Genotypic diversity in clinical and environmental isolates of Cryptococcus neoformans from India using multilocus microsatellite and multilocus sequence typing

Abstract

Summary Background Cryptococcus neoformans is the leading cause of cryptococcal meningitis in HIV/AIDS patients. As infections in humans are predominantly caused by the inhalation of basidiospores from environmental sources, therefore, analysing the population structure of both clinical and environmental populations of C neoformans can increase our understanding of the molecular epidemiology of cryptococcosis. Objective To investigate the genotypic diversity and antifungal susceptibility profile of a large collection of C neoformans isolates (n = 523) from clinical and environmental sources in India between 2001 and 2014. Materials and methods Cryptococcus neoformans isolates were genotyped by AFLP, microsatellite typing (MLMT) and MLST. In vitro antifungal susceptibility for standard antifungals was undertaken using CLSI M27‐A3. Results All isolates were C neoformans, AFLP1/VNI and exhibited mating‐type MATα. MLMT revealed that the majority of isolates belonged to microsatellite cluster (MC) MC3 (49%), followed by MC1 (35%), and the remaining isolates fell in 11 other MC types. Interestingly, two‐thirds of clinical isolates were genotype MC3 and only 17% of them were MC1, whereas majority of environmental strains were MC1 (54%) followed by MC3 (16%). Overall, MLST assigned 5 sequence types (STs) among all isolates and ST93 was the most common (n = 76.7%), which was equally distributed in both HIV‐positive and HIV‐negative patients. Geometric mean MICs revealed that isolates in MC1 were significantly less (P < .05) susceptible to amphotericin B, 5‐flucytosine, itraconazole, posaconazole and isavuconazole than isolates in MC3. Conclusions The study shows a good correlation between MLMT and MLST genotyping methods. Further, environmental isolates were genetically more diverse than clinical isolates.

Molecular Markers Reveal Epidemiological Patterns and Evolutionary Histories of the Human Pathogenic Cryptococcus

“…The application of MLMT to analyze CNSC and CGSC samples has resulted in the identification of a large number of genotypes within both CNSC and CGSC ( Garcia-Hermoso et al., 2010 ; Prakash et al., 2020 ). The power of MLMT in strain discrimination was also shown in a recently emerged human fungal pathogen Candida auris where results based on MLMT markers showed similar discrimination power as that based on single nucleotide polymorphisms at the whole genome level ( De Groot et al., 2020 ).…”

“…The study also identified a correlation between microsatellite genotypes with the original source of strains and their susceptibilities to 5-flucytosine ( Pan et al., 2012 ). Another MLMT analysis of 523 CNSC isolates collected from India showed that environmental isolates were genetically more diverse than clinical isolates ( Prakash et al., 2020 ). However, an MLMT study based on 89 CNSC isolates from Brazil found no differences in antifungal susceptibility and capsule size between major environmental and clinical MLMT types ( Zhu et al., 2010 ).…”

Cryptococcal Immune Reconstitution Inflammatory Syndrome: From Blood and Cerebrospinal Fluid Biomarkers to Treatment Approaches

“…Cultured isolates can subsequently be serotyped by real-time PCR assay, and mating type can be determined by amplified fragment length polymorphism (AFLP) or PCR-restriction fragment length polymorphism (PCR-RFLP) [ 34 , 50 ]. Molecular typing revealed that genotypes, drug susceptibility, and the virulence of Cryptococcus species varied between different continents and in different countries [ 51 ]. However, recent studies found no correlation between antifungal drug susceptibility and hazards of death for therapeutic outcomes in the cohort of severely immunosuppressed AIDS patients [ 52 ].…”

Síndrome inflamatória da reconstituição imune associada à meningite criptococócica: fatores de risco e biomarcadores

“…Os isolados cultivados podem ser subsequentemente sorotipados pelo ensaio de PCR (Reação em cadeia da polimerase) em tempo real e a diversidade genotípica podem ser desvendadas pelo polimorfismo dos fragmentos amplificados (AFLP) ou polimorfismo do comprimento do fragmento de restrição (RFLP) 16,30 . A tipagem molecular revelou que os genótipos, a suscetibilidade às drogas e a virulência das espécies de Cryptococcus variaram entre os diferentes continentes e em diferentes países 31 . Entretanto, estudos recentes não encontraram correlação entre a suscetibilidade aos antifúngicos e os riscos de morte para resultados terapêuticos na coorte de pacientes com AIDS gravemente imunossuprimidos 32 .…”