Microsatellite typing and susceptibilities of serial Cryptococcus neoformansisolates from Cuban patients with recurrent cryptococcal meningitis

Abstract

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.

Cryptococcus neoformans isolates from Yaoundé human immunodeficiency virus-infected patients exhibited intra-individual genetic diversity and variation in antifungal susceptibility profiles between isolates from the same patient

“…grubii serotype A. Microsatellite analysis [(CT)n, (TG)n, (TA)n, (CTA)n, (TCT)n, (CCA)n, (TTAT)n, (ATCC)n and (TATT)n] of 19 Cuban C. neoformans var. grubii serotype A isolates provided 14 molecular types (Illnait-Zaragozí et al, 2010b). In the present study, 24 % (6/25) of patients were infected by allowed identification of genotypes named 1 to 15.…”

“…The molecular type VNI has been described recently in Cameroon (Bertout et al, 2013). Moreover, it has been hypothesized that a given patient could be infected by more than one strain during an episode of the disease (Desnos-Ollivier et al, 2010Illnait-Zaragozí et al, 2010b). It was also observed that colonies from the same patient may present genetic variabilities (Bertout et al, 2013).…”

“…(Desnos-Ollivier 2015). Another speculation is that genetic variations in an initial strain allow for a better adaptation within the host organism (Illnait-Zaragozí et al, 2010b). This process may allow the fungal population to change and escape eradication by the immune system, and thus cause chronic infections as suggested by Jain et al (2005).…”

“…Microsatellites are increasingly popular moleculartyping targets, since they provide cost-effective genotyping with fast turnaround times. Like MLST data, microsatellitetyping data are transportable and exchangeable (Illnait-Zaragozí et al, 2010b). This study describes the intra-individual genetic diversity of C. neoformans isolates from Yaounde HIV-infected patients assessed by using microsatellite length polymorphism analysis, and also evaluates the antifungal-susceptibility testing of these isolates with seven antifungal drugs.…”

“…Multiple molecular-typing methods have been described to study the epidemiology of the C. neoformans species complex. The most commonly used approaches to date involve AFLP, PCR fingerprinting and PCR-RFLP (Meyer et al, 2009;Illnait-Zaragozí et al, 2010b;Hagen et al, 2012). PCR fingerprinting and PCR-RFLP have proven useful in discriminating between the different serotypes and mating types, but have some limits for discrimination within specific C. neoformans species complex members.…”