Identification Of Fungal Species Research Articles

719. Susceptibility Testing of Oteseconazole (VT-1161) Against Clinical Isolates from Phase 3 Clinical Studies in Subjects with Recurrent Vulvovaginal Candidiasis

Abstract Background Oteseconazole (VT-1161) is a novel, investigational oral therapy that is currently under FDA review for the treatment of recurrent vulvovaginal candidiasis (RVVC). Susceptibility testing was performed on all viable clinical isolates collected from three Phase 3 studies to determine the susceptibility of causative pathogens to oteseconazole versus the current treatment standard of care, fluconazole. Methods Vaginal cultures were obtained at screening and at all subsequent study visits throughout the duration of the studies (approx. 48 Weeks) and submitted to a central mycology laboratory for fungal species identification and storage. Susceptibility testing was conducted in accordance with the CLSI Reference Method for Broth Dilution Antifungal Susceptibility Testing of Yeast M27-Ed4. Results Candida albicans was identified as the primary causative pathogen in 87% of women with RVVC presenting with an acute infection, followed by C. glabrata (8%). Other non-albicans species including; C. parapsilosis, C. tropicalis, C. krusei, C. dubliniensis, C. kefyr and Saccharomyces cerevisiae were responsible for < 1% of infections. A total of 1910 isolates were collected. The MIC range, MIC50 and MIC90 values against all isolates for oteseconazole were ≤ 0.0005 to > 0.25, 0.002 and 0.06 µg/mL, respectively. In comparison, the MIC range, MIC50 and MIC90 values for fluconazole were, ≤ 0.06 to > 32, 0.25 and 8 µg/mL respectively. The MIC range, MIC50 and MIC90 values against all C.glabrata isolates for oteseconazole were 0.002 to > 0.25, 0.03 and 0.125 µg/mL, respectively. In comparison, the MIC range, MIC50 and MIC90 values for fluconazole were, ≤ 0.06 to 32, 2 and 8 µg/mL respectively. Conclusion Oteseconazole demonstrated very low MIC values against most Candida strains, including fluconazole resistant isolates, aligning with clinical study outcomes. Oteseconazole MICs against C. glabrata strains were approximately 6-fold lower than fluconazole. Disclosures Mahmoud Ghannoum, PhD, Mycovia Pharmaceuticals (Grant/Research Support, Research Grant or Support) Thorsten Degenhardt, Ph.D, Mycovia Pharmaceuticals (Employee, Shareholder) Karen Person, M.S., Mycovia Pharmaceuticals, Inc. (Employee)Mycovia Pharmaceuticals, Inc. (Employee) Stephen Brand, Ph.D, Mycovia Pharmaceuticals (Employee)

A Systematic Study of the Antibacterial Activity of Basidiomycota Crude Extracts.

The excessive consumption of antibiotics in clinical, veterinary and agricultural fields has resulted in tremendous flow of antibiotics into the environment. This has led to enormous selective pressures driving the evolution of antimicrobial resistance genes in pathogenic and commensal bacteria. In this context, the World Health Organization (WHO) has promoted research aiming to develop medical features using natural products that are often competitive with synthetic drugs in clinical performance. Fungi are considered an important source of bioactive molecules, often effective against other fungi and/or bacteria, and thus are potential candidates in the search of new antibiotics. Fruiting bodies of sixteen different fungal species of Basidiomycota were collected in the Italian Alps. The identification of fungal species was performed through Internal Transcribed Spacer (ITS) sequencing. Most species belong to genera Cortinarius, Mycena and Ramaria, whose metabolite contents has been scarcely investigated so far. The crude extracts obtained from the above mushrooms were tested for their inhibition activity against five human pathogens: Candida albicans ATCC 14053, C. glabrata ATCC 15126, Staphylococcus aureus NCTC 6571, Pseudomonas aeruginosa ATCC 27853 and Klebsiella pneumoniae ATCC 13883. Twelve crude extracts showed activity against P. aeruginosa ATCC 27853. Highest activity was shown by some Cortinarius species, as C. nanceiensis.

Unique host effect of Tilia japonica on ectomycorrhizal fungal communities independent of the tree’s dominance: A rare example of a generalist host?

Trees of the genus Tilia (Malvaceae) are known to establish symbiosis with ectomycorrhizal (EM) fungi, however, most of what is known about their EM relations is based on few European Tilia species, despite the highest diversity of the genus found in the Asian subcontinent. To the authors’ knowledge, this is the first report on EM fungal communities hosted by Tilia in Japan. Three study sites, on Hokkaido (H) where Tilia was a dominant forest component, Honshu (C) and Kyushu (K) islands where Tilia was extremely rare, were established. From each site 30 root samples of T. japonica were collected after tracing from the trunk, and additional 30 random soil core samples near the traced trees. EM root tips in the samples were subjected to molecular identification of EM fungal species and hosts. The study identified 30 EM fungal lineages (246 OTUs), with 28 lineages (164 OTUs) recorded from T. japonica EM communities. In sites H, C, and K, Tilia communities comprised of 19, 20, and 15 lineages, respectively. 127 EM fungal OTUs were never reported from Japan, and 69 OTUs had no previous records in the UNITE database. Contrarily, about 25% of the recorded OTUs have been exclusively reported from Japan. The observed EM richness hosted by T. japonica exceeded those of cooccurring EM trees as identified from the random soil samples and of other previously studied Tilia species. In each study site, T. japonica was found to exert a consistent “generalist” host effect on its EM fungal communities. About 36% of the EM fungal taxa found in random soil samples were also confirmed in T. japonica root samples, indicating a high connectance of Tilia in the local EM networks. The studied EM communities showed nested structure, with T. japonica as the main contributor to the local community nestedness.

Identification of Fungal Species Associated with Crown and Root Rots of Wheat and Evaluation of Plant Reactions to the Pathogens in North Dakota

Common root rot (CRR) and crown rot (CR), caused by Bipolaris sorokiniana and Fusarium species, respectively, can cause significant yield losses in cereal crops. To assess the prevalence, incidence, and severity of these diseases in North Dakota, wheat samples were collected from spring wheat fields across the state in 2012, 2013, and 2014. Based on sub-crown internode symptoms, a greater incidence and severity of CRR was observed in 2012 (warm and dry year) than in 2013 and 2014. Also, the Northwestern Glaciated Plains and Northwestern Great Plains ecoregions showed greater CRR incidence and severity compared to the Northern Glaciated Plains and Lake Agassiz Plains ecoregions in the state. Bipolaris sorokiniana and Fusarium species including F. acuminatum, F. avenaceum, F. culmorum, F. graminearum, F. equiseti, F. pseudograminearum, F. oxysporum, F. redolens, F. sporotrichioides, and F. solani were isolated and identified from the root and crown tissues of the wheat samples. B. sorokiniana was isolated more frequently than other fungal species in all sampled years and ecoregions of North Dakota. F. acuminatum, F. avenaceum, F. culmorum, F. equiseti, F. graminearum, F. pseudograminearum, and F. redolens were pathogenic causing infections on seedlings of the two wheat genotypes (ND652 and Alsen), whereas isolates of F. oxysporum and F. solani were non-pathogenic and considered as secondary invaders associated with the root and crown rot diseases. Evaluation of some spring wheat genotypes for reactions to one B. sorokiniana isolate at seedling and adult plant stages, and one F. culmorum isolate at the seedling stage indicated that susceptibility to these pathogens varied among different wheat genotypes tested. This study provides useful information on fungal species associated with root and crown rots of wheat in North Dakota and on resistant/susceptible reactions of some spring wheat lines to the different fungal isolates evaluated.

Effectiveness and safety of topical amphotericin B in 30% dimethyl sulfoxide cream versus 30% dimethyl sulfoxide cream for nondermatophyte onychomycosis treatment: A pilot study.

Background Although topical amphotericin B cream is effective for the treatment of nondermatophyte mold onychomycosis in vitro, studies of its effectiveness and safety in vivo are limited. Objectives We studied the effectiveness and safety of topical 0.3% amphotericin B in 30% dimethyl sulfoxide cream (amphotericin B cream) in nondermatophyte mold onychomycosis using the vehicle cream 30% dimethyl sulfoxide cream as control. Methods This randomized controlled study was conducted between January 2019 and November 2020. Patients diagnosed with nondermatophyte mold onychomycosis were randomly divided into two groups of ten patients each: one treated with amphotericin B cream and the other with the vehicle cream. Clinical and mycological cure as well as safety were evaluated. Results Ten patients each treated with amphotericin B cream and the vehicle cream were included in the study, but only nine patients in the vehicle cream group were available for follow up. All the 19 evaluable patients had distal lateral subungual onychomycosis and the great toenails were affected in 18 (94.7%) of these. Mycological cure was achieved in 8 (80%) patients treated with amphotericin B cream and in 4 (44.4%) patients using the control (vehicle) cream. Clinical cure was achieved in 7 (70%) patients treated with amphotericin B cream, but only in 2 (22.2%) patients on the control cream. No adverse events were observed. Limitations The small sample size and the fact that PCR fungal identification that provides accurate identification of fungal species was not performed are limitations of our study. Conclusion Topical amphotericin B cream was both very effective and safe in the treatment nondermatophyte mold onychomycosis. The control (vehicle) cream containing 30% dimethyl sulfoxide also demonstrated some antifungal activity.

Study of some risk factors for fungal contamination of dental unit waterlines in Alexandria, Egypt.

Monitoring the microbial quality of water in dental unit waterlines is an important part of infection control measures carried out in dental clinics. Fungal contamination of such waterlines has not been extensively studied, compared with bacterial contamination. This study aimed at assessing the magnitude and risk factors for fungal contamination of dental unit waterlines. This cross-sectional study included 82 dental units, randomly collected from 3 private clinics and 8 governmental hospitals in Alexandria, Egypt. A total of 204 water samples from dental unit waterlines output were membrane-filtered and cultured for fungal enumeration and species identification. The biofilm forming-ability was assessed for the most prevalent fungal species. The acceptability of samples was determined according to the Swedish drinking water guidelines. The acceptability of samples was 89.7%. The most common mould was Aspergillus flavus, while Candida spp. was the most common yeast (10 isolates), with unusual predominance of Candida dubliniensis (9 isolates). All isolates of Aspergillus flavus and Candida dubliniensis were biofilm-formers. The risk factors for fungal contamination of dental unit waterlines included: dental specialty (p = 0.042), time of sample collection (p < 0.001), older age of dental unit (p < 0.001) and use of 5-15% of sodium hypochlorite. The presence of biofilm-forming fungi in dental unit waterlines is a potential hazard, even when samples have acceptable levels of fungal counts. Risk factors for contamination are numerous and should be addressed.

​Isolation and Identification of Different Fungal Species from Major Kharif Vegetables of Sindh Province, Pakistan

Background: Altenaria species are posing major threat to vegetable crops nowadays, thus it is of utmost importance to identify its different species, so that potential control measures may be explored. Methods: The isolation and identification of different fungi was conducted from major Kharif vegetables, tomato, chilli and eggplant. Samples showing typical symptoms of fruit rot and leaf spot were collected from fields and then pathogens were isolated and identified at laboratory using standard procedures. Result: The total of 07 fungal species, Alternaria alternata, A. solani, Aspergillus flavus, A. niger, Fusarium oxysporum, Pencillium sp. and Rhizopus stolonifer isolated from tomato fruit rot. Similarly, 07 fungi viz; A. alternata, A. tenuissima, A. flavus, A. niger, Colletotrichum capisi, Penicillium sp. and R. stolonifer from chilli fruit rot and 06 fungi viz; A. alternata, A. flavus, A. niger, F. solani, Penicillium sp. and R. stolonifer from eggplant leaf spots were isolated. Significantly highest infection frequency was recorded for A. solani (48.83%) and A. tenuissima (44%) from tomato and chilli fruit rot, respectively. From eggplant leaf spot it was significantly highest for A. alternata (34.5%). Study concludes that 03 species, A. solani, A. tenuissima and A. alternata, dominantly damaged tomato, chilli fruits and eggplant leaves.

Identification and characterization of fungal species associated with the finger millet pathogen Pyricularia oryzae in Kenya

Identification and characterization of fungal species associated with the finger millet pathogen Pyricularia oryzae in Kenya

Drug Sensitivity Profile of Fungi Isolated from Onychomycosis Patients and Evaluation of Squalene Epoxidase Mutation in One Terbinafine-Resistant Trichophyton mentagrophytes Species.

Background: The resistance to treatment of onychomycosis is increasingly reported. The present study aimed to assess the antifungal activity of itraconazole, terbinafine, luliconazole, and efinaconazole against dermatophytes, molds, and also yeast isolated from patients with onychomycosis. Furthermore, the mechanism of resistance to terbinafine in resistant Trichophyton mentagrophytes species was evaluated using the squalene epoxidase (SQLE) gene sequence. Methods: A total of 71 fungal isolates were collected from 97 patients with suspected onychomycosis. The identification of fungal species was performed using conventional and molecular approaches. In vitro drug susceptibility for itraconazole, terbinafine, luliconazole, and efinaconazole was carried out using the broth microdilution method according to the CLSI-M60 and CLSI-M38 3rd ed., respectively. The SQLE gene of one terbinafine-resistant T. mentagrophytes was amplified using the specific primers. Results: Efinaconazole and luliconazole demonstrated higher effectiveness against all isolates in the study. One mismatch was detected at position 1177, which showed A → C change associated with Phe397Leu amino acid substitution of the SQLE protein in terbinafine-resistant T. mentagrophytes. Conclusion: The occurrence of resistant strains of organisms causing onychomycosis should be considered and evaluated. Furthermore, the identification of amino acid changes responsible for resistance to antifungals is a useful consideration in drug-target interaction.

Molecular identification of fungal species and evaluation of Ochratoxin A contamination in spreading flour in Hamedan bakeries

Background: The presence of toxicogenic fungi and the production of ochratoxin in flour are dangerous to human health. Therefore, identification of these fungi by molecular and precise methods is essential. The aim of this study was molecular identification of fungal species and investigation of ochratoxin A contamination in flours used in bakeries in Hamadan Methods: In this descriptive-analytical study, 60 flour samples were collected from bakeries in Hamadan. At first, fungi were identified using phenotypic methods such as slide culture. Then, the genus and species of fungi were confirmed by PCR and sequencing of PCR products. ELISA method was used to detect ochratoxin A. Data were analyzed with GraphPad software version 6. Results: Of the 60 samples, 28 flour samples (46.66%) were free of fungal contamination and 32 flour samples (53.33%) were over fungal contamination (104 colonies/gr). Aspergillus and penicillium were the most abundant in samples. The prevalence of fungi in flour of Lavash bread, Sangak Bread, Barbari bread, Taftoon bread (Handmade), Taftoon bread (Machinal made), Stokbrood bread and Loaf bread were 50%, 46%, 35%, 31%, 26%, 18% and 11%, respectively. However, the highest levels of ochratoxin A were reported in flour of Lavash bread (3.98 ppb) and the lowest in Loaf bread (0.66 ppb). There was also a significant relationship between the fungal species and the amount of ochratoxin A production in flour of beards. Conclusion: While phenotypic and genotypic methods did not show the same sensitivity, the presence of ochratoxin in the studied flours indicated the necessity of modification in wheat storage and bakery flours.

Morphological and genetic identification of fungal genus/species in formalin-fixed, paraffin-embedded specimens obtained from patients with histologically proven fungal infection.

Although fungi are found relatively easily by microscopic examination of pathological samples, identification of fungal genus and species in pathological samples is not easy because the morphological features of fungi are similar among genera and species. A multiple real-time PCR was developed for identification of fungal genus/species, and morphological characterizations of fungi were analysed in pathological samples. Seventy-five formalin-fixed paraffin-embedded (FFPE) samples morphologically proven to contain any fungus were examined. A multiple real-time PCR system was developed to identify 25 fungal genus/species in pathological samples. Morphology of fungus in the specimens was re-reviewed retrospectively based on the results of real-time PCR. Real-time PCR identified fungal genus/species in 56 of 75 (74.6%) specimens with histologically proven fungal infection. In 53 specimens of filamentous fungi, Aspergillus spp. (22 specimens), Cladosporium (8), Scedosporium apiospermum (4), Malassezia sympodialis (1) and Candida albicans (1) were identified. Pseudohyphae of Candida were confused with filamentous fungus in a case. Morphological observation suggested differences in the presence of septated or non-septated hyphae, the filament size, and the branch angle among genus/species of filamentous fungi; however, genus/species was not able to be determined by their morphological features. In 22 specimens of yeasts, real-time PCR allowed for the identification of Candida albicans (12 specimens), Candida glabrata (2), Cladosporium (2), Scedosporium apiospermum (2), Pichia kudriavzevii (1) and Aspergillus sydowii (1). These data suggest that it is difficult to identify fungal genus/species by morphological features alone. Real-time PCR is useful to identify fungal genus/species in pathological samples.

Molecular characterization of Fusarium solani and Fusarium oxysporum phyto-pathogens causing mango maturity malconformation

Mango (Mangifera indica L.) is a significant fruit on global scale and is vulnerable to varied kinds of the decline disorders, subsequently leading to the immense economic loss of the mango growers in different countries. Pakistan is a renowned exporter of auspicious varieties of the mango fruit. However, the mango fruit in Pakistan is also exposed to a number of phyto-pathogens. Current work has carried out the task of the fungal species identification involved in post-harvest rotting of mango in orchards by cultural and pathological characteristics as well as molecular technique. By molecular analysis, the genetic diversity of Fusarium species (Fusarium solani, Fusarium oxysporum) was isolated from the rotten mango fruits of the selected districts and evaluated by 5.8S and two ITS flanking regions ITS1 and ITS2 gene of rDNA. Molecular analysis facilitated the identification of the varied kinds of the isolated pathogens and also determined the relationship of various Fusarium strains involved in post-harvest diseases leading to the malconformation of mangoes. Prior to the current research, anthracnose has been reported to be the major mango rotting disease caused by Colletotrichum gloeosporides in Pakistan. However, the results of our work are indicative of the profound involvement of the F. solani and F. oxysporum in destroying the mango crop. Current work is indicative of the opportunist nature of the F. solani and F. oxysporum pathogens infecting post-harvest mangoes through wounds and cause injuries.

New Method for Identifying Fungal Kingdom Enzyme Hotspots from Genome Sequences.

Fungal genome sequencing data represent an enormous pool of information for enzyme discovery. Here, we report a new approach to identify and quantitatively compare biomass-degrading capacity and diversity of fungal genomes via integrated function-family annotation of carbohydrate-active enzymes (CAZymes) encoded by the genomes. Based on analyses of 1932 fungal genomes the most potent hotspots of fungal biomass processing CAZymes are identified and ranked according to substrate degradation capacity. The analysis is achieved by a new bioinformatics approach, Conserved Unique Peptide Patterns (CUPP), providing for CAZyme-family annotation and robust prediction of molecular function followed by conversion of the CUPP output to lists of integrated “Function;Family” (e.g., EC 3.2.1.4;GH5) enzyme observations. An EC-function found in several protein families counts as different observations. Summing up such observations allows for ranking of all analyzed genome sequenced fungal species according to richness in CAZyme function diversity and degrading capacity. Identifying fungal CAZyme hotspots provides for identification of fungal species richest in cellulolytic, xylanolytic, pectinolytic, and lignin modifying enzymes. The fungal enzyme hotspots are found in fungi having very different lifestyle, ecology, physiology and substrate/host affinity. Surprisingly, most CAZyme hotspots are found in enzymatically understudied and unexploited species. In contrast, the most well-known fungal enzyme producers, from where many industrially exploited enzymes are derived, are ranking unexpectedly low. The results contribute to elucidating the evolution of fungal substrate-digestive CAZyme profiles, ecophysiology, and habitat adaptations, and expand the knowledge base for novel and improved biomass resource utilization.

Year, Location, and Variety Impact on Grape-Associated Mycobiota of Arkansas-Grown Wine Grapes for Wine Production.

Wine grape berries (Vitis spp.) harbor a wide variety of yeasts and filamentous fungi that impact grapevine health and the winemaking process. Identification of these fungi could be important for controlling and improving wine production. The use of high-throughput sequencing (HTS) strategies has enabled identification and quantification of bacterial and fungal species in vineyards. The aims of this study were to identify mycobiota from Cabernet Sauvignon and Zinfandel (V. vinifera), Carlos and Noble muscadines (V. rotundifolia), Cynthiana (V. aestivalis), and Vignoles hybrid (cross of different Vitis spp.) grapes, and investigate the effect of grape variety, location, and year on grape fungal communities. Grape berries were collected in 2016 and 2017 from four vineyards located in Arkansas. The HTS of the Internal Transcribed Spacer 1 region was used to identify grape indigenous epiphytic and endophytic fungal communities. The predominant genera identified on the Arkansas wine grapes were Uwebraunia, Zymoseptoria, Papiliotrema, Meyerozyma, Filobasidium, and Curvibasidium. Overall, the data suggested that grape fungal community distribution and relative abundance were influenced by grape variety, year, and location, but each was influenced to a different extent. Not only were grape mycobiota influenced by year, variety, and location but also it appeared that communities from the previous year impacted microbial communities the following year. For example, an increase of the mycoparasite Ampelomyces quisqualis was noticed in 2017 on grapes that carried the causal agent of powdery mildew, Erysiphe necator, in 2016, thus, amplifying the importance of vineyard microbiota knowledge for disease management and winemaking.

In honor of John Bissett: authoritative guidelines on molecular identification of Trichoderma

Modern taxonomy has developed towards the establishment of global authoritative lists of species that assume the standardized principles of species recognition, at least in a given taxonomic group. However, in fungi, species delimitation is frequently subjective because it depends on the choice of a species concept and the criteria selected by a taxonomist. Contrary to it, identification of fungal species is expected to be accurate and precise because it should predict the properties that are required for applications or that are relevant in pathology. The industrial and plant-beneficial fungi from the genus Trichoderma (Hypocreales) offer a suitable model to address this collision between species delimitation and species identification. A few decades ago, Trichoderma diversity was limited to a few dozen species. The introduction of molecular evolutionary methods resulted in the exponential expansion of Trichoderma taxonomy, with up to 50 new species recognized per year. Here, we have reviewed the genus-wide taxonomy of Trichoderma and compiled a complete inventory of all Trichoderma species and DNA barcoding material deposited in public databases (the inventory is available at the website of the International Subcommission on Taxonomy of Trichodermawww.trichoderma.info). Among the 375 species with valid names as of July 2020, 361 (96%) have been cultivated in vitro and DNA barcoded. Thus, we have developed a protocol for molecular identification of Trichoderma that requires analysis of the three DNA barcodes (ITS, tef1, and rpb2), and it is supported by online tools that are available on www.trichokey.info. We then used all the whole-genome sequenced (WGS) Trichoderma strains that are available in public databases to provide versatile practical examples of molecular identification, reveal shortcomings, and discuss possible ambiguities. Based on the Trichoderma example, this study shows why the identification of a fungal species is an intricate and laborious task that requires a background in mycology, molecular biological skills, training in molecular evolutionary analysis, and knowledge of taxonomic literature. We provide an in-depth discussion of species concepts that are applied in Trichoderma taxonomy, and conclude that these fungi are particularly suitable for the implementation of a polyphasic approach that was first introduced in Trichoderma taxonomy by John Bissett (1948–2020), whose work inspired the current study. We also propose a regulatory and unifying role of international commissions on the taxonomy of particular fungal groups. An important outcome of this work is the demonstration of an urgent need for cooperation between Trichoderma researchers to get prepared to the efficient use of the upcoming wave of Trichoderma genomic data.

Using DNA Barcoding in Fungal Taxonomy

Totally, 1.5 million fungal species estimated, of which less than 10 percent have been described to date. Recent advances in molecular biology as well as gene sequencing technologies facilitate the discovery and identification of new species in various groups of living organisms, especially fungi. Morphological methods do not have efficiency for the identification of fungal species, so, using new techniques based on DNA data has been considered in the rapid and accurate fungal species identification. DNA Barcoding is a new technique with several years’ backgrounds for detecting fungi and fungal like organisms. The Internal Transcribed Spacer in ribosomal RNA gene (ITS-rDNA) has been considered as a suitable barcode sequence. The efficacy of this region’s sequence data in most fungal groups has led to its use as a standard barcode by different mycologists. However, the ITS-rDNA region appears to be used as the primary barcode sequence for fungi of the genus or species group. The accurate species-specific identification is performed depending on the fungal group based on secondary barcodes. Due to the pleomorphic nature of fungi, the use of DNA barcoding is of importance. To date, hundreds of thousands of reference barcode sequences have been created for thousands of species of living organisms through DNA barcoding projects. The subsequent efforts of scientists are managing and automation of DNA barcoding in order to speed up the identification process.

Microbial and Fungal Contamination of Wheat Flour, Dough, and Bread Samples Collected from Isfahan, Iran

The present investigation was done to evaluate the microbial and fungal contamination and also molecular identification of fungal species in flour, dough and bread samples collected from Isfahan, Iran.Two-hundred and forty different types of cereals including flour, dough and bread were collected. Samples were subjected to microbial and fungal counting and also PCR-based identification of fungal species and sequencing of Aspergillus species. Ranges of total bacterial count of flour, dough and bread samples collected from breads were between 2.83±0.99 and 6.43±1.12 log cfu/g. Flour, dough and bread samples collected from bulk breads had the higher counts of total bacteria than those of flat breads. Ranges of mold count of flour, dough and bread samples collected from breads were between 0.00±0.00 and 1.63±0.63 log cfu/g. Flour, dough and bread samples collected from flat breads had the higher counts of mold than those of bulk breads.Distribution of Penicillium, Cladosporium, Mucor, Aspergillus, Alternaria, Canninghamella, Hyaline, Rhizopus, Epicoccum and Syncephalestrum amongst detected fungal colonies were 24.40%, 20.10%, 20.10%, 19.00%, 3.80%, 3.80%, 3.80%, 2.70%, 1.00% and 1.00%, respectively. Sequencing of isolated Aspergillus genus revealed that all strains were related to flavus, oryzea, terreus,andnijer species. Detected total count and mold count in studied bread samples were lower than limit standards announced by Institute of Standards and Industrial Research of Iran. However, considering the high consumption rate of these kinds of food samples among Iranian people, severe surveillance programs should perform to inhibit from their bacterial and fungal contamination.

The mycologist dilemma: Challenges in reporting and identifying histoplasma capsulatum in mycology laboratory, UMMC

Background: Histoplasmosis are endemic in the eastern and central states of United States. However it is not a very common fungal pathogen found and isolated in Malaysia. Disease presentation can be varied from very mild symptoms to a full blown disseminated disease. Diagnosis of this dimorphic fungus in mycology laboratory can be challenging. Case description: A 27 year old Indonesian with HIV positive results presented with intermittent fever for two months followed by generalized maculopapular rash upon admission. Patient was started on amphotericin B when fungal hypae was identified by direct smear from blood culture bottles that was flagged positive by the Bactec. The antifungal was started on the presumptive assumption that it may be a common opportunistic fungal pathogen, Tarolomyeses marneffei which is endemic in Malaysia Discussion: Histoplasma capsulatum which is rarely isolated in clinical specimen and needs to be identified precisely for prompt treatment. Histoplasma capsulatum can take up two to three weeks to grow in its mycelia form. Typical microscopic morphology of this pathogen depends on the maturity of mycelia. Tuberculated macrconidia an important microscopic identification feature for this pathogen in prepared slide culture . In this clinical case the slide culture which was prepared shown, only few single small capsulated like macroconidia without any tuberculation on the surface of conidia. It was very in-conclusive findings from micoroscopic point of view and unable to diagnose the pathogen just looking at this nontypical features. In the suspicious growth of gross morphology colonies, which appears white chalky on Sabouraud Dextrose Agar (SDA) agar, we randomly performed MALDI-TOF MS analysis on this colonies with special prepartion for mold identification. Surprisingly it was excellent scores, 99.9% identified as histoplasma capsulatum within 20 minutes. Conclusion: Identifying rare fungal pathogen in mycology laboratory needs experience and experts. Alternative methods of diagnosis need to be established if common conventional methods failed especially in mycology laboratory. Fungal Species Identification by MALDI-ToF Mass spectrometry is very good alternative method for fast and efficient compare to molecular method.

Rapid microbial identification and colistin resistance detection via MALDI-TOF MS using a novel on-target extraction of membrane lipids

Rapid infection diagnosis is critical to improving patient treatment and outcome. Recent studies have shown microbial lipids to be sensitive and selective biomarkers for identifying bacterial and fungal species and antimicrobial resistance. Practical procedures for microbial lipid biomarker analysis will therefore improve patient outcomes and antimicrobial stewardship. However, current lipid extraction methods require significant hands-on time and are thus not suited for direct adoption as a clinical assay for microbial identification. Here, we have developed a method for lipid extraction directly on the surface of stainless-steel matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) plates, termed fast lipid analysis technique or FLAT, which facilitates the identification of bacterial and fungal species using a sub-60-minute workflow. Additionally, our method detects lipid A modifications in Gram-negative bacteria that are associated with antimicrobial resistance, including to colistin.

Universal Multiplex Polymerase Chain Reaction-Restriction Fragment Length polymorphism (UMPCR-RFLP) for Rapid Detection and Species Identification of Fungal and Mycobacterial Pathogens

Fungal and mycobacterial skin infections are common in immunocompromised patients and patients with febrile neutropenia, since the patients’ ability to control localized infection is diminished by the disease. The similarity of the lesions caused by these organisms conduces to difficulty of differential diagnosis. Although a histopathological examination and a microbial culture are standard methods for laboratory diagnosis of skin infection, the methods have drawbacks. Histopathological examination yields low positive results, while microbial culture is time-consuming and might result in no growth, causing delayed treatment. This study aimed to develop and evaluate an in-house rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the detection and identification of fungal and mycobacterial pathogens in resource-limited laboratories. A total of 26 typed species of human pathogenic fungi and 12 species of mycobacteria were used. Strain differentiation was analyzed by using multiplex PCR-RFLP. The internal transcript spacer region (ITS) of fungi and heat-shock protein 65 (hsp65) gene of mycobacteria were amplified using ITS1-ITS4 and Tb11-Tb12 universal primers, respectively. The RFLP patterns were examined at genus-specific and species-specific level. No cross-amplification was observed between fungal and mycobacterial tested strains, nor any specific binding between primers and human DNA. It was concluded that the multiplex PCR-RFLP method developed in this study can be used as a molecular diagnostic test for fungal and mycobacterial species identification. In the future, this technique may be useful for detecting fungal and mycobacterial infection directly from clinical specimens.