Virulence factors, antifungal susceptibility and molecular mechanisms of azole resistance among Candida parapsilosis complex isolates recovered from clinical specimens.

BACKGROUND: The aim of this study was to determine the biofilm formation, the extracellular enzymatic activities of 182 clinical isolates of the Candida parapsilosis complex. METHODS: Molecular 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. RESULTS: 63.5% 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. CONCLUSIONS: A 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.

Changes in genotype and fluconazole susceptibility of isolates from patients with Candida glabrata in Tunisia.

UNLABELLED: Candida glabrata has emerged as an opportunistic pathogen of considerable importance in invasive and superficial infections. AIMS: To analyze the development of fluconazole resistance in patients under treatment through epidemiological survey in our hospital. PATIENTS AND METHODS: Twenty two patients (89 clinical strains) were collected. Molecular typing of isolates was performed by polymorphic markers. Analysis of gene expression was realized by reverse transcriptase-real time polymerase chain reactions (RT-qPCR). RESULTS: Genetic analysis showed that 63% persists with apparently unchanged strains (n=14). Among them, four showed fluconazole resistance development. A strain replacement was observed in 6 patients and two patients selected more resistant isolates during the course of treatment. An analysis of Candida glabrata cerebellar degeneration-related protein 1 (CgCDR1), Candida glabrata cerebellar degeneration-related protein 2 (CgCDR2) and Candida glabrata sterol 14 alpha-demetylase Erg 11 (CgERG11) expression revealed an over-expression in 10 resistant isolates. CONCLUSION: This study demonstrated that C. glabrata strain undergo frequent changes in vivo. The increase in CgCDR1 and CgCDR2 expression was the most mechanism associated with fluconazole resistance.

Molecular methods for colorectal cancer screening: Progress with next-generation sequencing evolution.

Currently, colorectal cancer (CRC) represents the third most common malignancy and the second most deadly cancer worldwide, with a higher incidence in developed countries. Like other solid tumors, CRC is a heterogeneous genomic disease in which various alterations, such as point mutations, genomic rearrangements, gene fusions or chromosomal copy number alterations, can contribute to the disease development. However, because of its orderly natural history, easily accessible onset location and high lifetime incidence, CRC is ideally suited for preventive intervention, but the many screening efforts of the last decades have been compromised by performance limitations and low penetrance of the standard screening tools. The advent of next-generation sequencing (NGS) has both facilitated the identification of previously unrecognized CRC features such as its relationship with gut microbial pathogens and revolutionized the speed and throughput of cataloguing CRC-related genomic alterations. Hence, in this review, we summarized the several diagnostic tools used for CRC screening in the past and the present, focusing on recent NGS approaches and their revolutionary role in the identification of novel genomic CRC characteristics, the advancement of understanding the CRC carcinogenesis and the screening of clinically actionable targets for personalized medicine.

Bacteremia due to Serratia rubidaea in intensive care unit: a case series.

INTRODUCTION: Bacteremia caused by Serratia rubidaea is seldom mentioned in comparison with other Enterobacteriaceae species. It primarily affects immunocompromised patients undergoing invasive procedures. Furthermore, the incidence, clinical features, and microbiological profile of this pathogen in the intensive care unit are rarely described. CASE PRESENTATION: We present four North African case studies of bacteremia in four young female patients admitted to the intensive care unit for ketoacidosis with a history of diabetes mellitus. All four patients developed catheter-related infections complicated by deep vein thrombosis. The catheter site was femoral in all cases, and the main clinical manifestation was poorly tolerated fever. The pathogen was isolated in multiple peripheral blood cultures (> 4) for each patient, showing a similar profile in all cases: resistance to third-generation cephalosporins and sensitivity to aminoglycosides, piperacillin, fluoroquinolones, and folate-pathway inhibitors. Targeted treatment consisted of a combination of ciprofloxacin 400 mg twice per day and trimethoprim/sulfamethoxazole 400/80 mg thrice per day for all four cases. However, in one case, this regimen was switched to amikacin due to adverse effects. The outcomes were favorable in the majority of cases. The patients described in this study were 21, 66, 22, and 27-year-old North African women. CONCLUSION: Most of the reported cases shared common risk factors and clinical aspects. Notably, a case of thrombosis complicating a catheter infection caused by Serratia rubidaea has not been previously reported in the literature. Furthermore, this bloodstream infection typically affects deeply immunocompromised patients. However, our four cases, admitted to the intensive care unit for ketoacidosis, only had a history of diabetes mellitus.

Invasive aspergillosis: resistance to antifungal drugs.

Although the arsenal of agents with anti-Aspergillus activity has expanded over the last decade, mortality due to invasive aspergillosis remains unacceptably high. Resistance of the Aspergillus spp. species to antifungal drugs increased in the last 20 years with the increase in antifungal drugs use and might partially account for treatment failures. Recent advances in our understanding of mechanisms of antifungal drug action in Aspergillus, along with the standardization of in vitro susceptibility testing methods, have brought resistance testing to the forefront of clinical mycology. Recent modifications in taxonomy and understanding of the acquired resistance mechanisms of Aspergilli to drugs should support a better management of Aspergillus infections. In this paper, we review the current knowledge on epidemiology and underlying mechanisms involved in antifungal resistance in Aspergillus.

High Frequency of Enterocytozoon bieneusi Genotype WL12 Occurrence among Immunocompromised Patients with Intestinal Microsporidiosis.

Microsporidiosis is an emerging opportunistic infection causing severe digestive disorders in immunocompromised patients. The aim of this study was to investigate the prevalence of intestinal microsporidia carriage among immunocompromised patients hospitalized at a major hospital complex in the Tunis capital area, Tunisia (North Africa), and perform molecular epidemiology and population structure analyses of Enterocytozoon bieneusi, which is an emerging fungal pathogen. We screened 250 stool samples for the presence of intestinal microsporidia from 171 patients, including 81 organ transplant recipients, 73 Human Immunodeficiency Virus (HIV)-positive patients, and 17 patients with unspecified immunodeficiency. Using a nested PCR-based diagnostic approach for the detection of E. bieneusi and Encephalitozoon spp., we identified 18 microsporidia-positive patients out of 171 (10.5%), among which 17 were infected with E. bieneusi. Microsporidia-positive cases displayed chronic diarrhea (17 out of 18), which was associated more with HIV rather than with immunosuppression other than HIV (12 out of 73 versus 6 out of 98, respectively, p = 0.02) and correlated with extended hospital stays compared to microsporidia-negative cases (60 versus 19 days on average, respectively; p = 0.001). Strikingly, internal transcribed spacer (ITS)-based genotyping of E. bieneusi strains revealed high-frequency occurrence of ITS sequences that were identical (n = 10) or similar (with one single polymorphic site, n = 3) to rare genotype WL12. Minimum-spanning tree analyses segregated the 17 E. bieneusi infection cases into four distinct genotypic clusters and confirmed the high prevalence of genotype WL12 in our patient population. Phylogenetic analyses allowed the mapping of all 17 E. bieneusi strains to zoonotic group 1 (subgroups 1a and 1b/1c), indicating loose host specificity and raising public health concern. Our study suggests a probable common source of E. bieneusi genotype WL12 transmission and prompts the implementation of a wider epidemiological investigation.

[Local humoral immunity in vulvovaginal candidiasis]. / Étude de l'immunité humorale locale au cours d'une candidose vulvo-vaginale.

Recurrent vulvo-vaginal candidiasis (RVVC) is a significant problem facing women of child bearing age. It is now accepted that RVVC is the consequence of local immunodeficiency. The aim of this study was to assess differential secretion of IgAs and IgG anti-C. albicans in vaginal secretions of patients with RVVC, VVC and asymptomatic women. Vaginal secretions collected from 3 groups of women: 14 patients with RVVC, 8 patients with VVC and 17 asymptomatic women. Overall analysis of vaginal secretions revealed that the prevalence of IgAs (73%) and IgG (33%) antibodies anti-C. albicans were significantly different. The prevalence of IgAs antibodies was 86% in patients with RVVC, 75% in women with VVC and 61% in asymptomatic women. IgG antibodies were detected in 43% of women with RVVC, in 37% of women with VVC and in 18% of asymptomatic women. Sensibility and specificity of detection of IgA in vaginal secretion were 54% and 83%, respectively. The prevalence of detection of IgAs and IgG were more important in patients than asymptomatic women. However, RVVC cannot be attributed only to the impairment of local humoral immunity and further proteomic investigations are needed.

Interactions between copy number and expression level of genes involved in fluconazole resistance in Candida glabrata.

OBJECTIVES: This study aimed to elucidate the relative involvement of drug resistance gene copy number and overexpression in fluconazole resistance in clinical C. glabrata isolates using a population-based approach. METHODS: Fluconazole resistance levels were quantified using the minimal inhibitory concentration (MIC) via Etest method. Both gene expression levels and gene copy number of CgCDR1, CgPDH1, CgERG11, and CgSNQ2 were assessed via quantitative real-time PCR. The influence of the main effects and first-level interactions of both the expression level and copy number of these genes on fluconazole resistance levels were analyzed using a multivariate statistical model. RESULTS: Forty-three C. glabrata isolates were collected from 30 patients during in a hospital survey. In the multivariate analysis, C. glabrata fluconazole MICs were independently increased by CgSNQ2 overexpression (p < 10(-4)) and the interaction between CgPDH1 gene copy number and CgPDH1 expression level (p = 0.038). In contrast, both CgPDH1 overexpression (p = 0.049) and the interaction between CgSNQ2 and CgERG11 expression (p = 0.003) led to a significant decrease in fluconazole MICs. CONCLUSION: Fluconazole resistance in C. glabrata involves complex interactions between drug resistance gene expression and/or copy number. The population-based multivariate analysis highlighted the involvement of the CgSNQ2 gene in fluconazole resistance and the complex effect of the other genes such as PDH1 for which overexpression was associated with reduced fluconazole resistance levels, while the interaction between PDH1 overexpression and copy number was associated with increased resistance levels.

Microsatellite analysis of Candida isolates from recurrent vulvovaginal candidiasis.

Candida albicans and Candida glabrata are the most common causative agents of both vulvovaginal candidiasis (VVC) and recurrent vulvovaginal candidiasis (RVVC). Studying the population structure and genotype differentiation of Candida species that cause RVVC may lead to a significant improvement in clinical management. A total of 106 isolates were collected from 55 patients who were subdivided into three groups. Group I comprised 15 patients with RVVC (n=50 isolates); group II comprised 16 patients, who had a history of at least two episodes of VVC in the last year (n=32 isolates, two from each patient); and group III comprised 24 patients (n=24 isolates) who had experienced a single episode of VVC in the previous 1 year period. C. albicans microsatellite markers CAI, CAIII and CAIV and C. glabrata RPM2, MTI and ERG3 microsatellites were amplified in a multiplex PCR. All isolates were subjected to population genetic analysis, which provided evidence that there is a predominantly clonal population structure of C. albicans in each group. However, recombination was detected to some degree in C. albicans isolates in group III. A genetic homogeneity between the different C. albicans groups was observed. Although, C. glabrata isolates showed an important genetic differentiation between group I and group III (F(ST)=0.207). Genotype analysis revealed that the dominant genotypes of C. glabrata and C. albicans strains were more prevalent in patients with RVVC. The frequent scenario for cases of recurrent infection in our study was strain replacement (53.3%). In conclusion, the identification of recurrence-associated genotypes and a specific C. glabrata population structure in the RVVC group could be a significant marker for further investigations of virulence factors and RVVC management.

[Methods for studying the in vitro susceptibility of Candida spp. to antifungals]. / Méthodes d'étude de la sensibilité in vitro de Candida spp. aux antifongiques.

In recent years, an increase of systemic Candida infections was noted. Thus, identification and susceptibility testing to antifungals became of considerable importance. The technique of dilution in liquid medium developed by « National committee for clinical laboratory standards ¼ NCCLS or more recently named CLSI « Clinical and laboratory standards institute ¼ is the reference method most used. The European committee "European committee on antibiotic susceptibility testing" or EUCAST has made progress by determining the susceptibility of strains within a shorter time. However, the use of these techniques is limited especially in clinical microbiology laboratories. Other techniques for determining antifungal sensitivity have been developed such as those based on agar diffusion (E-test and disk diffusion), on microdilution (Sensititre yeastOne, Vitek 2 AST-YS01), on flow cytometry techniques and the MALDI-TOF.