Conventional Mycological Methods Research Articles

Comparison of diagnostic techniques in patients with COVID-19-associated pulmonary aspergillosis (CAPA) in a Venezuelan population

Background: Invasive aspergillosis related to COVID-19 or CAPA is defined by the association of a group of histological, mycological, radiological and clinical criteria that establish this disease as a secondary infection of high morbidity without timely diagnosis. Clinical and histological methods, due to the non-specific characterization of the disease, have been supported by techniques based on microbiological methods, such as culture and direct examination, with a sensitivity close to 50%, allowing the identification of biomarkers such as the GM of the pathogen, being the immunological techniques of greater sensitivity and specificity, the use of radial immunodiffusion and ELISA with antigens secreted by Aspergillus spp. are recommended. The objective of this article was to compare three different serological diagnostic methods used in the mycology laboratory to diagnose CAPA in a Venezuelan population. Methods: Based on a longitudinal study, 77 patient samples diagnosed as positive for SARS-CoV-2 by RT-PCR were analyzed, of which 24 CAPA BAL samples were subsequently evaluated by conventional mycological diagnostic methods (direct examination and culture) and corresponding CAPA serum samples by three serological methods: DID, ELISA with Aspergillus pool antigen, and the Galactomannan detection with Lateral flow (GM- LFA) essay. Results: By microscopy, fungal structures were observed in 18/24 patients and 16/24 were positive for GM-LFA. Regarding the DID assays, precipitating bands were obtained in 21/24 patients with 86% sensitivity; and 97% specificity. To use ELISA technique, shows that 16/24 sera from patients with aspergillosis were positive, which represents 66% sensitivity and 97% specificity. As for GM- LFA, all sera were tested and 16/24 positive sera were obtained with the commercial kit, representing 66% sensitivity and 97% specificity. Conclusions: Finally, when comparing the three tests studied, all techniques were found to be highly sensitive and have excellent specificity, recommending that the diagnosis of CAPA and IPA be based on a combination of diagnostic assays.

Molecular determination of the microbial diversity associated with vaginitis and testing their sensitivity to selected antimicrobials

Abstract. Al-Zaidi MHH, Al-Tamimi WH, Saleh AAA. 2023. Molecular determination of the microbial diversity associated with vaginitis and testing their sensitivity to selected antimicrobials. Biodiversitas 24: 4253-4261. Millions of women suffer from bacterial vaginitis is associated with several serious health problems. The present study aimed to investigate the microbial diversity and their sensitivity to different antimicrobial agents. Bacterial isolates were identified by genotyping using the 16S rRNA gene sequencing and BLASTn analysis, while conventional mycological methods identified Candida spp. The disc diffusion method was used to study the antimicrobial susceptibility patterns. The molecular identification showed that Escherichia coli (20.8%) and Staphylococcus haemolyticus (20.8%) were the most frequent species, followed by Klebsiella pneumoniae (16.7%). At the same time, Macrococcus caseolyticus, Streptococcus agalactiae, Staphylococcus aureus, and Bacillus cereus were the least prevalent bacteria (4.16%)—all Candida sp. against the selected antimicrobial agents. Thus, molecular assays are important in monitoring microorganisms associated with vaginitis. Prospective genotyping studies are needed to determine these microbes' resistant genes and understand their mode of action and response to drug therapy.

P114 Clinical and microbiological spectrum of dermatophytosis from a tertiary care institute

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesThere is an increasing incidence of recalcitrant dermatophytosis in India due to irrational use of antifungals, inappropriate treatment, and also in vitro resistance of the organism by itself. This study is done to determine the clinic-mycological profile, antifungal susceptibility, and outcome of patients with dermatophytosis in our institute.MethodsAll patients with culture-proven dermatophytosis attending the outpatient department of our hospital from January 2019 to December 2019 were included in the study. Detailed clinical data of all the patients were collected.Morphological Identification of the dermatophytes was done by conventional mycological methods. The isolates were sent to PGIMER Chandigarh for further identification by MALDI-TOF and antifungal susceptibility testing. Antifungal susceptibility testing was done for 47 isolates of Trichophyton species.ResultsOf the 155 clinical suspected cases, growth of dermatophytes was observed in 55 (35.4%) of the cases. Tinea corporis 39/55(70.9%) was the predominant clinical type The duration of infection was less than 6 months in 22/55(40%) of cases and >6 months in 33(60%) of the cases. Majority of the patients were in the age group of 20-30 years and were male. A total of 36/55 (65.4%) of the patients belong to middle socio-economic status and 19/55 to lower socioeconomic status (34.5%). In all, 10/55(19.2%) of the patients were students and 10/55 (19.2%) housewives; others include auto drivers, mechanics, teachers, cashiers, etc. All except 4 patients were from urban areas. Comorbid conditions noted were diabetes mellitus in 7/55 (12.7%), hypertension in 6/55 (10.9%), systemic steroid usage in 3/55 (5.4%), post-renal transplant status in 1/55 (1.8%), and SLE in 1/55 (1.8%). Sharing of personal use items was found in 12/55 (21.8%) of the patients and 9/55 (16.3%) of patients complained of excessive sweating.Previous therapy with topical and systemic antifungals was given in 32/55 (58.8%) of the patients, other modes of treatment like homeopathy, and ayurveda in 4/55 (7.2%). Topical steroids were given to 4 patients and 19/55 (34.5%) of the patients were not treated for the infection. Trichophyton mentagrophytes complex (69%) was the predominant species complex isolated followed by T. rubrum, M. gypseum, M.canis, and T. tonsurans.Of the 47 Trichophyton isolates subjected to AFST, all the isolates showed MIC >1ug/ml for fluconazole and griseofulvin. Majority of the isolates showed MIC of <1 ug/ml for other antifungals; high MICs (MIC >1) were exhibited by 5 isolates for terbinafine and naftifine, 2 isolates for sertoconazole, and 1 isolate for voriconazole. Molecular detection of terbinafine resistance done in 15/55 isolates showed mutation in the squalene epoxidase (SE) gene leading to F397L substitutions in 2 isolates. In the present study, the patients were treated with both oral and topical antifungalsOf the 55 cases, complete cure was observed in 21 (38%), partial cure in 9 (16.3%), and relapse in 5 (9%) on 2 years follow-up. However, 20/55 (36.3%) of the cases were lost to follow-up.Conclusion Trichophyton mentagrophytes complex was the predominant species isolated and Tinea corporis was the commonest clinical presentation. Resistance to terbinafine, griseofulvin, and fluconazole has been noted. Dermatophytosis has become a difficult to treat disease due to antifungal resistance, and chronicity/recurrence of the lesions. Early diagnosis followed by rational antifungal therapy are essential for improved outcome.

P157 Entomophthoromycosis: a rare fungal infection case series

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectivesTo present a case series of entomophthoromycosis (a rare fungal infection of the skin and subcutaneous tissue with a protracted and chronic clinical evolution), which would aid in the early recognition and management of this condition.Materials and MethodsA total of 7 laboratory-confirmed cases of entomophthoromycosis, diagnosed and treated between May 2018 and March 2022 at our center, were included in the series. Diagnostic criteria were based on the results of conventional mycological diagnostic methods, including direct epifluorescence microscopy and visual identification of isolates obtained by culture on solid media. Clinical histories were collected retrospectively by chart review and correlated with laboratory findings.ResultsOf the seven patients, six were male and one was female, with ages ranging from 2.5 to 42 years. Most of the patients presented with chronic progressive nasal or rhinofacial swelling and nasal obstruction. Aseptate or pauciseptate hyphae were observed in direct epifluorescence microscopy of tissue samples from all the patients. Conidiobolus spp was isolated from samples from five patients, and Basidiobolus spp from the remaining two. Although growth in culture was obtained several days after sample inoculation, specific antifungal treatment for entomophthoromycosis (using a combination of potassium iodide and itraconazole) was initiated early based on concurrence between clinical features, typical fungal elements seen in direct microscopy and histopathological findings. All the patients improved rapidly and made full recoveries.ConclusionEntomophthoromycosis is a rare condition whose identification requires a high index of clinical suspicion. The treatment for this condition is specific and different from the treatment for other clinically significant invasive fungal infections, but confirmation of diagnosis by culture may take weeks. Therefore, early diagnosis supported by a combination of suggestive clinical features as well as mycological and histopathological evidence, is vital to ensure successful outcomes.

How do Time, Tannin, and Moisture Content Influence Toxicogenic Fungal Populations during the Storage of Sorghum Grains?

Cereal grains are usually ensiled to improve their nutritional value and are one of the main sources of feed for dairy cattle. However, during storage, grains can be contaminated with toxicogenic fungi. Sorghum is one of the most economically important cereals in the world. Therefore, the aim of this work was to evaluate the influence of storage duration and tannin and moisture content (MC) on toxicogenic fungal populations in sorghum grain storage. Samples that were prepared with varieties high in tannins (genotypes Morgan 108 and ACA 558, >5 g/kg dry matter) and with varieties low in tannin content (genotypes Flash 10 and ACA 546, <1 g/kg dry matter) were collected and manually compacted in experimental laboratory silos where they received different MC treatments: low (15 to 25%), medium (26 to 32%), and high (33 to 42%). Freshly harvested grains were analyzed at time 0, and stored grains were analyzed at 30, 90, and 180 days. Fungal isolation and identification were performed following conventional mycological methods. Penicillium citrinum (34%), Aspergillus flavus (60%), and Fusarium nygamai (68%) were the most abundant species. Rapid detection of aflatoxins and fumonisins in each sample was performed using enzyme-linked immunosorbent assay according to the AOAC method, and the quantification of aflatoxin B1 was performed using high-performance liquid chromatography. In four samples of pre- and poststorage grains, aflatoxins were detected with levels of 6.7 to 28.8 μg/kg and aflatoxin B1 with a level of 2 to 14 μg/kg. Fumonisins were only detected in two freshly harvested samples, with levels of 500 to 900 μg/kg. In general, storage time favored the increase of Penicillium populations and reduced Aspergillus and Fusarium. Conversely the abundance of the three populations was not affected by the MC. The results of this study show that fungal populations must be analyzed at different times.

Antifungal susceptibility profile of Aspergillus species from patients with increased risk for aspergillosis

Aspergillosis is the most common fungal infection caused by molds, especially in high-risk patients. The treatment of these diseases is based on the use of polyene and azole antifungal drugs. Resistance rates of Aspergillus species to antifungal drugs vary widely across medical centers around the world. Antifungal susceptibility testing of Aspergillus species to antifungal agents could provide useful information for clinicians to make decision regarding the patient therapy. The aim of the study was to evaluate the antifungal susceptibility profile of Aspergillus species towards amphotericin B, itraconazole, voriconazole and caspofungin, isolated from patients with increased risk for aspergillosis. During a 2-year period, clinical specimens from 125 patients divided into 4 groups according to clinical diagnosis and EORTC/MSG criteria, were analysed at the Institute of Microbiology and parasitology, Faculty of Medicine, Skopje, Republic of North Macedonia. These groups included patients with primary immune deficiency, critically ill patients treated in intensive care units, patients with chronic aspergillosis and cystic fibrosis. All specimens (from respiratory tract and blood culture) were investigated with conventional mycological methods, by inoculation of specimens on media for support of fungal growth. Identification of Aspergillus was performed with macroscopic analysis of mold colonies and additional microscopic analysis of their conidia with lactophenol blue method. E-test strips of voriconazole, itraconazole, amphotericin B and caspofungin (AB bioMerieux, France) were used for determination of the antifungal susceptibility profile of Aspergillus species. Seventy-one isolates of Aspergillus species were confirmed in our patients. Four isolates of A.fumigatus (5.6%) were confirmed in blood cultures, from patients with primary immune deficiencies, and 67 isolates (94.4%) originated from respiratory specimens from patients with different underlying diseases. A.flavus was identified in 11 patients and A.terreus in 3 patients. Resistance to amphotericin B was detected in 6 isolates (2 isolates of A.fumigatus, 1 isolate of A.flavus and 3 isolates of A.terreus). Only one isolate of A.fumigatus showed resistance to itraconazole. All isolates of Aspergillus species were sensitive to voriconazole and caspofungin. In vitro antifungal susceptibility testing with E-test demonstrated resistance to amphotericin B in 6 isolates of Aspergillus species. Only one isolate of A.fumigatus was resistant to itraconazole. All isolates of Aspergillus species showed sensitivity to voriconazole and caspofungin. Antifungal susceptibility testing of Aspergillus species with E-test provides useful information for clinicians for appropriate choice of antifungal agents for treatment of aspergillosis. Keywords: Aspergillus, mold, infection, susceptibility, E-test

Epidemiology of infections due to zoophilic dermatophyte trichophyton simii, an update

Trichophyton simii is an important zoophilic dermatophyte. It has two different names, one for the asexual form (the anamorph state) that occurs in the vertebrate host, and the other for the sexual form (teleomorph also called “perfect state”) produced by mating between anamorphs. The sexual state of T. simii belongs to the genus Arthroderma in the family Arthrodermataceae of the phylum Ascomycota of the Kingdom fungi. Zoophilic Trichophyton species include Trichophyton equinum, T. bullosum, members of the T. mentagrophytes complex, T. simii, and T. verrucosum. The clinical lesions caused by T. simii in humans and animals are usually inflammatory and erythematous. It can be distinguished from other Trichophyton species by its faster growth on agar media, forming finely granular colonies with white-to-pale yellow color on reverse and distinctive to fusiform 3–7 septate macroconidia converting into chlamydospores in older cultures, and pyriform microconidia, and inability to perforate hair in-vitro, and produce the enzyme urease . Trichophyton simii is known to infect monkeys, chickens, dogs, and humans worldwide, though infections are sporadic and epidemic potential and zoonotic risk for humans is low; this dermatophyte is also known to occur as a geophilic species in several countries. The literature search generated a lot of data on T. simii infections from several countries, namely India, Sri Lanka, Japan, Iraq, Iran, Saudi Arabia, Belgium, France, the USA, and Brazil; many of the reports lacked details of clinical lesions and did not mention about treatment/outcome of infections. The results are analyzed and presented concisely in the tables. There is need for investigating the epidemiology of T. simii infections in countries from where, human T. simii infections have been reported, and occurrence of this dermatophyte in soil by employing conventional mycological methods and a newly developed PCR technique based on ITS genomic sequences of this dermatophyte.

Isolation and Identification of Natural Colorant Producing Soil-Borne Aspergillus niger from Bangladesh and Extraction of the Pigment.

Natural colorants have been used in several ways throughout human history, such as in food, dyes, pharmaceuticals, cosmetics, and many other products. The study aimed to isolate the natural colorant-producing filamentous fungi Aspergillus niger from soil and extract pigments for its potential use specially for food production. Fourteen soil samples were collected from Madhupur National Park at Madhupur Upazila in the Mymensingh district, Bangladesh. The Aspergillus niger was isolated and identified from the soil samples by following conventional mycological methods (cultural and morphological characteristics), followed by confirmatory identification by a polymerase chain reaction (PCR) of conserved sequences of ITS1 ribosomal DNA using specific oligonucleotide primers. This was followed by genus- and species-specific primers targeting Aspergillus niger with an amplicon size of 521 and 310 bp, respectively. For pigment production, a mass culture of Aspergillus niger was conducted in Sabouraud dextrose broth in shaking conditions for seven days. The biomass was subjected to extraction of the pigments following an ethanol-based extraction method and concentrated using a rotary evaporator. Aspergillus niger could be isolated from three samples. The yield of extracted brown pigment from Aspergillus niger was 0.75% (w/v). Spectroscopic analysis of the pigments was carried out using a UV–VIS spectrophotometer. An in vivo experiment was conducted with mice to assess the toxicity of the pigments. From the colorimetric and sensory evaluations, pigment-supplemented products (cookies and lemon juice) were found to be more acceptable than the control products. This could be the first attempt to use Aspergillus niger extracted pigment from soil samples in food products in Bangladesh, but for successful food production, the food colorants must be approved by a responsible authority, e.g., the FDA or the BSTI. Moreover, fungal pigments could be used in the emerging fields of the food and textile industries in Bangladesh.

Combining urine antigen and blood polymerase chain reaction for the diagnosis of disseminated histoplasmosis in hospitalized patients with advanced HIV disease.

In this prospective cohort study carried-out in a referral center in São Paulo, Brazil, we found a high frequency of AIDS-related disseminated histoplasmosis (8/106, 7.5%). We used urine antigen test and blood PCR assay to improve the diagnosis of this opportunistic disease.

The History of Botrytis Taxonomy, the Rise of Phylogenetics, and Implications for Species Recognition.

Botrytis is one of the oldest, most well studied, and most economically important fungal taxa. Nonetheless, many species in this genus have remained obscured for nearly 300 years because of the difficulty in distinguishing these species by conventional mycological methods. Aided by the use of phylogenetic tools, the genus is currently undergoing a taxonomic revolution. The number of putative species in the genus has nearly doubled over the last 10 years and more species are likely to be discovered in the future. The implementation of phylogenetic species recognition concepts in Botrytis is providing for more resolution on the relatedness among species than ever before, and this has helped to overcome issues in historical species recognition using morphology, sexual crosses, and pathogenicity tests. Meanwhile, the use of genetic tools is helping to reveal surprising insight into this archetypal necrotroph's behavior, making these approaches increasingly important in species recognition and identification. As Botrytis taxonomy continues to evolve at a rapid pace, researchers should be encouraged to continue to employ the powerful tool of phylogenetics while considering how it fits into a larger framework of classical Botrytis species recognition. Starting points for discussion on how to move forward with Botrytis species recognition are included herein, with an emphasis on the implications and utility of new species descriptions.

Genotyping and Antifungal Susceptibility of C. albicans Isolated from Infected women

The aim of this study was to determine the genotypic and antifungal susceptibility of C. albicans obtained from vulvaginities women The study included 100 vulvaginitis patients who exhibited candidiases infection in uterus. vaginal swabs from women patients and healthy persons screened for the occurrence of C. albicans. Isolates were identified by the conventional mycological methods. Genotypes were determined using 25S rDNA PCR analysis. Vaginal C. albicans was detected in(39.6) % women patients and For the 21 isolates of C. albicans: 14 (66.7 %), 4 (19 %), and 3 (14.3%) isolates were recognized as genotypes A, B and C, respectively …in vitro susceptibility tests were performed using the agar inhibition zone method recommended by the Clinical and Laboratory Standard, the results showed that all C. albicans genotypes (100)%were sensitive to Miconazole. In the case of Nystatin (95)% of isolates were sensitive and (4.8)% were resistant. For ketoconazole (71.4)% of isolates were sensitive (23.8)%were resistant, and (4.8)%showed dose dependent sensitivity. For Fluconazole antifungal (66.7) % of isolates showed sensitivity to fluconazole, (28.6)% were resistant and (4.7)%were dose dependent.

Genotyping and Antifungal Vulnerability of Candida albicans Isolated from Cancer Patients in Dewaniyeh Governorate.

Background: Candida infections are one of the major causes of morbidity in cancer patients.&#x0D; Aim: To determine the genotype and antifungal susceptibility of C. albicans isolated from cancer patients and compared with that from healthy individuals.&#x0D; Materials and methods: Oral swabs collected from cancer patients and healthy subjects were screened for the occurrence of C. albicans. Isolates were identified to species level by the conventional mycological methods. Genotypes were determined with use of 25S rDNA PCR analysis. Susceptibility testing was performed using HiCombo MIC technique.&#x0D; Results: Phenotypic examination showed that oral C. albicans was detected in 90% of cancer patients and 50% of healthy controls. PCR shows that the isolates of A, B, C and T genotypes, among which genotype A C. albicans was the predominant genotype. Genotype A C. albicans recognized the entirely isolates in healthy group. Isolates were most sensitive to amphotericin B. Occurrence of resistance to amphotericin B was the lowest followed by itraconazole. However, the isolates demonstrated a high rate of resistance to ketoconazole and fluconazole.&#x0D; Conclusion: Based on these results, 25S rDNA have been shown to be a useful criterion for distinguishing among various isolates of C. albicans. Amphotericin B is effective antifungal agents that can be used against isolates.

Prevalence and antifungal susceptibility profiles of Candida glabrata, Candida parapsilosis and their close-related species in oral candidiasis

ObjectiveTo evaluate the importance of Candida glabrata, Candida parapsilosis and their close-related species, Candida bracarensis, Candida nivariensis, Candida metapsilosis and Candida orthopsilosis in patients with oral candidiasis and, to determine the in vitro activities of antifungal drugs currently used for the treatment. MethodsOne hundred fourteen isolates of C. glabrata and 97 of C. parapsilosis, previously identified by conventional mycological methods, were analysed by molecular techniques. In vitro antifungal susceptibility to fluconazole, itraconazole, miconazole, and nystatin was evaluated by CLSI M44-A2 disk diffusion test, and by CLSI M27-A3 microdilution for fluconazole. ResultsAll C. glabrata isolates were identified as C. glabrata sensu stricto, 93 out of 97 C. parapsilosis isolates as C. parapsilosis sensu stricto, three as C. orthopsilosis and one as C. metapsilosis. Candida glabrata was mainly isolated in mixed cultures but C. parapsilosis complex was more frequent in pure culture. Candida metapsilosis and C. orthopsilosis were isolated as pure culture and both species were susceptible to all antifungal agents tested. Most C. glabrata isolates were susceptible to miconazole and nystatin, but resistant to fluconazole and itraconazole. Azole cross resistance was also observed. Candida parapsilosis isolates were susceptible to fluconazole although azole cross resistance to miconazole and itraconazole was observed. ConclusionThis study highlights the importance of accurate identification and antifungal susceptibility testing of oral Candida isolates in order to have an in-depth understanding of the role of C. glabrata and C. parapsilosis in oral candidiasis.

Fungal Pollution in the Homes of Respiratory Allergic Patients in Mashhad City, Northeast Iran

Background: Fungal spores are ubiquitous constituents indoors and outdoors and are now generally identified as important causes of respiratory allergies.Objectives: This study aims to evaluate the frequency and prevalence of allergens with fungal origin at homes of allergic patients with respiratory symptoms in the city of Mashhad, Iran.Methods: In this cross-sectional study, a group of patients with allergic rhinitis (n = 50) were selected based on positive skin prick test. Healthy volunteers with no respiratory allergy were included in control group with the same age as diseased group. Samples from nasal cavity and different parts of bedroom were collected and cultured. The fungal agents were recognized by conventional mycological methods based on the cultural and microscopic appearance as properties.Results: Colonization with fungi was obtained in 26% - 64% and 86-98% of the patient and healthy groups, respectively. Among all species isolated from specimens of the patients, A.flavus was dominant which followed by A. niger, Penicillium and Cladosporium whereas the most commonly isolated fungi from the whole specimens of healthy subjects were A. flavus, A. fumigatus, A. niger, Penicillium, Yeast, Alternaria and Cladosporium. Conclusions: Fungi can be hazardous for health. Furthermore, prevention of fungal spores’ growth is important, and also identifying and determining the most common allergen fungi are key steps to provide the necessary recommendations to the patients in controlling and preventing disease.

Phospholipase and proteinase activities of Candida spp. isolates from vulvovaginitis in Iran

This study aims to characterize phospholipase and proteinase activities of Candida isolates from 82vulvovaginal candidiasis (VVC) and to study the relationship of these activities with vulvovaginitis. Totally 82Candida isolates from vagina samples of VVC patients were randomly collected over the period between September and December 2014 from hospitalized patients at the general hospitals of Lorestan province, Iran. Isolates were previously identified by conventional mycological methods. The phospholipase and proteinase activities were evaluated by Egg yolk agar, Tween 80 opacity medium and agar plate methods. The most common Candida species was identified Candida albicans (n=34, 41.5%), followed by Candida famata (n=13, 15.8%), Candida tropicalis (n=11, 13.4%), and Candida parapsilosis (n=9, 11%). The most phospholipase activity was observed in Candida colliculosa (40%), followed by C.famata (38.5%), and Candida krusei (33.3%). The findings revealed that the correlation between phospholipase production by Candida spp. and the presence of VVC was not found to be statistically significant (P=0.91). All Candida spp. exhibited considerable proteinase activity; so that 100% of C.colliculosa, C.parapsilosis, Candida kefyr, and Candida intermedia isolates produced high proteinase activity with Pz 4+ scores. There was a significant correlation between proteinase production by Candida spp. and the presence of VVC (P=0.009). The obtained findings revealed that Candida spp. isolates may produce both virulence factors, phospholipase and proteinase. Although the phospholipase production was only observed in <40% of the isolates; however there was a significant association between proteinase production by Candida spp. and VVC.

Determination of Candida colonization and Candida score in patients in anesthesia intensive care unit

The colonization rate of Candida spp. reaches up to 80% in patients who reside in intensive care units (ICUs) more than a week, and the mean rate of development of invasive disease is 10% in colonized patients. Since invasive candidiasis (IC) in ICU patients presents with septic shock and high mortality rate, rapid diagnosis and treatment are crucial. The aim of this study was to assess the relationship between invasive infection and the determination of Candida colonization index (CI) and Candida score (CS) in patients admitted to ICU who are at high risk for IC and likely to benefit from early antifungal therapy. A total of 80 patients (34 female, 46 male; age range: 12-92 years, mean age: 69.57 ± 16.30) who were in ICU over seven days or longer of Anesthesia Department of Kayseri Education and Research Hospital between April, 2014 and July, 2015 were included in the study. None of the patients were neutropenic. After admission, throat, nose, skin (axillary region), urine, rectal swab and blood cultures have been collected weekly beginning from day zero. Isolation and identification of Candida strains were performed by using conventional mycological methods. CI was calculated as the ratio of the number of culture-positive distinct body sites (except blood culture) to the total number of body sites cultured. CI> 0.2 was considered as fungal colonization, while CI≥ 0.5 as intensive colonization. CS value was calculated according to the components including total parenteral nutrition (TPN) (plus 0.908 points), surgery (plus 0.907 points), colonization in multiple areas (plus 1.112) and severe sepsis (plus 2.038 points), and cut-off value for CS was accepted as >2.5. In our study, overall 1009 cultures (mean: 13 cultures per patient) were taken from 80 patients, and yeast growth was detected in 365 (36.2%) of them. Accordingly, among 68 (85%) of 80 patients included, in at least one sample, yeast growth was determined. No yeast growth was observed in the blood cultures. Of 365 yeast-positive cultures, C.albicans was isolated from 184 (50.4%), C.glabrata from 66 (18%), C.parapsilosis from 42 (11.5%), C.tropicalis from 12 (3.3%), C.kefyr from three (0.8%), and C.krusei from one (0.3%) samples, whereas six (1.6%) samples yielded other yeasts (3 Saprochaete capitata, 3 Trichosporon spp.) and 51 (13.9%) samples yielded multiple yeast growth. The highest colonization rates were detected in rectal swabs (27.4%), urine (23.3%) and throat (22.5%) samples. CI value was found as >0.2 in 65% (52/80), and ≥0.5 in 25% (20/80) of the patients, whereas CS value was >2.5 in only 2.5% (2/80) of the patients. In the statistical evaluation, significant correlations were found between fungal colonization (CI> 0.2) and gender (p=0.032) and length of stay in ICU (p=0.004), and between intensive colonization (CI≥ 0.5) and gender (p=0.008) and age (p=0.012). However, the correlation between Candida colonization and the presence of underlying diseases, APACHE II score, Glasgow coma scale, invasive procedures, use of extended-spectrum antibiotics, presence of bacterial infections, haemodialysis, transfusion and history of previous hospitalization was not statistically significant. Our results have also indicated a statistically significant relationship between fungal colonization and the positivity of C.albicans, C.glabrata, C.parapsilosis ang C.albicans/C.glabrata (p=0.001, p=0.002, p=0.008 and p=0.028, respectively), emphasizing the importance of species-level identification of Candida isolates. The reason of lacking of IC development in our patients may be explained by their low CI and CS values. In conclusion, monitoring of ICU patients who are at high risk for IC in terms of CI and CS would be beneficial. However it is clear that our data need to be supported by multi-center and high-scale studies.

Study of Inhibitory Effect of Echinops cephalotes on Candida Spp. Isolated from Vulvovaginal Candidiasis Patients in Isfahan

Background: Candida spp. has been considered as the agents of acute and recurrent vulvovaginal candidiasis. Objectives: The aim of current study was the evaluation of antifungal activity of Echinops cephalotes (Leaves and stem, manna) plant against species of Candida isolated from patients with vulvovaginal candidiasis. Materials and Methods: In this research study identification of clinical isolates (50 cases) was inducted to the species level by means of conventional mycological methods, morophology on corn meal agar and chromogenic agar, germ tube production and biochemical methods. Antifungal activity of the ethanolic, methanolic and aqueous extracts of E. cephalotes was studied against isolated Candida using agar well diffusion and microdilution methods. Results: Candida spp. which isolated from patients was C. albicans, C. glabrata, C. tropicalis and C. parapsilosis. The inhibition zone of ethanolic extract was 16.6, 13.3, 14, and 22 mg/mL respectively. Minimum inhibitory concentration (MIC) for most the cases were 15.6 mg/mL. The inhibition zone of aqueous extract was 16.8, 16.7, 15 and 15 mg/mL respectively. MIC for most the cases were 15.6-31.2 mg/mL. The inhibition zone of methanolic extract was 15.4, 13.2, 12 and 18 respectively. MIC for most of the cases was 7.8 mg/mL. Among the different extracts, ethanolic extract has the highest and aqueous extract has the lowest anti-Candida activity. Ethanolic, methanolic and aqueous extracts of trehala manna did not show any antifungal activity. Conclusions: This research is the first study on antifungal activity of E. cephalotes. Hence, this plant may be used further as medicinal plant against Candida spp.

Are the classic diagnostic methods in mycology still state of the art?

The diagnostic workup of cutaneous fungal infections is traditionally based on microscopic KOH preparations as well as culturing of the causative organism from sample material. Another possible option is the detection of fungal elements by dermatohistology. If performed correctly, these methods are generally suitable for the diagnosis of mycoses. However, the advent of personalized medicine and the tasks arising therefrom require new procedures marked by simplicity, specificity, and swiftness. The additional use of DNA-based molecular techniques further enhances sensitivity and diagnostic specificity, and reduces the diagnostic interval to 24-48 hours, compared to weeks required for conventional mycological methods. Given the steady evolution in the field of personalized medicine, simple analytical PCR-based systems are conceivable, which allow for instant diagnosis of dermatophytes in the dermatology office (point-of-care tests).

Distribution and antifungal susceptibility of Candida species in patients with increased risk for fungal infections

Candida species are opportunistic yeasts that can be a serious threat for immunocompromised and critically ill patients, and a cause for increased morbidity and mortality in hospitalized patients. The aim of this study was to determine the frequency and distribution of different Candida species in clinical specimens in patients with increased risk for fungal infections, and to determine the antifungal susceptibility profile of invasive Candida species to antifungal agents. During a two year period, clinical specimens from 120 patients divided into 4 groups were analysed at the Institute of microbiology and parasitology, Faculty of Medicine, Skopje, Republic of Macedonia. Each of these 4 groups consisted of specimens from 30 patients, with primary immune deficiency, critically ill patients treated in the intensive care units (ICU), patients with mucosal candidiasis only, and patients with cystic fibrosis. All specimens were investigated with conventional mycological methods. Identification of Candida species was performed with VITEK-2 system (bioMérieux, France). E-test strips of fluconazole, voriconazole, amphotericin B and caspofungin (AB bioMerieux, France) were used for determination of the antifungal susceptibility profile. In this study, a total of 115 isolates of Candida species were confirmed in different clinical specimens (91 isolates from mucosal surfaces and 24 isolates from blood culture). Colonisation of mucosal membranes of gastrointestinal, respiratory and/or urinary tracts was registered in 56.67% (17/30), 56.67% (17/30), 90% (27/30) and 100% (30/30) of the specimens in the first, second, third and fourth group respectively. In all four groups of patients, the following Candida species were confirmed: C. albicans - 55%, C. glabrata - 17.6%, C. parapsilosis - 7.7%, C. tropicalis - 6.6%, unidentified Candida species - 4.4%, C. dubliniensis - 3.3%, C. kefyr - 2.2%, and one isolate of C. rugosa, C. pelliculosa and C. krusei each. Positive blood culture was registered in 23.33% specimens from the first group, 43.33% in the second group, 23.08% of the third group, and in one specimen of the fourth group. The most frequent isolates from blood culture were C. tropicalis and C. krusei, followed by C. albicans, C. parapsilosis and C. tropicalis, and in the second group C. albicans and C. pelliculosa were equally distributed, followed by C. parapsilosis and C. glabrata. All invasive isolates of Candida species were susceptible to amphotericin B, voriconazole and caspofungin. Resistance to fluconazole was registered in 8.3% (2/24) of all confirmed Candida species. Dose-dependent susceptibility to fluconazole was confirmed in 46% (11/24) of the isolates. Our study confirms high prevalence of colonisation and candidemia with non-albicans Candida species. Resistance to antifungal agents was registered only in two isolates of C. krusei. An epidemiological study is necessary for surveillance of dynamics of candidemia and antifungal susceptibility profile of invasive isolates of Candida species in our patients.

In vitro susceptibility of amphotericin-B, voriconazole and caspofungin against Candida guilliermondii biofilms, isolated from dentals units water pipes, under different growth phases

The dental units water pipes are a favorable medium for biofilms formation because of the small diameter of the pipe and the duration of water stagnation, but the question which arises is the nature of the biofilms which are formed inside? This article gives a progress report on the nature of this microbial contamination and precisely the fungal biofilms formation by examining their susceptibility to antifungal agents under different growth phases. Sixteen samples of dental units water pipes were taken from public dental clinic and from stomatology unit at the university hospital of Tlemcen (Algeria). The isolated strains were identified by the conventional mycological methods and were analyzed to determine their minimal concentrations inhibiting their growth (planktonic and sessile forms) using three antifungal agents. Five strains type Candida guilliermondii were identified and analyzed for their resistance to antifungal agents. Antifungal susceptibility testing demonstrated the sensitivity of all planktonic Candida guilliermondii cells against amphotericin-B, voriconzole and caspofungin but the sessile cells of these strains revealed a less susceptibility to antifungal agents and even a resistance when the biofilm made mature. Several types of yeast contaminated the dental units water pipes and especially Candida guilliermondii that was the most founded. This specie was susceptible to antifungal agents under planctonic forms and resistance where the biofilm made mature.