Abstract
Cryptococcus neoformans and Candida species are the main causative agents of yeast opportunistic infections among HIV infected persons. However, information on molecular their epidemiology and antifungal susceptibility are scarce in Cameroon. The main objective of this work was to study the genetic diversity and the antifungal susceptibility against antifungal drugs and plant extracts of C. neoformans and Candida isolates from Yaoundé HIV patients. C. neoformans (25) and Candida (317 among which 113 C. albicans) Isolates were obtained, from 171 and 402 HIV patients at the Yaoundé Central Hospital respectively. They were identified by phenotypic and biochemical characters, by mass spectrometry and quantitative PCR. The genetic diversity of 150 C. neoformans isolates (25 initial isolates and 125 colonies) was carried out by serotyping, microsatellite length polymorphism and PCR-RFLP. The genetic diversity of the 113 C. albicans isolates was performed by genotyping and microsatellite length polymorphism. The identification of C. albicans complex species was achieved by PCR amplification of the Hwp1 gene. The antifungal susceptibility testing of C. neoformans against posaconazole, voriconazole, ketoconazole, itraconazole, fluconazole, amphotericin B and 5-fluorocytosine was carried out by the broth microdilution test using the « Sensititre YeastOne® » kit. The CLSI M27-A3 protocol was used for the determination of the C. albicans isolate's susceptibility against amphotericin B, ketoconazole, fluconazole and itraconazole which are frequently used in Cameroon. The antifungal activity of extracts from Terminalia mantaly, Terminalia catappa and Monodora tenuifolia was performed by a preliminary screening with the determination of minimal inhibitory concentrations (MIC) of crude extracts. Selected extracts were therefore submitted to the bio-guided fractionation. Selected subfractions were submitted to combination assays. C. neoformans var grubii was the lonely Cryptococcus species isolated in cerebrospinal fluids. Fifteen Candida species were isolates from mucosae with C. albicans remaining the most frequent. C. africana has been isolated for the first time in Cameroon. C. neoformans and C. albicans provided 14 and 65 major molecular types respectively. It was also found that a patient can be infected by 2 different molecular types of C. neoformans. C. albicans genotype A was the most frequent. The PCR amplification of the Hwp1 gene allowed the identification of a novel molecular profile among the C. albicans complex and named H (H1-H6). C. neoformans isolates were susceptible to the tested drugs. However, one isolate exhibited reduced susceptibility to fluconazole and one another to 5-fluorocytosine. C. albicans isolates expressed various susceptibility profiles similar to what described in the literature. Furthermore, there was a relationship between the H-typing and the antifungal susceptibility of C. albicans isolates against itraconazole (p-value<0.05). T. mantaly, T. catappa and M. tenuifolia extracts exhibited antifungal activity against tested yeasts. Bioguided fractionation allowed improves of the antifungal activity from crude extracts to subfractions. Synergism was observed, and the most active combination from T. mantaly and M. tenuifolia was also fungicidal on tested yeasts. Conclusively, the present work brings new tools for the comprehension and the better management of C. neoformans and Candida infections among Yaoundé HIV positive patients. The antifungal resistance emergence of yeasts isolates could be compensated by the development of a new antifungal medicine from subfractions combinations of T. mantaly and M. tenuifolia.