PHENOTYPIC, GENOTYPIC, VIRULENCE AND AZOLE RESISTANCE CHARACTERIZATION OF CANDIDA SPECIES FROM CLINICAL SPECIMENS IN NAIROBI COUNTY, KENYA

Abstract

Invasive and systemic candidiasis are prevalent mycoses complicating immune suppressive conditions and leading to high mortality. The development and spread of azole antimycotic drug resistance hampers their effective utilisation in therapy and prophylaxis of candidiasis. Candida albicans (CA), consisting of genotypes A-H with varying virulence, is the most prevalent species. Several virulence factors including biofilm formation, haemolysins, proteases, and phospholipase (PLs) activities confer the fungi with invasiveness and dissemination of disease. This study determined the distribution and anti-azole resistance of CA and non-albicans candida (NAC), including genotypes and virulence factor expression in CA isolates. This cross sectional research was conducted on clinical samples (n=141) of patients presenting with different candidal mycoses at various metropolitan health facilities in Nairobi city, Kenya. Candida isolate identification was done via mycological culture, morphological, biochemical, and VITEK® 2-YST methods. Itraconazole (ITZ) and voriconazole (VOR) resistance were evaluated by the disk diffusion and broth micro dilution tests. Genotyping and virulence factor expression were determined for the CA isolates (n=94) using PCR-RFLP and virulence expression culture methods, respectively. Overall, 141 isolates were recovered, consisting of CA (66.7%) and NAC (C. glabrata, 16.3%; C. parapsilosis, 7.8%; C. tropicalis, 3.6%; C. krusei, 2.8%; C. guilliermondii, 1.4%; and C. famata, 1.4%). Anti-azole sensitivity was low for ITZ (21.3%) and VOR (24.5%) with high cross-resistance (74.5%) in the CA isolates. In the NAC isolates, sensitivity to ITZ, VOR, and cross-resistance were: C. glabrata (8.7%, 13.0% and 87.0%); C. parapsilosis (36.4%, 45.5% and 54.5%); C. tropicalis (40.0%, 60.0% and 40.0%); C. krusei (0.0%, 33.3% and 66.7%); C. famata (50.0%, 50.0% and 50.0%); and C. guilliermondii (0.0%, 50.0% and 50.0%), respectively. The mean (range) mg/ml minimum inhibitory concentrations (MIC) at MIC50 and MIC90 of sensitive CA isolates were 0.125 and 0.250 (0.031-0.500) for ITZ; and 0.250 and 0.250 (0.031-1.000) for VOR, respectively. For NAC, the MIC50 and MIC90 mean (range) mg/ml were 0.063 and 0.125 (0.025-0.500) for ITZ; and 0.094 and 0.250 (0.031-1.250) for VOR, respectively. CA A (66.0%) versus B (4.3%), and C (19.1%) genotypes was more frequent. All CA isolates had higher activities of biofilm production (75.5%) and PLs (73.4%) compared to haemolysin (21.3%) and proteinase (36.2%). Higher expressions of biofilm (95.2% vs. 42.9%; P<0.0001), PLs (96.8% vs. 28.6%; P<0.0001), and proteinase (52.4% vs. 9.5%; P=0.001) activities were also noted in A relative to non-A genotypes, respectively. Additionally, PLs activity was higher in ITZ resistant vs. sensitive strains (84.8% vs. 61.1%; P=0.044). Altogether, these results demonstrate that CA and its genotype A are the most prevalent isolates. Candida isolates exhibit low sensitivity rates to ITZ and VOR. Production of biofilm and PLs are the most expressed virulence factors in CA isolates linked to genotype A and ITZ resistance. Implications of these results include adoption of a combination and/or use of different antifungal agents for candidiasis treatment and prophylaxis