformans var. neoformans). One isolate, obtained from a building in Rio de Janeiro inhabited by pigeons, belonged to the AD hybrid AFLP genotype 3. All isolates from trees of C. neoformans var. gattii (= C. gattii) belonged to AFLP genotype 6, and their banding patterns showed relatively low genetic homogeneity with a similarity value of about 76%. Isolates of this genotype occupy an environmental niche in the Americas, and they may cause disease in non-AIDS and AIDS patients as well.

Prevalence of Candida dubliniensis in the BCCM/IHEM Biomedical Fungi/Yeasts culture collection (isolates before 1990).

The BCCM/IHEM Biomedical Fungi/Yeasts collection hosts 1200 Candida albicans strains of the Vanbreuseghem mycotheque isolated between 1951 and 1997. From this collection, 469 freeze-dried C. albicans strains, producing chlamydospores, germ tubes and forming green colonies on CHROMagar, all isolated before 1990, were screened to identify the Candida dubliniensis isolates. Screening was performed in different steps using the growth at 45 degrees C, the assimilation of xylose, the intracellular beta-glucosidase activity test and C. dubliniensis-specific polymerase chain reaction (PCR) with primers from ACT1 intron sequence. Five isolates (1%) were identified as C. dubliniensis: one isolate was not documented, the others were of oropharyngeal origin of which two (1987 and 1990) were from proven human immunodeficiency virus patients.

Possible primary ecological niche of Cryptococcus neoformans.

To study hollows of living trees as natural habitats of Cryptococcus neoformans in an endemic area of cryptococcosis in the northeastern region of Brazil, samples of decaying wood were collected inside 32 hollows of living trees and plated on niger seed agar. Identification of C. neoformans was based upon morphological and physiological tests. Canavanine-glycine-bromothymol medium was used to screen the varieties and Crypto Check Iatron Kit to serotype the isolates. A total of 123 C. neoformans colonies were recovered from samples of six (18.5%) out of 32 hollow trees. C. neoformans var. neoformans and C. neoformans var. gattii were found occurring alone (pink shower tree, fig tree and pottery tree) or sharing the same hollow (pink shower tree). Long lasting positivity (19-36 months) and significant number of cfu of C. neoformans per gram of decaying wood (0.15-21.7 x 10(3) cfu g(-1)) inside hollows of pink shower tree, fig tree and pottery tree were observed, indicating colonization of these habitats by the fungus. For the first time, C. n. var. neoformans and C. n. var. gattii were found sharing the same natural biotope, thus establishing a possible link between them in their life cycle in nature and suggesting the primary natural niche for the species.

Cryptococcus neoformans var. gattii--evidence for a natural habitat related to decaying wood in a pottery tree hollow.

To study hollows of living trees as the natural habitat of Cryptococcus neoformans in an endemic area of cryptococcosis in the northeastern Brazilian region, samples of decaying wood were collected inside the hollows, plated on niger seed agar and inoculated into mice and hamsters. Identification of C. neoformans was based on morphological and physiological tests. Canavanine-glycine-bromothymol medium was used to screen the varieties and Crypto Check Iatron Kit to serotype the isolates. For a period of 29 months C. neoformans var. gattii serotype B was isolated repeatedly from the hollow of a pottery tree (Moquilea tomentosa), pointing to the natural occurrence of C. neoformans var. gatti in decaying wood forming hollows in living trees. Evidence for a natural habitat of the variety gattii other than that related to Eucalyptus camaldulensis are discussed.

Natural habitat of Cryptococcus neoformans var. neoformans in decaying wood forming hollows in living trees.

Cryptococcus neoformans var. neoformans was repeatedly isolated from decaying wood forming hollows in living trees growing in urban areas of Rio de Janeiro, Brazil. A new natural habitat for C. neoformans var. neoformans has been found that is not associated with specific trees.