Evaluation of the Performance of the Dynamiker Fungus (1-3)-ß-D-Glucan and Fungitell Assay for Diagnosis of Candidemia: Need for New Cut-off Development and Test Validation.

(1-3)-Beta-D Glucan (BDG) detection has shown to be a highly effective tool to diagnose invasive fungal infections. Therefore, this study aimed to compare clinical characteristics of the Fungitell (FA) and Dynamiker Fungus (1-3)-ß-D-Glucan assay (DFA) for the diagnosis of candidemia. Using DFA and FA, the BDG levels of 57 serum samples from case and control groups were determined. The kappa coefficient (κ) and Spearman's rank correlation (rs) were used to examine the consistency of assays on a quantitative and qualitative level, respectively. The sensitivity, specificity, and accuracy were 94.6 %, 65.0 %, and 87.7% for DFA, and 94.6 %, 75.0 %, and 89.4 % for FA, respectively. The performance of the DFA for the diagnosis of candidemia was highly consistent with that of the FA, both quantitatively (rs: 0.9) and qualitatively (kappa = 0.78). Collectively, the DFA assay performed excellently in comparison to the FA for the diagnosis of candidemia.

Species distribution and antifungal susceptibility profiles of yeasts isolated from onychomycosis: a cross-sectional study with insights into emerging species.

Candida onychomycosis is a common fungal infection affecting the nails, primarily caused by Candida (C.) species. Regarding the increasing trend of Candida onychomycosis and the antifungal resistant phenomenon in recent years, this study aims to evaluate the epidemiological characteristics of Candida onychomycosis, the distribution of emerging species, and the antifungal susceptibility profiles of isolates. Onychomycosis caused by yeast species was confirmed through direct examination and culture of nail scraping among all individuals suspected to have onychomycosis and referred to a medical mycology laboratory between June 2019 and March 2022. Species of yeast isolates were identified using the multiplex PCR and PCR-RFLP methods. The antifungal susceptibility of isolates to common antifungal agents and imidazole drugs was evaluated according to the M-27-A3 CLSI protocol. Among 101 yeast strains isolated from onychomycosis, Candida parapsilosis complex (50.49%) was the most common species, followed by C. albicans (20.79%) and C. tropicalis (10.89%). Rare species of yeasts such as C. guilliermondii and Saccharomyces cerevisiae were also identified by molecular methods. Results obtained from antifungal susceptibility testing showed significant differences in MIC values of isoconazole, fenticonazole, and sertaconazole among different species. Overall, a fluconazole-resistant rate of 3% was found among Candida species. Moreover, there was a statistically significant difference in MICs of fenticonazole and clotrimazole between the two most prevalent causative species, C. parapsilosis complex and C. albicans. Correct identification of the causative agents of onychomycosis and performing susceptibility testing could be helpful in choosing the most appropriate antifungal therapy.

Direct molecular analysis of Malassezia species from the clinical samples of patients with pityriasis versicolor.

Background and Purpose: Species identification of Malassezia using culture-dependent methods is time-consuming due to their fastidious growth requirements. This study aimed to evaluate a rapid and accurate molecular method in order to diagnose the pityriasis versicolor (PV) and identify Malassezia species from direct clinical samples. Materials and Methods: Skin scraping or tape samples from patients with PV and healthy volunteers as the control group were collected. Diagnosis of PV was confirmed by direct microscopic examination. The DNA extraction was performed according to the steel-bullet beating method. Polymerase chain reaction-restriction fragment length polymorphism assay using HhaI restriction enzyme was applied for the identification and differentiation of Malassezia species. Results: The PCR method was able to detect Malassezia in 92.1% of specimens which were also confirmed with microscopic examination. Statistically, a significant association was observed between the results of the two assays (P < 0.001). Moderate agreement was identified between the two methods to diagnose the PV in both populations (Kappa: 0.55). Considering microscopic examination as the gold standard method for confirmation of PV, the sensitivity, specificity, positive predictive value, and negative predictive value values of the PCR assay for recognition of PV were 85%, 75%, 92%, and 60%, respectively. M. globosa and M. restricta were the most prevalent species isolated from patients. Conclusion: In this study, the two-step molecular method based on the amplification of the D1/D2 domain and digestion of the PCR product by one restriction enzyme was able to diagnose and identify Malassezia directly from clinical samples. Consequently, it can be said that the molecular-based method provides more facilities to identify fastidious species, such as M. restricta.

Evaluation of the features of cystic echinococcosis with concurrent super-infection: a retrospective study in Southern Iran.

OBJECTIVE: Superinfection of cystic echinococcosis (CE) is a life-threatening complication with significant morbidities, which can be prevented with early diagnosis and treatment. This study aims to examine the clinical characteristics, diagnostic methods, and treatment options for superinfected CE, as there is currently limited information available on the differences between superinfected and non-infected CE in terms of clinical features, serological and radiologic findings. METHODS: This cross-sectional study was conducted on hospital records of patients who were diagnosed with hydatid cysts in a 15-year period (2004 to 2018) in two main university-affiliated referral centers in Fars province, southern Iran. Patients' information regarding the demographical and clinical features related to CE, age, sex, previous history of CE or recurrence, size and location of CE, and length of hospital stay were collected. Moreover, the characteristics of concurrent infections with hydatid cysts were recorded. RESULTS: A total of 586 surgeries due to CE were performed on 501 patients, of which 67 (11.43%) had reoperations due to the recurrence of the disease. A total of 30 (5.99%) incidences of superinfection were observed. There were no statistically significant differences in terms of laboratory and imaging findings between CE patients with concurrent infections and other CE patients (p-value > 0.05). Among the patients with super-infection, four had fungal infections of the lungs. Aspergillus fumigatus was the causative pathogen in all four patients that were diagnosed with fungal superinfection. All patients underwent surgical excision with favorable long-term outcomes. CONCLUSION: Our study revealed a 5.99% incident rate of CE superinfection. Regarding the concurrent fungal infections in hydatid cysts, the patient's symptoms and laboratory and imaging findings are inconclusive and histopathological evaluation seems to be the most reliable option. Surgical resection is the gold-standard treatment option with favorable outcomes and potentially can be curative.

Molecular strategy for the direct detection and identification of the most common fungal community in cerumen specimens by multiplex PCR.

Introduction. Otomycosis is a superficial fungal infection that is responsible for approximately 9-27 % of otitis externa. However, fungal communities in otomycosis are varied, but Aspergillus spp. and Candida spp. are the most common causes of this infection.Hypothesis Statement. The multiplex PCR assay is postulated to be able to directly detect more than one fungal genus in cerumen specimens.Aim. This study aimed to develop and evaluate the role of the multiplex PCR assay in detecting the most common genus of fungi that cause otomycosis directly from the cerumen specimens.Methodology. To detect Candida and Aspergillus/Penicillium genera, three pairs of primers, including pan-fungal, pan-Candida, and pan-Aspergillus/Penicillium, were used in a multiplex PCR. In order to evaluate the performance and reproducibility of the multiplex PCR. the cerumen of 140 patients suspected of otomycosis were investigated.Results. Pan-Candida and pan-Aspergillus/Penicillium primers were designed to amplify the ITS1-5.8S-ITS2 region and the ß-tubulin gene, respectively. In the multiplex PCR assay, 64 (47.40 %) and 118 (87.40 %) specimens were positive with pan-Candida and pan-Aspergillus/Penicillium primers, respectively. Double amplicon bands of Candida and Aspergillus were obtained in 51 (37.77 %) specimens. In the culture method, yeast (n=18, 13.33 %) and mould (n=117, 86.66 %) were isolated from 135 cerumen specimens. The sensitivity, specificity, and positive and negative predictive values of the multiplex PCR assays using culture method results as the gold standard were determined to be 94, 33, 97, and 22 %, respectively.Conclusion. In our study, multiplex PCR assays enabled simultaneous detection of two common genera of the causative agent of otomycosis in a cerumen specimen. Regarding the high sensitivity of the first step of the multiplex PCR assay, this assay may be used for the direct detection of Candida and Aspergillus genera in other clinical specimens.

Quantitative analysis of in vitro biofilm formation by clinical isolates of dermatophyte and antibiofilm activity of common antifungal drugs.

BACKGROUND: The ability of dermatophytes to develop biofilm, as one of the virulence factors in fungal infections which contribute to antifungal resistance, is an outstanding aspect of dermatophytosis that has been noted recently. Because of the paucity of data about the biofilm formation by dermatophytes and their susceptibility to antifungal drugs, this study evaluated the biofilm formation by clinical isolates of dermatophytes and antibiofilm activity of common antifungals widely used to manage dermatophytosis. METHODS: The ribosomal DNA internal transcribed spacer (ITS) regions sequencing for species identification of 50 clinical dermatophyte isolates was performed. The ability of isolates to form biofilm and inhibitory activity of itraconazole, terbinafine, and griseofulvin against biofilm formation was assayed by the crystal violet staining method. Optical microscopy and scanning electron microscopy (SEM) were applied for the visualization of the biofilm structures. RESULTS: Trichophyton (T.) mentagrophytes (n: 14; 28%) and T. rubrum (n: 13;26%) were included in more than half of the dermatophyte isolates. Biofilm formation was observed in 37 out of 50 (74%) isolates that were classified as follows: nonproducers (n: 13; 26%), weak producers (n: 4; 8%), moderate producers (n: 16; 32%), and strong producers (n: 17; 34%) by comparison of the absorbance of biofilms produced by clinical strains with control. The mean IC50 values for terbinafine, griseofulvin, and itraconazole were 2.42, 3.18, and 3.78 µg/ml, respectively. CONCLUSIONS: The results demonstrated that most of the clinical dermatophyte isolates are capable to form biofilm in vitro with variable strength. Moreover, terbinafine can be suggested as the first-line choice for the treatment of biofilm-formed dermatophytosis.

Cytotoxic screening and in vitro effect of sodium chlorite against Leishmania major promastigotes.

Cutaneous leishmaniasis (CL) is one of the most important parasitic diseases in the world. Despite the existence of many therapeutic strategies, the treatment of this infection still faces problems. Sodium chlorite as an antimicrobial agent has been shown to have acceptable tissue regenerative and wound healing properties. Therefore, the present study aimed to analyze the in vitro effects of different concentrations of sodium chlorite on Leishmania major promastigotes and macrophage cells. The inhibitory and toxicity effect of various concentrations (0.0035, - 1.8 mg/ml) of sodium chlorite on the standard Iranian strain of L. major promastigotes were evaluated via counting the cells and flow cytometry. Furthermore, cytotoxicity on promastigotes and J774 macrophage cell line were performed by MTT assay. The results of the inhibitory test demonstrated that sodium chlorite had dose-dependent, anti-leishmanial activities. The half-maximal inhibitory concentration (IC50) for promastigotes and J774 cells by cytotoxicity test was detected at 0.17 mg/ml and 0.08 mg/ml after 48 h respectively. Flow cytometry results showed that 27.34% death of promastigotes was observed in 0.0035 mg/ml of sodium chlorite and 78.12% in 1.8 mg/ml. The results of the present study showed that sodium chlorite could be used as an effective treatment for CL, especially in cases resistant to treatment with pentavalent compounds. However, the toxicity of this substance in high concentrations should be considered in clinical setting.

Epidemiology, clinical features, therapeutic interventions and outcomes of mucormycosis in Shiraz: an 8-year retrospective case study with comparison between children and adults.

Mucormycosis is an invasive fungal infection with high morbidity and mortality rate despite the early diagnosis and proper therapeutic interventions. Given the importance of epidemiological data in reviewing the attitude toward infectious diseases in developing countries, the current retrospective case study aimed to compare the epidemiological aspects, risk factors, clinical characteristics, therapeutic interventions, and outcomes of mucormycosis between adults and children during eight years (2013-2021) in the main infectious disease referral centers in the southwest of Iran. The median age of 164 patients included in this study was 47 years (IQR 22-59). The median length of hospitalization was 33 days.The annual incidence of mucormycosis-related hospitalizations was estimated 1.76 per 10,000 admissions during the study period. Moreover, the incidence of infection was 2.4 times higher in males than females in children. Diabetes mellitus was the most frequent predisposing factor in adults (46.0%). The main risk factor in children was hematologic malignancy (52.6%), but a considerable proportion of them (28.9%) were immunocompetent.The most frequent antifungal agent used was liposomal amphotericin B (82.3%) as monotherapy. The combination therapy was used more in adults (15.8%) than children (7.9%). In addition, surgical intervention with antifungal therapy was considered the most effective therapeutic approach. The in-hospital mortality rate was 14.6% for adults, whereas it was zero for children. Our findings provide a recent epidemiologic analysis of mucormycosis among hospitalized patients in both children and adults. Mucormycosis mainly affects individuals with diabetes mellitus or hematological malignancies and presents as rhino-orbito-cerebral form. Proven diagnosis of mucormycosis according to clinical manifestations and histopathology observations accompanied by proper antifungal treatments may improve survival rates.

Evaluation of different DNA extraction methods based on steel-bullet beating for molecular diagnosis of onychomycosis.

BACKGROUND: Considering increased trends toward molecular methods for detection/identification of fungi causing onychomycosis, the aim of this study is comparison three DNA extraction methods based on steel-bullet beating to extract DNA from nail. METHODS: Ex -vivo onychomycosis model was developed using bovine hoof with Candida albicans and Aspergillus flavus. For two models, total DNA was extracted using the three different methods. In method 1, the extraction and purification were performed by steel-bullet beating and phenol chloroform protocol, respectively. In method 2, a freezing step were applied before beating. The purification step in method 3 was carried out using a commercial kit, although DNA extraction was done similarly to method 1 in that approach. To evaluate the efficacy of each method, the extracted genomic DNA was amplified with Polymerase Chain Reaction (PCR) using Internal Transcribed Spacer (ITS) regions. Moreover, 50 nail samples were evaluated for onychomycosis using direct microscopy examination as well as PCR in order to evaluate the diagnostic efficiency of the optimal DNA extraction method. RESULTS: Regarding the desirable quality of the extracted DNA, cost effectiveness, and simplicity, method 1 could be used to extract DNA effectively. Additionally, the obtained data showed that PCR had a higher detection rate of fungal agents in the nail samples than direct microscopic examination. CONCLUSIONS: This study demonstrated that the mechanical disruption of the cell wall by steel-bullet beating is a useful and practical method to improve the quantity and quality of fungal DNA thorough the extraction process.

Molecular characterization and antifungal activity against non-dermatophyte molds causing onychomycosis.

Onychomycosis is a fungal disease that caused by different types of fungi. Non-dermatophyte molds are a large saprophytic fungi group that live in nature and could affect traumatic nails. The aim of this study was to identify non-dermatophyte molds causing onychomycosis and evaluation of several antifungal activities against the isolates. The samples consisted of 50 non-dermatophyte molds isolated from patients with onychomycosis confirmed by direct and culture examination fungal. DNA was extracted, amplified, and sequenced. Disk diffusion method was used to evaluate itraconazole, fluconazole, ketoconazole, terbinafine, posaconazole, and econazole activity against the isolates. The species identified as: Aspergillus flavus 22 (44%), A. niger 12 (24%), A. fumigates, 3 (6%), A. sydowii 3 (6%), A. terreus 1 (2%), Penicillium commune 2 (4%), P. glabrum 2 (4%), P. chrysogenum, 1 (2%), Fusarium solani 3 (6%) and F. thapsinum 1 (2%). Most of the samples were sensitive to terbinafine, itraconazole, and econazole and 94% of the isolates were resistant to fluconazole. This study showed that Aspergillus species were the most common cause of non-dermatophyte mold onychomycosis and fluconazole was the most resistant antifungals. Care must be taken to choose the appropriate antifungal drug for a better cure.

Survey of aflatoxins and ochratoxin A contamination in spices by HPLC-based method in Shiraz, Southern of Iran.

Among food and agricultural products, spices play important roles in the diets of millions of people worldwide. These products may be colonized by fungi genus and subsequently mycotoxin production. Due to the large demand and supply of spice for cooking, preservative effects, or medicine purpose, it is essential that further investigation is designed to examine mycotoxins in spice. In the present study, the possible contamination of spices by aflatoxins (AFTs) and ochratoxin A (OTA) were analyzed. A total of 80 spice samples (curry, sumac, ginger, and saffron) were purchased and cultured on appropriate medium. Simultaneously mycotoxins from spices were extracted with immunoaffinity columns (IAC), and the occurrence of AFTs (B1 + B2 + G1 + G2) and OTA was then determined using high-performance liquid chromatography (HPLC) with a fluorescence detector (FD). The results depicted that 62 (77.5%) and 58 (72.5%) spice samples were contaminated with AFTs and OTA, respectively. Out of the 80 analyzed spices samples, the mean concentration of AFTs and OTA was higher in the curry samples. Among spices that contaminated with mycotoxins, 5 (6.25%) and 2 (10%) of the samples were above the acceptable limit of AFTs (≥ 10 µg/kg) and OTA (≥ 15 µg/kg), respectively. Aspergillus species were the predominant species isolated, followed by Penicillium, and finally Mucor species.Among the examined samples, only few curry samples were contaminated with mycotoxins above acceptable limit. Despite this low level of contamination, this spice is used daily in the cuisine of this region of the world, and consequently, even the small amount of these heat stable toxins for a long time may cause many adverse effects. Hence, it is recommended to monitor the toxicogenous fungi contamination and level of mycotoxins in the spices.

A simple multiplex polymerase chain reaction assay for rapid identification of the common pathogenic dermatophytes: Trichophyton interdigitale, Trichophyton rubrum, and Epidermophyton floccosum.

BACKGROUND AND PURPOSE: The most common etiological agents of human dermatophytosis in various parts of the world are Trichophyton rubrum, Trichophyton interdigitale, and Epidermophyton floccosum. The main aim of this study was to design and evaluate a simple and straightforward multiplex polymerase chain reaction (PCR) assay for reliable identification/differentiation of these species in clinical isolates. MATERIALS AND METHODS: The reliable sequences of several molecular targets of dermatophytes species were used to design a multiplex PCR for the identification of common pathogenic dermatophytes. The isolates and clinical specimens examined in this study included seven standard strains of dermatophytes, 101 isolates of dermatophytes and non-dermatophyte molds/yeasts which had already been identified by sequencing or PCR-restriction fragment length polymorphism (RFLP), and 155 clinical samples from patients suspected of cutaneous mycoses. RESULTS: Species-specific primer pairs for T. rubrum and T. interdigitale/T. mentagrophytes were designed based on the sequence data of the translation elongation factor 1-alpha gene, and the primers for E. floccosum targeted the specific sequence of the internal transcribed spacer region (ITS). The multiplex PCR successfully detected T. rubrum, T. interdigitale/T. mentagrophytes, and E. floccosum strains that were identified by sequencing or PCR-RFLP. However, the primer pairs selected for T. interdigitale/T. mentagrophytes cross-reacted with Trichophyton tonsurans. In testing the PCR system directly for clinical samples, the proportion of positive multiplex PCR was higher than positive culture (68.1% vs. 55.4%, respectively). CONCLUSION: The multiplex assay could detect three common agents out of several causal agents of dermatophytosis, namely T. rubrum, T. interdigitale, and E. floccosum. Therefore, by adding pan-dermatophyte primers it can be used as a comprehensive detection/identification test.

Molecular identification of Malassezia species isolated from neonates hospitalized in Neonatal intensive care units and their mothers.

Background and Purpose: Given the important role of Malassezia spp. in skin diseases and other associated infections in neonates, this study aimed to investigate the presence and frequency of Malassezia spp. in the skin of neonates hospitalized in neonatal intensive care units and their mothers using culture and accurate molecular-based methods. Materials and Methods: In total, 205 samples were collected from 130 neonates (>4-day-old) and 75 mothers. Isolation of Malassezia spp. from the skin was performed using Leeming-Notman agar and modified Dixon agar media. To compare the Malassezia microflora on the skin of the neonates and their mothers, a polymerase chain reaction-sequencing method was performed for spp. identification of 92 isolates obtained from neonates and their mothers. Moreover, possible associated risk factors for the colonization of Malassezia spp. on the skin were recorded. Results: Cultures from 62.3% of neonates and 77.3% of mothers were positive for Malassezia spp. growth. Malassezia globosa was the most prevalent isolated spp. found in the skin of the study population. It is noteworthy that a rare Malassezia spp., Malassezia arunalokei, was isolated from the skin of one neonate. There was a 76% similarity between the mother-neonate isolate sequences results. The statistical analysis showed that the type of feeding is a significant (P<0.001) associated factor for Malassezia skin colonization. Conclusion: The findings support the hypothesis that the colonization of Malassezia in neonates is significantly influenced by that of the mother, and this may be associated with breastfeeding.

Molecular identification and antifungal susceptibility among clinical isolates of dermatophytes in Shiraz, Iran (2017-2019).

Dermatophytosis is a common superficial mycotic infection affecting individual's quality of life worldwide. The present study aimed to perform species-level identification and evaluate the antifungal susceptibility patterns of dermatophytes isolated in Shiraz, Iran. This cross-sectional study was conducted on clinical samples collected during 2017-2019 from 307 patients suspected of having dermatophytosis. The isolates were identified by direct microscopy, culture and internal transcribed spacer ribosomal DNA sequencing, and their antifungal susceptibility patterns were determined by the microdilution method. Among 307 patients, dermatophytosis was diagnosed by microscopy in 190 (61.8%) subjects and confirmed in 130 (42.3%) cases by both microscopy and culture. It was found out tinea pedis was the most common clinical manifestation, and Trichophyton mentagrophytes was the most prevalent species (28.4%), followed by T tonsurans (23.8%), Microsporum canis (11.5%), T interdigitale (10%), T verrucosum (6.9%), T rubrum (6.9%), T benhamiae (4.6%), T violaceum (3%), T simii (3%), Epidermophyton floccosum (0.7%) and M ferrugineum (0.7%). Moreover, it was revealed that luliconazole with a geometric mean (GM) minimum inhibitory concentration (MIC) of 0.03 µg ml-1 was the most effective agent against all tested isolates. Regardless of species, 30% of isolates responded to high MICs of griseofulvin (MIC90  > 2 µg ml-1 ). The increasing prevalence of nonindigenous species of T simii, T benhamiae and M ferrugineum in Shiraz, Iran, was a notable finding. In addition, infections due to zoophilic species showed an increasing trend. These epidemiological data, along with antifungal susceptibility patterns, may have implications for clinical decision-making and successful treatment.

Coinfection of Strongyloides stercoralis and Aspergillus sp.

BACKGROUND: Strongyloides stercoralis has the ability to proliferate in its hosts for a long time. In most patients with a competent immune system, the infection remains asymptomatic. OBJECTIVES: Herein, we report a case of concomitant infection of Strongyloides and Aspergillus. Similar cases reported previously were reviewed in the literature and discussed in terms of diagnosis, clinical presentation, and treatment. METHODS: The patient was a 55-year-old man who had a medical history of two masses in his lung and was treated with corticosteroids six months before the presentation. RESULTS: Using the parasitological methods, massive actively motile larvae of S. stercoralis were seen in the patient's faecal sample. Aspergillus infection was isolated from his fresh bronchoalveolar lavage (BAL) sample and confirmed by observing the septate, dichotomously branched hyphae in direct microscopic examination and also the isolation of the fungus from the culture medium. Molecular analysis revealed that the fungal species isolated from the patient are A. flavus and A. niger. Conclusion. The case highlights the features of concomitant infection of S. stercoralis and Aspergillus in immunocompromised patients and the importance of screening patients for strongyloidiasis before initiation of immunosuppressive therapy.

Translation elongation factor 1-alpha gene as a marker for diagnosing of candidal onychomycosis.

BACKGROUND AND PURPOSE: Culture-based identification methods have been the gold standard for the diagnosis of candidal onychomycosis. Molecular technologies, such as polymerase chain reaction (PCR) assays, can provide an alternative for the rapid detection of Candida species. The present study was conducted to investigate a pan-Candida PCR assay based on the translation elongation factor 1-alpha (TEF-1α) gene for the detection of the most prevalent pathogenic Candida species. MATERIALS AND METHODS: For the purpose of the study, an optimized pan-Candida PCR primer pair was designed, and the target was amplified and sequenced. The analytical and clinical diagnostic performance of the designed primers was tested using 17 reference strains, 137 nail scrapings suspected of onychomycosis, and 10 healthy nail specimens. RESULTS: The use of the universal pan-Candida primers designed on TEF-1α gene resulted in the successful amplification of a 270-base pair fragment in all Candida species tested, except for C. glabrata, and reacted neither with other fungi nor with E. coli. The sequence difference count matrix showed poor insertion/deletion differences (0-2 nt) among Candida species. Among 137 nail specimens, 35% (n=48), 30.7% (n=42), and 40.1% (n=55) of the samples were found to be positive by direct microscopy, culture, and pan-Candida PCR, respectively. CONCLUSION: Based on the findings, the PCR-based detection targeting the DNA TEF-1α gene is a rapid and simple procedure for the diagnosis of candidal onychomycosis directly from nail sample.

Comparing real-time PCR and Calcofluor-white with conventional methods for rapid detection of dermatophytes: Across-sectional study.

In this study, the performance of conventional methods, Calcofluor-white, and Real-time PCR methods were compared to establish an effective method for screening dermatomycosis. Our results showed excellent agreement between direct examination with Calcofluor -white (kappa = 0.97) and real-time PCR (kappa = 0.89) in 307 clinical samples.

Chemical compositions and antifungal activities of Satureja macrosiphon against Candida and Aspergillus species.

BACKGROUND AND PURPOSE: Despite the various applications of Satureja species, there are limited data in this domain. Regarding this, the present study was conducted to investigate the essential oil (EO) biological activity of S. macrosiphon species in Iran. MATERIALS AND METHODS: The EO of S. macrosiphon flowers was obtained by hydrodistillation. Chemical compositions of the EO were analyzed using gas chromatography-mass spectrometry. In addition, minimum inhibitory concentrations (MIC) were measured by means of the broth microdilution method. The estimation of antibiofilm and cytotoxic activities was also accomplished using the tetrazolium salt and MTT assays, respectively. RESULTS: A total of 26 components were identified in the EO with linalool as the main constituent (28.46%). A MIC range value of 0.25-8 µL/mL was obtained against all of the tested fungi. The EO inhibited the biofilm development of the Candida tested strains at a concentration of 4-8 µL/mL. Cytotoxicity (IC50) of EO against the HeLa cell was greater than the MIC concentration (6.49 µL/mL). CONCLUSION: Based on the findings, it was concluded that the EO of S. macrosiphon has the potential for further use as an antifungal agent.

Characterization of beta-tubulin DNA sequences within Candida parapsilosis complex.

BACKGROUND AND PURPOSE: Candida parapsilosis is a common cause of candidemia in children and patients with onco-hematological diseases, septic arthritis, peritonitis, vaginitis, and nail and skin infections. Regarding this, the present study was condcuted to evaluate intra- and inter-species variation within beta-tubulin DNA sequence of C. parapsilosis complex in order to establish the utilization of this gene in the identification and phylogenetic analysis of the species. MATERIALS AND METHODS: A total of 23 isolates representing three different species of C. parapsilosis complex were used in this study, all of which were identifed by ITS-sequencing. For the successful amplification of beta-tubulin gene, a newly designed set of pan-Candida primers was used, followed by bilaterally sequence analysis for pairwise comparisons, determination of multiple alignments, evaluation of sequence identity levels, counting sequence difference, and construction of phylogenetic tree. RESULTS: The multiple alignment of 623-629 bp-long nucleotide (nt) sequences reflecting the beta-tubulin gene indicated an inter-species divergence ranging within 0-68 nt in C. parapsilosis, C. orthopsilosis, and C. metapsilosis with a mean similarity of 84.7% among the species. Meanwhile, the intra-species differences of 0-20 and 0-6 nt were found between the strains of C. parapsilosis and C. orthopsilosis, respectively. The phylogenetic tree topology was characterized by a clade made up by C. parapsilosis and C. orthopsilosis, while C. metapsilosis formed a related but separate lineage. CONCLUSION: Our data provided the basis for further discoveries of the relationship between the species belonging to C. parapsilosis complex. Furthermore, the findigns of the prsent study revealed the efficiency of beta-tubulin DNA sequence data in the identification and taxonomy of C. parapsilosis and other pathogenic yeasts.

Clinical evaluation of ß-tubulin real-time PCR for rapid diagnosis of dermatophytosis, a comparison with mycological methods.

Following our previous report on evaluation of the beta tubulin real-time PCR for detection of dermatophytosis, this study aimed to compare the real-time PCR assay with conventional methods for the clinical assessment of its diagnostic performance. Samples from a total of 853 patients with suspected dermatophyte lesions were subjected to direct examination (all samples), culture (499 samples) and real-time PCR (all samples). Fungal DNA was extracted directly from clinical samples using a conical steel bullet, followed by purification with a commercial kit and subjected to the Taq-Man probe-based real-time PCR. The study showed that among the 499 specimens for which all three methods were used, 156 (31.2%), 128 (25.6%) and 205 (41.0%) were found to be positive by direct microscopy, culture and real-time PCR respectively. Real-time PCR significantly increased the detection rate of dermatophytes compared with microscopy (288 vs 229) with 87% concordance between the two methods. The sensitivity, specificity, positive predictive value, and negative predictive value of the real-time PCR was 87.5%, 85%, 66.5% and 95.2% respectively. Although real-time PCR performed better on skin than on nail samples, it should not yet fully replace conventional diagnosis.