BackgroundHuman cryptococcal infections have been associated with bird droppings as a likely source of infection. Studies toward the local and global epidemiology of Cryptococcus spp. have been hampered by the lack of rapid, discriminatory, and exchangeable molecular typing methods.Methodology/Principal FindingsWe selected nine microsatellite markers for high-resolution fingerprinting from the genome of C. neoformans var. grubii. This panel of markers was applied to a collection of clinical (n = 122) and environmental (n = 68; from pigeon guano) C. neoformans var. grubii isolates from Cuba. All markers proved to be polymorphic. The average number of alleles per marker was 9 (range 5–51). A total of 104 genotypes could be distinguished. The discriminatory power of this panel of markers was 0.993. Multiple clusters of related genotypes could be discriminated that differed in only one or two microsatellite markers. These clusters were assigned as microsatellite complexes. The majority of environmental isolates (>70%) fell into 1 microsatellite complex containing only few clinical isolates (49 environmental versus 2 clinical). Clinical isolates were segregated over multiple microsatellite complexes.Conclusions/SignificanceA large genotypic variation exists in C. neoformans var. grubii. The genotypic segregation between clinical and environmental isolates from pigeon guano suggests additional source(s) of human cryptococcal infections. The selected panel of microsatellite markers is an excellent tool to study the epidemiology of C. neoformans var. grubii.

BackgroundCryptococcus neoformans is commonly associated with meningoencephalitis in immunocompromised patients and occasionally in apparently healthy individuals. Recurrence of infection after initial treatment is not uncommon. We studied C. neoformans isolates from 7 Cuban patients with recurrent cryptococcal meningitis. Antifungal susceptibility and genotyping with microsatellite molecular typing were carried out.MethodsIsolates (n = 19) were recovered from cerebrospinal fluid, blood, urine and semen. Antifungal susceptibilities for amphotericin B, fluconazole, flucytosine, itraconazole, voriconazole, posaconazole and isavuconazole were tested by CLSI M27A3 broth microdilution method. Genotyping was done using a panel of 9 microsatellite (STR) markers: (CT)n, (TG)n, (TA)n, (CTA)n, (TCT)n, (CCA)n, (TTAT)n, (ATCC)n and (TATT)n.ResultsThe average number of isolates/patient was 2.71. The mean time interval between the collection of any two isolates was 52.5 days. All strains were identified as C. neoformans var. grubii (serotype Aα). Although none of the strains were resistant to the studied drugs, in serial isolates from two patients, MICs values of triazoles increased 4-5 log2 dilutions over time. STR patterns showed 14 distinctive profiles. In three patients the recurrent infection was associated with genotypically identical isolates. The four other patients had relapse isolates which were genotypically different from the initial infecting strain.ConclusionRecurrences of cryptococcal meningitis in our series of patients was not associated with development of drug resistance of the original strain but by an initial infection with different strains or a reinfection with a new strain.

Summary Cryptococcus isolates from Cuban patients were identified as C. neoformans var. grubii. Although this species has since long been associated with bird droppings, a recent genotyping study provided strong evidence for additional origins of exposure. We sampled different species of trees in Havana, Cuba to identify other potential sources of exposure to this fungus. A total of 662 samples were collected from 331 trees and cacti from Havana, Cuba. Initial selection of the isolates was carried out by conventional techniques. Isolates were further characterised using a combination of AFLP analysis and DNA sequence analysis. Identification by conventional methods yielded 121 C. neoformans and 61 C. gattii isolates. Molecular analyses showed that none of these isolates was C. gattii and only one isolate proved to be C. neoformans var. grubii. A total of 27 different other species were identified. The most prevalent species was C. heveanensis (33%). Sixty‐five unidentifiable isolates segregated into ten potentially novel species. Conventional cultivation methods have a low specificity for C. neoformans complex and molecular analyses need to be applied to confirm identification of isolates from environmental sources. Environmental niches responsible for most of human cryptococcal infections in Cuba remain to be identified.

Cryptococcosis has emerged as an important public health problem in Africa, Asia and the Americas due to the increasing numbers of persons at risk of this infection and the adaptation of its aetiological agents to new environments. The proper management requires early recognition of Cryptococcus neoformans/C. gattii species complex infection, familiarity with the use and limitations of diagnostic tests and knowledge of the available treatment options. This review will address these issues with the goal of providing sufficient information to suspect, diagnose and treat patients with cryptococcosis based on Cuban data and review of the literature.

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