الفهرس 
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Abstract
Although C. albicans is the primary etiological agent of candidemia worldwide, non- albicans Candida (NAC) species are likely to overtake C. albicans in many hospital units. The incidence of infections caused by NAC has increased and some of the common species isolated are C. tropicalis.
The present study included a total of 71 non-duplicate C. tropicalis species isolated from various clinical specimens. Samples were collected from different ICU patients in Alexandria. All clinical isolates were identified to the species level by means of conventional mycological methods, chromogenic Candida speciation media, and the VITEK 2 compact ID system. Antifungal susceptibilities of all isolates were determined using the VITEK 2 system AST-YS07 card containing amphotericin B, caspofungin, fluconazole, flucytosine, micafungin and voriconazole.
The virulence factors studied were the production of haemolytic activity, extracellular hydrolytic enzymes (phospholipase and proteinase) and biofilm formation. Biofilm formation was determined by both visual and spectrophotometric methods.
Molecular detection of possible azole resistance mechanisms included: (i) Relative quantitative analysis of CDR1 and MDR1 genes expressions by real time PCR using SYBR GREEN for the tested C. tropicalis clinical isolates. (ii) Sequence analysis and detection of point mutations (amino acid substitutions) in Erg11 gene were investigated among the tested isolates.
The present study showed the following results: the majority of isolates were collected from urine 49 (69.01%), followed by respiratory samples 13 (18.31%), 7 (9.86%) from blood while 2 (2.82%) were isolated from skin & soft tissue infections.
Out of the 71 collected isolates, 30 (42.25%) were non-susceptible –MIC 4≤ µg/ml– (resistant or dose-dependent susceptibility) to fluconazole, of which 28 isolates showed non-susceptibility –MIC 0.25≤ µg/ml– (resistance or dose-dependent susceptibility) to voriconazole and only two isolates were sensitive to voriconazole. Cross-resistance to azoles - resistance to the both azoles (fluconazole and voriconazole) - was found in 7.04% of isolates. All isolates were sensitive to amphotericin B, flucytosine and echinocandins (caspofungin & micafungin).
All isolates showed both hemolysin and proteinase activities. No statistical significant differences in proteinase activities of C. tropicalis isolates were observed among different infection sites. Only few numbers of isolates (9 isolates) showed phospholipase production
Biofilm formation was demonstrated in 98.59% (70/71). No relationship could be detected between biofilm production and azole resistance. Strong biofilm production was higher among blood culture isolates (85.71%), followed by respiratory and urinary isolates (61.54 % and 46.94 % respectively).
In the current study, it was found that relative CDR1 gene expression in fluconazole non-susceptible isolates was statistically significantly higher than that in fluconazole susceptible isolates (p ≤ 0.05). This finding was in contrast to relative MDR1 gene expression in fluconazole non-susceptible isolates which was statistically not significantly higher than that in fluconazole susceptible isolates (p = 0.589).
A positive correlation between CDR1 and MDR1 genes expressions was found in fluconazole non-susceptible isolates, which was statistically significant (P =0. 001), however, No significant correlation was found between CDR1 and MDR1 genes expressions in both fluconazole susceptible and total isolates (p=0.464, 0.143) respectively.
The analysis of the chromatograms of the 30 fluconazole non-susceptible isolates revealed that 4 isolates showed sequencing failure and consequently no mutations could be detected. The analysis of the remaining 26 isolates showed that seven different mutations took place, 2 of them were missense mutations: A395T and G1390A leading to amino acid substitutions: Y132F and G464S respectively. The other 5 mutations were silent mutations: T225C, G264A, G1362A, C1464T and T1554C.
It was observed that three different mutations consistently appeared together in 24 different isolates: G1362A, G1390A and T1554C. G1362A and T1554C mutations consistently appeared together in 25 different isolates. One missense mutation was detected in each isolate except in one isolate (S13RR) all the detected mutations were silent.