BackgroundThe aim of this study was to determine the biofilm formation, the extracellular enzymatic activities of 182 clinical isolates of the Candida parapsilosis complex. MethodsMolecular identification of the C. parapsilosis species complex was performed using PCR RFLP of SADH gene and PCR sequencing of ITS region. The susceptibility of ours isolates to antifungal agents and molecular mechanisms underlying azole resistance were evaluated.Results63.5% of C. parapsilosis were phospholipase positive with moderate activity for the majority of strains. None of the C. metapsilosis or C. orthopsilosis isolates was able to produce phospholipase. Higher caseinase activities were detected in C. parapsilosis (Pz = 0.5 ± 0.18) and C. orthopsilosis (Pz = 0.49 ± 0.07) than in C. metapsilosis isolates (Pz = 0.72 ± 0.1). 96.5% of C. parapsilosis strains and all isolates of C. metapsilosis and C. orthopsilosis produced gelatinase. All the strains possessed the ability to show haemolysis on blood agar. C. metapsilosis exhibited the low haemolysin production with statistical significant differences compared to C. parapsilosis and C. orthopsilosis. The biofilm forming ability of C. parapsilosis was highly strain dependent with important heterogeneity, which was less evident with both C. orthopsilosis and C. metapsilosis.Some C. parapsilosis isolates met the criterion for susceptible dose dependent to fluconazole (10.91%), itraconazole (16.36%) and voriconazole (7.27%). Moreover, 5.45% and 1.82% of C. parapsilosis isolates were respectively resistant to fluconazole and voriconazole. All strains of C. metapsilosis and C. orthopsilosis were susceptible to azoles; and isolates of all three species exhibited 100% of susceptibility to caspofungin, amphotericin B and 5-flucytosine.ConclusionsA combination of molecular mechanisms, including the overexpression of ERG11, and genes encoding efflux pumps (CDR1, MDR1, and MRR1) were involved in azole resistance in C. parapsilosis.

We investigated six microsatellite markers to type 85 unrelated and 118 related isolates of Candida glabrata from 36 patients. Three new markers were selected from the complete sequence of CBS138 and three previously described markers, RPM2, MTI and ERG3 were used. We found a genetic diversity of 0.949 by combining four of them. By applying the new microsatellite markers GLM4, GLM5 and GLM6 we were able to discriminate 29 isolates, originally identified by the more established markers, RPM2, MTI and ERG3. When epidemiologically closely related isolates from 36 patients were typed, 25 patients (72%) exhibited identical or highly related multilocus genotypes. We noted a microvariation in 4 of the patients. This minor change of one locus could be explained by a single step mutation. Since one of these patients had not received antifungal treatment; thus, the relationship between genome variation and antifungal therapy remains controversial. We can conclude from our analysis of these new microsatellite markers that they are highly selective and therefore should be considered as a useful typing system for differentiating related and unrelated isolates of C. glabrata, as well as being able to detect microvariation.

Whether in vitro antifungal susceptibility findings correlate with the outcome of patients with invasive aspergillosis (IA) remains debated. This study aimed to test whether IA patients' outcomes were associated with in vitro susceptibility results. To do so, we determined the in vitro susceptibility to amphotericin B (AMB) of 37 Aspergillus flavus isolates from 14 patients with haematological malignancies diagnosed with proven or probable IA, of which 13 were treated with AMB deoxycholate. Minimal inhibitory concentrations (MICs) were determined by Etest with the isolates classified as in vitro sensitive (AMB-S) or resistant (AMB-R) if their MICs were < 2 or ≥ 2 mg/l, respectively. The association of the patients' death with primary disease, administered antifungal treatment, and infection with AMB-R A. flavus was tested using generalized estimating equations logistic regression. We assessed AMB-R in 31/37 (84%) isolates. In the patients treated with AMB, the survival rate was 2/3 (67%) and 2/9 (22%) for those infected with AMB-S or AMB-R A. flavus, respectively. Both infection with AMB-R A. flavus (P = 0.014) strain and acute myelocytic leukaemia as the underlying primary disease (P = 0.036) were independent predictors of death. Our findings indicate that in vitro resistance predicts a poor outcome in patients with A. flavus invasive disease treated with AMB. Recent advances in non-culture-based microbiological methods should not discourage efforts to obtain in vitro antifungal susceptibility results, which are critical for the choice of antifungal therapy in patients with IA.

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This study aimed at comparing a real-time PCR assay and a PCR-ELISA assay of both serum and bronchoalveolar lavage (BAL) samples for the diagnosis of invasive aspergillosis (IA) in patients with hematological malignancies. Using a nested case-control design, 163 patients at risk were prospectively monitored and PCR assays were performed on frozen aliquots of 459 sera which were prospectively sampled twice weekly and 42 BAL specimens sampled from 43 probable and one proven IA cases and 47 matched controls. The data from three patients classified as possible IA were excluded from the nested case-control study. The sensitivity of real-time PCR and PCR-ELISA assays in serum was 73% and 86%, respectively and specificity was 100% for both. In BAL, sensitivity was 64% for real-time PCR, 71% for PCR-ELISA and 86% for Galactomannan antigen (GMA) assays with specificities of 96%, 96%, and 93%, respectively. While slightly less sensitive, the real time-PCR assay was highly specific and considerably faster and more workable than PCR-ELISA. Combining real-time PCR and GMA detection for both serum and BAL samples enhances routine laboratory IA diagnosis.