Investigation of Azole Resistance Involving cyp51A and cyp51B Genes in Clinical Aspergillus flavus Isolates.

This study aimed to investigate azole resistance mechanisms in Aspergillus flavus, which involve cyp51A and cyp51B genes. Real-time Reverse Transcriptase qPCR method was applied to determine the overexpression of cyp51A and cyp51B genes for 34 A. flavus isolates. PCR sequencing of these two genes was used to detect the presence of gene mutations. Susceptibility test found sensitivity to voriconazole (VOR) in all strains. 14.7% and 8.8% of isolates were resistant to itraconazole (IT) and posaconazole (POS), respectively, with a cross-resistance in 5.8%. For the double resistant isolates (IT/POS), the expression of cyp51A was up to 17-fold higher. PCR sequencing showed the presence of 2 mutations in cyp51A: a synonymous point mutation (P61P) in eight isolates, which did not affect the structure of CYP51A protein, and another non synonymous mutation (G206L) for only the TN-33 strain (cross IT/POS resistance) causing an amino acid change in the protein sequence. However, we noted in cyp51B the presence of the only non-synonymous mutation (L177G) causing a change in amino acids in the protein sequence for the TN-31 strain, which exhibits IT/POS cross-resistance. A short single intron of 67 bp was identified in the cyp51A gene, whereas three short introns of 54, 53, and 160 bp were identified in the cyp51B gene. According to the models provided by PatchDock software, the presence of non-synonymous mutations did not affect the interaction of CYP51A and CYP51B proteins with antifungals. In our study, the overexpression of the cyp51A and cyp51B genes is the primary mechanism responsible for resistance in A. flavus collection. Nevertheless, other resistance mechanisms can be involved.

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.

Genotypic Analysis of the Population Structure in Malassezia globosa and Malassezia restricta.

The molecular characterization of Malassezia spp. isolates from animals and humans has not been thoroughly studied. Although a range of molecular methods has been developed for diagnosing Malassezia species, they have several drawbacks, such as inefficiency in differentiating all the species, high cost and questionable reproducibility. The present study aimed to develop VNTR markers for genotyping Malassezia isolated from clinical and animal samples. A total of 44 M. globosa and 24 M. restricta isolates were analyzed. Twelve VNTR markers were selected on seven different chromosomes (I, II, III, IV, V, VII and IX), six for each Malassezia species. The highest discriminatory power for a single locus was obtained with the STR-MG1 marker (0.829) and STR-MR2 marker (0.818) for M. globosa and M. restricta, respectively. After the analysis of multiple loci, 24 genotypes were noted among 44 isolates in M. globosa, with a discrimination index D of 0.943 and 15 genotypes were noted among 24 isolates in M. restricta, with a discrimination index D of 0.967. An endogenous infection was detected in two patients. Different genotypes of M. globosa strains colonized one patient. Interestingly, VNTR markers analysis revealed a carriage between a breeder and his dog in three cases for M. globosa and two for M. restricta. The FST (0.018 to 0.057) values indicate a low differentiation between the three populations of M. globosa. These results suggest a dominant clonal mode of reproduction in M. globosa. The typing of M. restricta showed a genotypic diversity of the strains, which can cause various skin pathologies. However, patient five was colonized with strains having the same genotype collected from different body parts (back, shoulder). VNTR analysis was capable of identifying species with high accuracy and reliability. More importantly, the method would facilitate monitoring Malassezia colonization in domestic animals and humans. It was shown that the patterns are stable and the method is discriminant, making it a powerful tool for epidemiological purposes.

Production and Quantification of Virulence Factors in Malassezia Species.

Seventy-seven strains of Malassezia were included in this study. Biofilm and hydrolytic enzyme production were studied by using specific solid media. The Real-Time reverse transcriptase qPCR method was applied to determine the overexpression of genes encoding the extracellular enzymes. All included Malassezia species produced biofilms. No statistically significant difference was observed between Malassezia species in biofilm formation (p = 0.567). All Malassezia species produced lipase, and 95% of Malassezia globosa showed a strong enzymatic activity (Pz = 0.55 ± 0.02). A statistically significant difference was observed between the mean keratinase indices of Malassezia slooffiae and the other Malassezia species (p = 0.005). The overexpression of one or more genes was observed in 100% of strains isolated from patients with folliculitis, 87.5% - with pityriasis versicolor, and 57.14% of the control group isolates. A statistically significant difference in the lipase gene expression (p = 0.042) was between the strains from patients with folliculitis and the control group. This investigation provides more information about the frequency of the production of the major enzymes considered virulence factors of Malassezia species. Interestingly, the overexpression of one or more genes was observed in strains isolated from patients with Malassezia disorders.

Epidemiological Study with Evaluation of Molecular and Immunologicalassay for Detection of Toxoplasma gondii in aborted women.

Toxoplasmosis, caused by Toxoplasma gondii, is one of the most prevalent parasite illnesses in humans. Although primary infection in a pregnant woman is usually asymptomatic, it has the potential to cause significant harm to the fetus, including miscarriage. In this study, we investigate the usefulness of the PCR to confirm the etiology of the abortion. A prospective study in the Al-Diwaniyah maternity and pediatric teaching hospital in Iraq was conducted. The research comprised 94 aborted women. We have reported a new internal primer for the nested PCR protocol to detect toxoplasmosis. In the 94-aborted women, 30 samples (31.9 %) were positive by the nPCR using the G529 repeat gene and qPCR using B1 gene primers. We have shown that three women carry the parasite in their placentas, and at the same time, they do not carry antibodies in their blood. We recommend that women should be aware of the risk of toxoplasmosis and the importance of preventing measures. In addition, PCR should be done in the case of abortion to enhance sensitivity even if serology is negative.

Molecular Genotyping of Candida parapsilosis Species Complex.

BACKGROUND: The Candida parapsilosis complex species has emerged as an important cause of human disease. The molecular identification of C. parapsilosis isolates at the species level can be helpful for epidemiological studies and then for the establishment of appropriate therapies and prophylactic measures. METHODS: The present study was undertaken to analyze 13 short tandem repeat (STR) markers (7 minisatellites and 6 microsatellites) in a global set of 182 C. parapsilosis complex isolates from different origins including invasive and superficial clinical sites. RESULTS: Upon the analysis of 182 strains of C. parapsilosis complex species, 10-17 haplotypes were detected for each minisatellite marker. The combination of 7 minisatellite markers yielded 121 different genotypes with a 0.995 D value. Upon the analysis of 114 isolates (68 from invasive infections and 46 from superficial infections), 21-32 genotypes were detected for each microsatellite marker. The combination of all 13 markers yielded 96 different genotypes among 114 isolates with a high degree of discrimination (0.997 D value). The same multilocus genotype was shared by isolates recovered from some patients and from the hand of theirs correspondent healthcare worker. For another patient, the same multilocus genotype of C. metapsilosis was detected in blood and skin confirming that candidemia usually arises as an endogenous infection following prior colonization. CONCLUSIONS: These STR markers are a valuable tool for the differentiation of C. parapsilosis complex strains, to support epidemiological investigations especially studies of strain relatedness and pathways of transmission.

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.

First genotype identification of Trichosporon asahii in Sfax, Tunisia.

PURPOSE: The objectives of our study were species identification and genotyping of Trichosporon isolates collected at the Parasitology and Mycology Laboratory in Sfax, Tunisia. METHODOLOGY: Molecular identification was carried out by analysing the IGS1 regions of the rDNA of 30 Trichosporon isolates. RESULTS: Trichosporon asahii was the most frequent species detected. Furthermore, four genotypes were identified in Tunisia: 1 (46.4 %), 4 (35.7 %), 7 (14.3 %) and 3 (3.6 %). In vitro antifungal susceptibility testing of the isolates showed that voriconazole exhibited the highest activity. CONCLUSION: This is the first reported study of genotype identification of T. asahii in Tunisia and even in the African continent.

Molecular study of the Candida parapsilosis complex in Sfax, Tunisia.

Candida parapsilosis, which was previously considered to be a complex of three genetically distinct groups, has emerged as a significant agent of nosocomial infections. Recently, this complex was separated into three species: C. parapsilosis sensu stricto, C. orthopsilosis, and C. metapsilosis In Tunisia, data pertaining to these fungi are limited. Thus, the purpose of our study was to determine by BanI PCR-RFLP and ITS sequencing, the occurrence of Candida parapsilosis complex among 182 isolates identified as C. parapsilosis by phenotypical methods. C. parapsilosis sensu stricto represented 94.5% of all isolates, while C. metapsilosis and. C. orthopsilosis were identified in 3.3% and 2.2%, respectively. Sequence analysis of internal transcribed spacer region confirmed and revealed only one genotype among the C. parapsilosis sensu stricto strains, three genotypes among six C. metapsilosis strains and two genotypes among four C. orthopsilosis strains.

Molecular characterization of strains of the Trichophyton verrucosum complex from Tunisia.

Trichophyton verrucosum is the most frequent etiologic agent of cattle dermatophytosis. Throughout the world, it was the second most common agent of zoophilic dermatophytes in human. The aim of our study was to evaluate the efficacy of the PCR- RFLP and PCR-sequencing methods for the identification and differentiation of T. verrucosum strains.Thirty-six clinical strains identified by morphological characteristics as T. verrucosum were isolated from patients referred to parasitology-mycology laboratory of Sfax University Hospital. Identification of our strains by conventional methods was confirmed by molecular methods in 94.4% of cases. Two strains were reclassified as T. violaceum PCR products digested with HinfI produced three profiles and two patterns with MvaI. Sequence analysis revealed a polymorphism in the ITS1and 5.8S regions. Analysis and alignment of consensus sequences has distinguished two types of genotypes among our T. verrucosum strains. The ITS type I was the dominant genotype (93.7%). Phylogenetic study showed that one cluster comprised T. verrucosum strains with ITS type I and species of T. mentagrophytes complex. It was related to Arthroderma vanbreuseghemii complex. The other cluster contained the two T. verrucosum strains with ITS type II, and was related to Arthroderma benhamiae complex. In this study, most of T. verrucosum isolates were type I, dissimilar to others rare studies where type II has been the most common. Specie and strain differentiation is relevant because it helps in prescribing the correct treatment and determining the source of the infection.

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.

Highly discriminatory variable-number tandem-repeat markers for genotyping of Trichophyton interdigitale strains.

Trichophyton interdigitale is the second most frequent cause of superficial fungal infections of various parts of the human body. Studying the population structure and genotype differentiation of T. interdigitale strains may lead to significant improvements in clinical practice. The present study aimed to develop and select suitable variable-number tandem-repeat (VNTR) markers for 92 clinical strains of T. interdigitale. On the basis of an analysis of four VNTR markers, four to eight distinct alleles were detected for each marker. The marker with the highest discriminatory power had eight alleles and a D value of 0.802. The combination of all four markers yielded a D value of 0.969 with 29 distinct multilocus genotypes. VNTR typing revealed the genetic diversity of the strains, identifying three populations according to their colonization sites. A correlation between phenotypic characteristics and multilocus genotypes was observed. Seven patients harbored T. interdigitale strains with different genotypes. Typing of clinical T. interdigitale samples by VNTR markers displayed excellent discriminatory power and 100% reproducibility.

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.

[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.

Genetic diversity of Pneumocystis jirovecii strains based on sequence variation of different DNA region.

Pneumocystis jirovecii is an important opportunistic pathogen that causes severe pneumonia in immunocompromised patients. The aim of the present study was to investigate the genetic diversity of P. jirovecii strains by direct sequencing and analysis of the Upstream Conserved Sequence (UCS) region, mitochondrial large-subunit (mtLSU) rRNA and dihydrofolate reductase (DHFR) genes. We identified the polymorphisms in P. jirovecii strains of 15 immunocompromised patients, as well as detecting a new tandem repeat of 5 nucleotides in UCS region. The following three different types of repeat unit were found: type a GCCCA; type b GCCCT; and type c GCCTT. In addition, we identified the repeat unit which consisted of 10 nucleotides and three different patterns of UCS repeats with 3 and 4 repeats, i.e., 1, 2, 3 (86.7%), 1, 2, 3, 3 (6.6%) and a new genotype 2, 2, 3, 3 (6.6%). The polymorphism in the mtLSUrRNA gene was seen primarily at position 85 where we detected three different genotypes. Genotype 3 and genotype 2 were the most abundant with frequencies of 53.3% and 40%, respectively. With regard to the DHFR gene, only two (20%) patients had nucleotide substitution in position 312. In conclusion, the multilocus analysis facilitated the typing of P. jirovecii strains and proved the important genetic biodiversity of this fungus.

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.

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.

A rapid and easy method for the DNA extraction from Cryptococcus neoformans.

DNA isolation from C. neoformans is difficult due to a thick and resistant capsule. We have optimized a new and rapid DNA isolation method for Cryptococcus using a short urea treatment followed by a rapid method using a chelex resin suspension. This procedure is simpler than previously reported methods.

A review molecular typing methods for Aspergillus flavus isolates.

Aspergillus flavus is the second most important Aspergillus species causing human infections. The importance of this fungus increases in regions with a dry and hot climate. Small phylogenetic studies in Aspergillus flavus indicate that the morphological species contains several genetically isolated species. Different genotyping methods have been developed and employed in order to better understand the genetic and epidemiological relationships between environmental and clinical isolates. Understanding pathogen distribution and relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. Typing techniques can also give us a deeper understanding of the colonization pattern in patients. Most of these studies focused on Aspergillus fumigatus because it is medically the most isolated species. To date, there has not been any publication exclusively reviewing the molecular typing techniques for Aspergillus flavus in the literature. This article reviews all these different available methods for this organism.

Two new media Pinus halepensis seed agar and blackberry agar for rapid identification of Cryptococcus neoformans.

Cryptococcus neoformans is an encapsulated yeast-like fungus that causes life-threatening infections, particularly in immunocompromised patients. The formation of brown pigment on many media described in the literature, such as that in Niger seed (Guizotia abyssinica) agar, has been used to identify C. neoformans. The present study compares melanin production by clinical and environmental isolates of C. neoformans and other medically important yeast on two new media, Pinus halepensis seed (PHS) agar and blackberry (BlaB) agar, and the classic medium Niger seed agar. Results obtained after the culture of 46 strains of C. neoformans, for 4, 24 and 48 h at 37 °C on these three media, showed that at 24 h, 100% of strains were pigmented on BlaB agar, 91.3% on PHS agar but only 34.8% on Niger seed agar. In conclusion, PHS and BlaB agar are two interesting new media for the rapid identification of C. neoformans isolates.