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.

Chemical Composition and Antifungal and Antibiofilm Effects of Vitex pseudo-negundo Essential Oil against Pathogenic Fungal Strains.

Background: Vitex pseudo-negundo is a plant of the Lamiaceae family that grows in different parts of the world and the vicinity of seasonal rivers in Iran. Methods: The chemical composition of the Vitex pseudo-negundo essential oils was distilled and evaluated using gas chromatography/mass spectrometry. The antifungal activity of the essential oils against the fungal strains was analyzed by broth microdilution methods as suggested by the Clinical and Laboratory Standards Institute. Furthermore, the antibiofilm activity of the Vitex pseudo-negundo essential oils was assessed using the XTT reduction assay. Results: Based on GC/MS analysis, the major components of the Vitex pseudo-negundo essential oils were α-pinene, α-terpinyl acetate, limonene, and (E)-caryophyllene. The growth of tested yeasts was inhibited at concentrations ranging from 2 to 64 µl/mL. Vitex pseudo-negundo fruit essential oil was the most effective in inhibiting yeast growth. Moreover, the essential oils exhibited antifungal activity against filamentous fungi strains. Additionally, the biofilm formation of Candida albicans was inhibited by the leaf, flower, and fruit of the essential oils. Conclusion: Considering the significant antifungal activities of these essential oils, they can be considered a potential source for formulating novel agents to control fungal infections.

Phenotypes characterization and ABC genotypes distribution of clinical Candida albicans isolates.

BACKGROUND: Candidemia and vaginitis are the most common types of candidiasis mostly caused by Candida albicans species. C. albicans has several genotypes and the potential ability to form different phenotype colonies on specific media. This study aimed to evaluate the genotype distribution of blood and vaginal C. albicans isolates and phenotype characteristics on Spider and yeast peptone dextrose agar medium. METHODS: A total of 40 clinical Candida albicans isolates comprising vagina (20) and blood (20) were used. ABC typing using CA-INT-R and CA-INT-L primers was performed to span the transposable group I intron of the 25S rDNA gene. For colony phenotypic characteristics, the Spider and YPDA media were used. RESULTS: Among the blood and vaginal isolates, genotype A (12/60%) and genotype C (10/50%) were the most common types, respectively. The highest phenotype shape frequency of the colonies in blood and vaginal samples was the ring and the lowest was the hat/ring. The dominant color phenotype in blood and vaginal samples was gray. There was a significant relationship between genotype and phenotype forms in the blood sample on YPDA medium (p = 0.02). In the Spider medium, there were no significant differences between genotypes and phenotypes. CONCLUSION: In this study, genotype A and genotype C were predominant in blood and vaginal samples, respectively. In both groups, YPD agar medium demonstrated the most variety of phenotypes that was related to genotypes A and C. The variety of phenotypes in both groups was the same in genotypes A and C on the Spider medium.

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.

Iranian National Survey on Tinea Capitis: Antifungal Susceptibility Profile, Epidemiological Characteristics, and Report of Two Strains with a Novel Mutation in SQLE Gene with Homology Modeling.

BACKGROUND: The data on the epidemiological and antifungal susceptibility profile of tinea capitis (TC) in Iran has not been updated in recent decades. This report presents the Iranian epidemiological and drug susceptibility data regarding the distribution of dermatophytes species isolated by six national mycology centers for a period of one year (2020-2021). MATERIAL AND METHODS: A total of 2100 clinical samples from individuals suspeted to TC were subjected to mycological analysis of direct microscopy and culture. For definite species identification, the culture isolates were additionally subjected to PCR-RFLP and PCR-sequencing of the ITS ribosomal DNA (ITS-rDNA) region. Antifungal susceptibility profiles for eight common antifungal drugs were determined by CLSI M38-A3 guidelines. The SQLE gene was partially amplified and sequenced in two terbinafine-resistant and two susceptible T. mentagrophytes isolates to elucidate probable substitutions involved in resistance. RESULTS: TC (n = 94) was diagnosed in 75 children (79.8%) and 19 adults (20.2%) by direct microscopy and culture. Frequency of TC was significantly more among males (66 males = 70.2% vs 28 females = 29.8%). The prevalent age group affected was 5-9 years (39.36%). Thirty-two (34.04%) T. mentagrophytes, 27 (28.7%) T. tonsurans, 14 (14.9%) M. canis, 13 (13.8%) T. violaceum, 5 (5.32%) T. indotineae, 2 (2.1%) T. benhamiae, and 1 (1.1%) T. schoenleinii were identified as the causative agents. MIC values of isolates showed susceptibility to all antifungal agents, except for fluconazole and griseofulvin with GM MIC of 11.91 µg/ml and 2.01 µg/ml, respectively. Terbinafine exhibited more activity against isolates, with GM MIC 0.084 µg/ml followed by ketoconazole (0.100 µg/ml), econazole (0.107 µg/ml), itraconazole (0.133 µg/ml), butenafine (0.142 µg/ml), and miconazole (0.325 µg/ml). Two resistant T. mentagrophytes isolates harbored missense mutations in SQLE gene, corresponding to amino acid substitution F397L. Remarkably, one unique mutation, C1255T, in the SQLE sequence of two terbinafine-susceptible T. mentagrophytes strains leading to a change of leucine at the 419th position to phenylalanine (L419F) was detected. CONCLUSIONS: T. mentagrophytes, T. tonsurans, and M. canis remained the main agents of TC in Iran, however less known species such as T. indotinea and T. benhamiae are emerging as new ones. Terbinafine could still be the appropriate choice for the treatment of diverse forms of TC.

Development of a high-resolution melt-based assay to rapidly detect the azole-resistant Candida auris isolates.

Background and Purpose: Candida auris is a multidrug-resistant yeast that rapidly spreads, making it the leading Candidate for the next pandemic. One main leading cause of emerging resistant C. auris isolates is nonsynonymous mutations. This study aimed to detect the Y132F mutation, one of the most important azole resistance-associated mutations in the ERG-11 gene of C. auris, by developing a reliable high-resolution melt (HRM)-based method. Materials and Methods: Five C. auris isolates from Iran, plus three control isolates from other Clades were used in the study. The antifungal susceptibility testing through micro broth dilution was performed to recheck their susceptibility to three azole antifungals, including fluconazole, itraconazole, and voriconazole. Moreover, the polymerase chain reaction (PCR) sequencing of the ERG-11 gene was performed. Following the bioinformatic analysis and HRM-specific primer design, an HRM-based assay was developed and evaluated to detect ERG-11 mutations. Results: The minimum inhibitory concentrations of fluconazole among Iranian C. auris isolates ranged from 8 to 64 µg/mL. The PCR-sequencing of the ERG-11 gene and bioinformatic analyses revealed the mutation of Y132F, a substitution consequence of A to T on codon 395 in one fluconazole-resistant isolate (IFRC4050). The developed HRM assay successfully differentiated the targeted single nucleotide polymorphism between mutant and wild types (temperature [Tm]: 81.79 ℃ - cycle threshold [CT]: 20.06 for suspected isolate). For both mutant and non-mutant isolates, the mean Tm range was 81.79-82.39 °C and the mean CT value was 20.06-22.93. These results were completely in accordance with the findings of DNA sequencing. Conclusion: The fast-track HRM-based method successfully detected one of the most common mechanisms of resistance in the ERG-11 gene of C. auris within 3 h. Finally, the development of more panels of HRM assays for the detection of all azole resistance mutations in C. auris ERG-11 is recommended to expand the scope of the field and facilitate the elaboration of rapid and accurate methods of antifungal resistance assessment.

Molecular identification and antifungal susceptibility profiles of etiologic agents of oral candidiasis among HIV-positive patients: A multicenter study.

Background and Purpose: Human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) is a serious risk factor for oral candidiasis (OC). In this regard, the present study aimed to investigate the frequency of Candida species collected from the oropharyngeal cavity of HIV-positive patients and the sensitivity of these isolates to antifungal drugs. Materials and Methods: Oral samples were collected from 169 HIV-positive patients. In addition to culture-based methods, a molecular assay via the polymerase chain reaction-restriction fragment length polymorphism method was applied to identify isolates using the MspI restriction enzyme. The disk diffusion method determined the susceptibility of isolated yeasts to common antifungal drugs according to the CLSI M44-A2 protocol. Results: In total, 81 participants (47.92%) were positive for OC, and Candida albicans was the most prevalent yeast (53.98%). The median age of patients was 36 years old (IQR=10.5; 17-59), and it was found that women are 27% more susceptible to HIV-associated OC (OR=1.268; 95% CI: 0.685-2.348). Patients who received antifungal therapy had a 97.3% reduced chance for OC (OR: 0.027; 95% CI: 0.008-0.091; P-value: 0.000). Antifungal therapy reduced the risk of OC by 97.3% (OR=0.027; 95% CI=0.008-0.091; P=0.000), and antiretroviral therapy decreased the chance of OC 4.42 times (OR=4.423; 95% CI=1.697-11.528; P=0.002). The resistance rates for antifungals, namely fluconazole, ketoconazole, itraconazole, amphotericin B, and nystatin were 15.93%, 8.85%, 7.96%, 5.31%, and 4.42%, respectively. Conclusion: Although several decades have passed since the emergence of HIV/AIDS, little information is available about fungal colonization and infections in this population. Further investigations are suggested using novel and reference molecular identification methods, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry and sequencing, respectively. In addition, more reliable methods for antifungal susceptibility testing are recommended.

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.

Anisakis spp, DNA detection in paraffin-embedded tissue biopsies recovered from patients with gastritis using real-time PCR in Bushehr, Persian Gulf, Iran.

Anisakiasis is a zoonotic fish-born parasitic disease caused by anisakid nematodes. Paraffin-embedded blocks containing biopsy samples taken from patients suffering gastritis with unknown causes were investigated by real-time PCR, in the Bushehr region, Iran; where human anisakiasis has not been reported, so far. A total of 50 paraffin-embedded blocks were randomly selected from 250 archived blocks of the patients with gastritis. A SYBER green-based real-time PCR targeting the ITS1 region was developed for the identification of Anisakis genus. An 86 bp partial fragment of the Anisakis spp. ITS1 gene was amplified successfully. A total of 3 out of 50 samples (6 %) had positive amplification in the samples and their pathology reports showed a significant finding of moderate chronic gastritis with or without ulcers. In conclusion, the developed qPCR could be used for detecting Anisakis spp. larval DNA in human biopsy blocks. This study showed the hidden human cases of anisakiasis in the Bushehr for the first time.

Global prevalence and subgroup analyses of coronavirus disease (COVID-19) associated Candida auris infections (CACa): A systematic review and meta-analysis.

BACKGROUND: Increased hospitalisation rates in the Coronavirus disease 19 (COVID-19) era lead to a new wave of hospital-acquired infections such as emerging multidrug-resistant Candida auris. We aimed to evaluate and estimate the global prevalence of coronavirus-associated C. auris infection (CACa). METHODS: We searched related databases between December 2019 and April 2022 for studies that reported data about CACa. Meta-analysis was performed using MedCalc software version 20.104 according to the DerSimonian and Laird method applying the random-effects model. We evaluated heterogeneity using the χ2 -based Q statistic (significant for p-value < .1) and the I2 statistic (>75% indicative of 'notable' heterogeneity). Moreover, if possible, an odds ratio (OR) analysis was performed for eligible data. RESULTS: Our meta-analysis includes ten eligible studies, including 1942 patients hospitalised with COVID-19; 129 were C. auris cases. The overall pooled prevalence of CACa was estimated at 5.7%. The mortality rate of CACa was estimated at 67.849%. Hypertension was the most prevalent comorbidity (59.374%), followed by diabetes mellitus (52.898%) and cardiovascular diseases (31.392%). Men with a prevalence rate of 80.012% were 3.27 (OR) times more prone to getting infected by C. auris. CONCLUSION: We concluded that the prevalence of C. auris infections decreased during the COVID-19 pandemic and the prevalence gradient changed from Asia to America. Unfortunately, there are many descriptive studies with duplicate content in the field of epidemiology of C. auris infections which are increasing every day. We suggest further non-descriptive studies to accurately establish the cause-and-effect relationships between C. auris and COVID-19 infections.

Candida auris: outbreak fungal pathogen in COVID-19 pandemic: a systematic review and meta-analysis.

Background and Objectives: Candida auris (C. auris) is the first fungal pathogen considered a global health threat. Because, C. auris is associated with multidrug resistance and associated diseases such as diabetes, sepsis, lung and kidney disease. This study investigated the prevalence and mortality of C. auris infection during Covid-19 pandemic. Materials and Methods: Databases were searched for peer-reviewed articles published in the English language up to Jan 18, 2022. Heterogeneity across studies was evaluated using Cochrane's Q test and the I2 index. The pooled point prevalences and their corresponding 95% confidence intervals (CIs) were estimated usingthe random-effects model. Results: In our meta-analysis, 11 eligible articles were included. The total pooled prevalence estimation of C. auris infection among COVID-19 patients was 13% (95% CI: 8%, 19%). The estimated pooled mortality rate of C. auris infection was 37% (95% CI: 15%, 61%). In terms of specific conditions, the pooled risk of mortality was higher in people with diabetes 65% (95% CI: 0.45%, 83%), in cases with >21 days admission inintensive care unit (ICU) 44% (95% CI: 21%, 0.68%), and after receiving steroids 43% (95% CI: 18%, 69%). Conclusion: Our study highlights the high prevalence rate of C. auris infection, particularly among people with a history of metabolic disorders.

Prevalence of superficial-cutaneous fungal infections in Shiraz, Iran: A five-year retrospective study (2015-2019).

BACKGROUND: Superficial and cutaneous fungal infections are common in tropical areas. The aim of this study was to provide a basic database of superficial and cutaneous mycoses and the most common etiological agents among patients. METHODS: Between 2015 and 2019, a total of 1807 patients suspected of superficial and cutaneous mycosis referring to the mycology laboratory of Shiraz medical school, Fars, Iran were evaluated. Specimens were taken from the patients' affected area, and clinical samples were examined by direct microscopy and culture. The epidemiological profile of the patients was collected. RESULTS: A total of 750 patients were confirmed with mycoses. Positive samples totaled 750 cases consisting of the nail (373/49.7%), skin (323/43%), head (47/6.26%), and mucosal membrane (4/0.5%). The yeasts group included 304 Candida spp. (70.3%), 123 Malassezia spp. (28.47%), and 5 Rhodotorula spp. (1.1%). The filamentous fungi were distributed as 34.8% dermatophytes and 7.5% non-dermatophyte. The clinical types of dermatophytosis were tinea unguium (110/261), tinea capitis (50/261), tinea pedis (48/261), tinea corporis (37/261), and tinea cruris (16/261). Non-dermatophyte molds included A. flavus 17, A. niger 4, Aspergillus spp. 15, Penicillium. 10, Fusarium 6, Mucor 2, Stemphylium 1, and Alternaria 1. CONCLUSION: This study provides useful data for the study trends of superficial and cutaneous fungal infections in a specific area. The mycological data confirmed higher incidence of candidiasis (mainly onychomycosis) and dermatophytosis in patients affected by fungal pathogens, which helped to better understand the epidemiological aspects of these mycoses.

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.

Comparative Analysis of Virulence Factors of Homozygous and Heterozygous Strains of Candida albicans Vaginal Isolates.

Although the epidemiology of pathogenic Candida species is changing due to invasive diseases, Candida albicans has become the common cause of human infections worldwide. Candida albicans is a diploid yeast with a mostly clonal mode of reproduction and without known complete sexual cycle. This species has two heterozygous and homozygous strains at hyphal wall protein 1 gene locus (hwp1). Little is known about virulence factors of these strains. The aim of this study was to evaluate the exoenzyme activity of heterozygous and homozygous C. albicans strains. A total of 60 stock Candida albicans species isolates, which consisted of 30 homozygous and 30 heterozygous strains, were used for exoenzyme activities. We used egg yolk agar, Sabouraud blood agar, and bovine serum albumin agar for evaluation of phospholipase, hemolysin, and proteinase activity, respectively. Homozygous strains of Candida albicans had more phospholipase and proteinase activity than heterozygous strains. However, there were no significant statistical differences between the two strains in the severity of exoenzymes production. Beta hemolysin activity was seen in 100% and 96.7% of the homozygous and heterozygous strains, respectively. The results of this study indicated that both of the strains exhibited exoenzyme activities in different ranges. There were no significant statistical differences in virulence factors between the homozygous and heterozygous strains.

Development a hydrolysis probe-based quantitative PCR assay for the specific detection and quantification of Candida auris.

BACKGROUND AND PURPOSE: Candida auris is an emerging multidrug-resistant pathogen. The identification of this species with the conventional phenotypic or biochemical mycological methods may lead to misidentification. Molecular-based species-specific identification methods such as quantitative real-time polymerase chain reaction (qPCR) facilitate a more reliable identification of C. auris than mycological methods. Regarding this, the present study aimed to develop a hydrolysis probe-based qPCR assay for the rapid, accurate identification of C. auris. MATERIALS AND METHODS: The internal transcribed spacer 2 regions in the nuclear ribosomal DNA of C. auris and other related yeasts were assayed to find a specific PCR target for C. auris. A 123-base-pair target was selected, and primers and a probe were designed for hydrolysis probe-based real-time PCR with TaqMan chemistry. Ten-fold serial dilutions of C. auris ranging from 106 to 100 CFU/mL were prepared to establish a standard curve to quantify the yeast. RESULTS: The qPCR assay was able to identify and quantify C. auris with a detection limit of 1 C. auris CFU per reaction. Specificity was confirmed by the non-amplification of the sequences belonging to other Candida species, yeasts, molds, bacteria, or human DNAs. The standard curve of the assay showed a highly significant linearity between threshold values and dilution rates (R2=0.99; slope=-3.42). CONCLUSION: The applied qPCR assay facilitated the rapid and accurate identification and quantification of emerging opportunistic C. auris. Therefore, considering the promising test validation results, we succeeded to develop a rapid and accurate hydrolysis probe- based qPCR assay for the screening and identification of C. auris.

Optical Coherence Elastography-Based Corneal Strain Imaging During Low-Amplitude Intraocular Pressure Modulation.

Purpose: Optical coherence elastography (OCE) is a promising technique for high-resolution strain imaging in ocular tissues. A major strain-inducing factor in the eye is intraocular pressure (IOP), with diurnal physiological fluctuations reaching up to 5 mmHg. We study herein low-amplitude IOP modulation to assess local corneal strain patterns. Methods: Ex vivo porcine eye globes were adjusted to an initial IOP of 15 mmHg and subsequently 25 mmHg. Corneal strain was induced by two subsequent pressure cycles, in which IOP was first increased and then decreased, each by a total of 5 mmHg. Two-dimensional optical coherence tomography (2D-OCT) B-scans were recorded after each loading step. Axial strain maps were obtained from magnitude and phase changes and supra-pixel displacements from cross-correlation. The strain detection sensitivity was evaluated in an isotropic material. Results: Deformations arising from a single 1-mmHg step could be resolved. The largest strain amplitudes (5.11·10-3) were observed in the posterior stroma at a low initial IOP. Strain amplitude was 1.34 times higher at 15 mmHg than at 25 mmHg (p = 0.003). Upon IOP increase, the anterior cornea was compressed, whereas the posterior cornea showed axial expansion. Both morphological images and strain maps were sensitive to postmortem time. Strains that are larger than 2.44·10-5 could be reliably measured. Conclusions: Low-amplitude IOP modulation, similar to diurnal physiological changes, induced measurable deformations in corneal tissue. Axial strain maps permit a localized comparison of the corneal biomechanical response. Small-strain OCE can likely be extended to other domains.

Evaluation of Exoenzyme Activities, Biofilm Formation, and Co-hemolytic Effect in Clinical Isolates of Candida parapsilosis Species Complex.

Candida parapsilosis species complex is considered as important emerging pathogens and little is known about their pathogenicity factors and co-hemolytic activity with different bacteria species. The aim of this study was to determine in vitro exoenzyme activities, biofilm formation, and co-hemolytic effect of different bacteria species on clinical C. parapsilosis complex isolates. In total, 67 C. parapsilosis complex isolates consist of C. parapsilosis sensu stricto 63/67 and Candida orthopsilosis 4/67 were used in this study. To determine the hemolytic activity of these species, Sabouraud dextrose sheep blood agar was used. Evaluation of the CAMP-like phenomenon carried out in the presence of Staphylococcus aureus, Staphylococcus saprophyticus, Staphylococcus epidermidis, and Streptococcus agalactiae. Tube test method with ethylenediaminetetraacetic acid-rabbit plasma was used to determine coagulase activity, and biofilm formation was assessed by the tube method in assist of Sabouraud glucose broth (8%) medium. Fisher's exact tests were used for data statistical analysis. Sixty-six of 67 (98.5%) and 3/67 (4.5%) of the species showed hemolysin and coagulase activity, respectively. Fifty-five of 67 (82.1%) of species had ability for biofilm formation, and none of the samples exhibited co-hemolytic effect in the presence of four mentioned bacteria. No significant difference was found between the level of enzyme production and biofilm formation among the isolates.

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.

Utilization of size polymorphism in ITS1 and ITS2 regions for identification of pathogenic yeast species.

PURPOSE: Despite the existence of a variety of available yeast-identification strategies, easier and more cost-effective methods are required for routine use in clinical laboratories. The internal transcribed spacer (ITS) regions of fungal rRNA genes exhibit variable sizes depending on the yeast species. In the present study, fragment size polymorphism (FSP) analysis of the ITS1 and ITS2 regions for identification of the clinically most important yeast species was assessed. METHODOLOGY: The ITS1 and ITS2 regions of 190 strains, including isolates of 31 standard strains and 159 clinical isolates, were separately PCR amplified with two primer sets: ITS1-ITS2 and ITS3-ITS4. PCR products were mixed and the two-band electrophoretic pattern of each sample was analysed according to the size of the ITS regions as predicted from the GenBank database. RESULTS: Using this method and avoiding expensive tools such as sequencing or capillary electrophoresis, we were able to differentiate nearly all pathogenic yeast species, including Candida albicans, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, Candida guilliermondii, Candida kefyr, Candida lusitaniae, Candida rugosa, Cryptococcus neoformans and Saccharomyces cerevisiae. The method showed limited discriminatory power to differentiate species of the Candida parapsilosis complex. Differentiation of Candida albicans and Candida tropicalis needs already identified controls. CONCLUSION: FSP method benefits from advantages such as lower cost, higher speed and wider range of species than some commercial yeast-identification methods. We consider this method as one of the easiest molecular approaches for identifying a wide range of human pathogenic yeast species, applicable to both diagnostic and epidemiological purposes.

Evaluation of CAMP-Like Effect, Biofilm Formation, and Discrimination of Candida africana from Vaginal Candida albicans Species.

Candida africana as a species recovered from female genital specimens is highly close to C. albicans. The present study was conducted to discriminate C. africana from presumptive vaginal C. albicans strains by molecular assay and evaluate their hemolysin activity, biofilm formation, and cohemolytic effect (CAMP) with vaginal bacterial flora. A total of 110 stock vaginal C. albicans isolates were examined by HWP1 gene amplification. Hemolysin activity and the ability of biofilm formation were evaluated by blood plate assay and visual detection methods, respectively. Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus agalactiae were used to evaluate the CAMP-like effects in Sabouraud blood agar media. Based on the size of the amplicons (941 bp), all isolates were identified as C. albicans. All samples were able to produce beta-hemolysin. Moreover, 69 out of 110 of the isolates (62.7%) were biofilm-positive, 54 out of 110 Candida isolates (49%) demonstrated cohemolytic effects with S. agalactiae, and 48 out of 110 showed this effect with S. aureus (43.6%). All isolates were CAMP-negative with S. epidermidis. We detected all isolates as Candida albicans and almost half of the isolates were CAMP-positive with S. aureus and S. agalactiae, suggesting that these bacteria increase the pathogenicity of Candida in vaginal candidiasis.