Journal of Fungi

Research

17 pages, 1433 KiB

Open AccessArticle

Phenotypic and Genotypic Identification of Dermatophytes from Mexico and Central American Countries

by Angélica Pérez-Rodríguez, Esperanza Duarte-Escalante, María Guadalupe Frías-De-León, Gustavo Acosta Altamirano, Beatriz Meraz-Ríos, Erick Martínez-Herrera, Roberto Arenas and María del Rocío Reyes-Montes

J. Fungi 2023, 9(4), 462; https://doi.org/10.3390/jof9040462 - 11 Apr 2023

Cited by 2 | Viewed by 1770

Abstract

Dermatophytes are fungi included in the genera Trichophyton, Microsporum, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, and Arthroderma. Molecular techniques have contributed to faster and more precise identification, allowing significant advances in phylogenetic studies. This work aimed to identify [...] Read more.

Dermatophytes are fungi included in the genera Trichophyton, Microsporum, Epidermophyton, Nannizzia, Paraphyton, Lophophyton, and Arthroderma. Molecular techniques have contributed to faster and more precise identification, allowing significant advances in phylogenetic studies. This work aimed to identify clinical isolates of dermatophytes through phenotypic (macro- and micromorphology and conidia size) and genotypic methods (sequences of ITS regions, genes of β tubulin (BT2), and elongation factor α (Tef-1α)) and determine the phylogenetic relationships between isolates. Ninety-four dermatophyte isolates from Costa Rica, Guatemala, Honduras, Mexico, and the Dominican Republic were studied. The isolates presented macro- and micromorphology and conidia size described for the genera Trichophyton, Microsporum, and Epidermophyton. Genotypic analysis classified the isolates into the genera Trichophyton (63.8%), Nannizzia (25.5%), Arthroderma (9.6%), and Epidermophyton (1.1%). The most frequent species were T. rubrum (26 isolates, 27.6%), T. interdigitale (26 isolates, 27.6%), and N. incurvata (11 isolates, 11.7%), N. gypsea and A. otae (nine isolates, 9.6%), among others. The genotypic methods clarified the taxonomic status of closely related species. For instance, the ITS and BT2 markers of T. rubrum/T. violaceum did not differ but the Tef-1α gene did. On the other hand, the three markers differed in T. equinum/T. tonsurans. Therefore, the ITS, BT2, and Tef-1α genes are useful for typing in phylogenetic analyses of dermatophytes, with Tef-1α being the most informative locus. It should be noted that isolate MM-474 was identified as T. tonsurans when using ITS and Tef-1α, but when using BT2, it was identified as T. rubrum. On the other hand, no significant difference was found when comparing the methods for constructing phylogenies, as the topologies were similar. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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12 pages, 1802 KiB

Open AccessArticle

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

by Ines Hadrich, Nahed Khemakhem, Amin Ilahi, Houaida Trabelsi, Hayet Sellami, Fattouma Makni, Sourour Neji and Ali Ayadi

J. Fungi 2023, 9(2), 263; https://doi.org/10.3390/jof9020263 - 15 Feb 2023

Cited by 1 | Viewed by 1364

Abstract

The molecular characterization of Malassezia spp. isolates from animals and humans has not been thoroughly studied. Although a range of molecular methods has been developed for diagnosing Malassezia species, they have several drawbacks, such as inefficiency in differentiating all the species, high cost [...] Read more.

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

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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14 pages, 2054 KiB

Open AccessArticle

Characterization of the Fungal Community in Fritillariae Cirrhosae Bulbus through DNA Metabarcoding

by Jingsheng Yu, Wenjuan Zhang, Yujie Dao, Meihua Yang and Xiaohui Pang

J. Fungi 2022, 8(8), 876; https://doi.org/10.3390/jof8080876 - 19 Aug 2022

Cited by 2 | Viewed by 1336

Abstract

Fritillariae Cirrhosae Bulbus (FCB) is a well-known and precious traditional Chinese medicine with a medicinal history spanning thousands of years. In recent years, it has been reported that fungal and mycotoxin contamination influenced the safety and quality of FCB. It is essential to [...] Read more.

Fritillariae Cirrhosae Bulbus (FCB) is a well-known and precious traditional Chinese medicine with a medicinal history spanning thousands of years. In recent years, it has been reported that fungal and mycotoxin contamination influenced the safety and quality of FCB. It is essential to systematically study the fungal community for the early warning of fungal and mycotoxin contamination in this herb. A total of 15 FCB samples were collected from five provinces in China, and the fungal communities in the FCB samples were analyzed via amplifying the internal transcribed spacer 2 region through the Illumina Miseq PE300 platform. Furthermore, we compared the differences in fungal community in five groups based on collection areas. Results showed that Ascomycota (41.58–99.66%) and Mucoromycota (0–57.42%) were dominant at the phylum level. Eurotiomycetes (8.49–63.93%), Eurotiales (8.49–63.53%), and Aspergillaceae (8.49–63.51%) were the most abundant at the class, order, and family levels. Aspergillus (8.49–63.41%), Rhizopus (0–57.42%), Fusarium (0–22.81%), Cladosporium (0.16–9.14%), and Alternaria (0.06–17.95%) were the main genera in FCB samples. A total of 34 fungal taxa were identified at the species level, including five potentially toxigenic fungi namely Penicillium brevicompactum, P. citrinum, P. oxalicum, Trichothecium roseum, and Aspergillus restrictus. The differences in fungal community between the five groups were observed. Our findings provide references for the safe utilization and quality improvement of FCB. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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21 pages, 3483 KiB

Open AccessArticle

Morphology, Phenotype, and Molecular Identification of Clinical and Environmental Fusarium solani Species Complex Isolates from Malaysia

by Jasper E. James, Jacinta Santhanam, Latiffah Zakaria, Nuraini Mamat Rusli, Mariahyati Abu Bakar, Satinee Suetrong, Jariya Sakayaroj, Mohd Fuat Abdul Razak, Erwin Lamping and Richard D. Cannon

J. Fungi 2022, 8(8), 845; https://doi.org/10.3390/jof8080845 - 11 Aug 2022

Cited by 6 | Viewed by 2551

Abstract

Fusarium infections in humans (fusariosis) and in economically important plants involve species of several Fusarium species complexes. Species of the Fusarium solani species complex (FSSC) are the most frequent cause of human fusariosis. The FSSC comprises more than 60 closely related species that [...] Read more.

Fusarium infections in humans (fusariosis) and in economically important plants involve species of several Fusarium species complexes. Species of the Fusarium solani species complex (FSSC) are the most frequent cause of human fusariosis. The FSSC comprises more than 60 closely related species that can be separated into three major clades by multi-locus sequence typing (MLST) using translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II (RPB2) DNA sequences. The MLST nomenclature for clade 3 of the FSSC assigns numbers to species types (e.g., FSSC 2) and lowercase letters to identify unique haplotypes. The aim of this study was to analyse the genotypic and phenotypic characteristics of 15 environmental and 15 clinical FSSC isolates from Malaysia. MLST was used for the genotypic characterisation of FSSC isolates from various locations within Malaysia, which was complemented by their morphological characterisation on potato dextrose and carnation leaf agar. MLST identified eight different FSSC species: thirteen Fusarium keratoplasticum (i.e., FSSC 2), six Fusarium suttonianum (FSSC 20), five Fusarium falciforme (FSSC 3+4), two Fusarium cyanescens (FSSC 27), and one each of Fusarium petroliphilum (FSSC 1), Fusarium waltergamsii (FSSC 7), Fusarium sp. (FSSC 12), and Fusarium striatum (FSSC 21). Consistent with previous reports from Malaysia, most (11 of 15) clinical FSSC isolates were F. keratoplasticum and the majority (9 of 15) of environmental isolates were F. suttonianum (5) or F. falciforme (4) strains. The taxonomic relationships of the isolates were resolved phylogenetically. The eight Fusarium species also showed distinct morphological characteristics, but these were less clearly defined and reached across species boundaries. Although TEF1-α and RPB2 sequences were sufficient for the species identification of most FSSC isolates, a more precise MLST scheme needs to be established to reliably assign individual isolates of the species-rich FSSC to their geographically-, epidemiologically-, and host-associated sub-lineages. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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13 pages, 2197 KiB

Open AccessArticle

Genetic Diversity Analysis based on the Virulence, Physiology and Regional Variability in Different Isolates of Powdery Mildew in Pea

by Parthasarathy Seethapathy, Subbiah Sankaralingam, Deepu Pandita, Anu Pandita, Kousalya Loganathan, Shabir Hussain Wani, Diaa O. El-Ansary, Hanoor Sharma, Ryan Casini, Eman A. Mahmoud and Hosam O. Elansary

J. Fungi 2022, 8(8), 798; https://doi.org/10.3390/jof8080798 - 29 Jul 2022

Viewed by 1931

Abstract

Powdery mildew is an omnipresent disease that reduces the yield and quality of pea crops (Pisum sativum L.). To examine the powdery mildew pathogen’s morphological, molecular, and genetic diversity, we collected samples of powdery mildew-affected pea crops from ten distinct locations in [...] Read more.

Powdery mildew is an omnipresent disease that reduces the yield and quality of pea crops (Pisum sativum L.). To examine the powdery mildew pathogen’s morphological, molecular, and genetic diversity, we collected samples of powdery mildew-affected pea crops from ten distinct locations in the Nilgiris district of Tamil Nadu, India. The pathogen Erysiphe pisi was identified morphologically based on anamorphic characters. Molecular identification of E. pisi isolates was befitted by targeting the internal transcribed spacer (ITS) region of rDNA and specific primers of powdery mildew fungi. The genetic variation between ten different E. pisi isolates collected from topographically distinct mountainous areas was studied using random amplified polymorphic (RAPD). Based on its morphological characteristics, the powdery mildew fungus presented high similarities to E. pisi. Molecular characterization of the ITS rDNA of E. pisi produced 650 bp nucleotides, PMITS (powdery mildew-internal transcribed region) primers produced 700 bp nucleotides, and an Erysiphe specific ITS primer pair amplified and synthesized 560 bp nucleotides. According to the findings, the collected E. pisi strains exhibited a low level of genetic diversity and only a slight differential in virulence on the host. In the study, E. pisi isolates from Anumapuram, Emerald Valley, Indira Nagar, and Thuneri showed a greater disease incidence in the natural field conditions and shared the same genetic lineage with other isolates in UPGMA hierarchical cluster analysis based on RAPD markers. There was no evidence of a link between the occurrence of the disease and these grouped populations. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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8 pages, 1188 KiB

Open AccessArticle

Analytical Performance of the Commercial MucorGenius^® Assay as Compared to an In-House qPCR Assay to Detect Mucorales DNA in Serum Specimens

by Théo Ghelfenstein-Ferreira, Laura Verdurme and Alexandre Alanio

J. Fungi 2022, 8(8), 786; https://doi.org/10.3390/jof8080786 - 27 Jul 2022

Cited by 5 | Viewed by 1361

Abstract

Standardized, reproducible and validated Mucorales quantitative PCR (qPCR) assays are needed in the context of routine testing in diagnostic labs. We, therefore, compared the commercial MucorGenius^® assay (PathoNostics, Maastricht) targeting five genera of Mucorales to our in-house qPCR targeting Rhizomucor spp., Lichtheimia [...] Read more.

Standardized, reproducible and validated Mucorales quantitative PCR (qPCR) assays are needed in the context of routine testing in diagnostic labs. We, therefore, compared the commercial MucorGenius^® assay (PathoNostics, Maastricht) targeting five genera of Mucorales to our in-house qPCR targeting Rhizomucor spp., Lichtheimia spp. and Mucor/Rhizopus spp. To assess their analytical sensitivity, 25 frozen leftover serum specimens, which had already tested positive based on our in-house assay, were selected. These sera were from 15 patients with probable or proven mucormycosis. For analytical specificity, 0.5 pg from 15 purified fungal DNAs from nine different Mucorales genera were spiked into pooled qPCR-negative leftover serum specimens. All samples were tested in parallel with both assays and the quantitative cycles (Cq) were compared. A total of 13/25 (52%) serum samples were amplified by one of the two assays with only four of them detected with the MucorGenius^® assay. In spiked specimens, all targeted strains were successfully amplified by our in-house qPCR. The MucorGenius^® assay was not able to detect Lichtheimia corymbifera but successfully amplified all other species targeted by the kit and two additional non-targeted species (Syncephalastrum monosporum and Saksenaea vasiformis). The MucorGenius^® assay showed lower analytical sensitivity compared to our in-house assay. Indeed, the MucorGenius^® assay amplified more species, as expected, but showed a decreased detection of the frequent species Lichtheimia corymbifera. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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15 pages, 7821 KiB

Open AccessArticle

Selection of Polymorphic Patterns Obtained by RAPD-PCR through Qualitative and Quantitative Analyses to Differentiate Aspergillus fumigatus

by Omar E. Valencia-Ledezma, Carlos A. Castro-Fuentes, Esperanza Duarte-Escalante, María Guadalupe Frías-De-León and María del Rocío Reyes-Montes

J. Fungi 2022, 8(3), 296; https://doi.org/10.3390/jof8030296 - 13 Mar 2022

Cited by 2 | Viewed by 2227

Abstract

The objective of this work was to use the random amplification of the polymorphic DNA–polymerase chain reaction (RAPD-PCR) technique to select polymorphic patterns through qualitative and quantitative analyses to differentiate the species A. flavus, A. fumigatus, A. niger and A. tubingensis [...] Read more.

The objective of this work was to use the random amplification of the polymorphic DNA–polymerase chain reaction (RAPD-PCR) technique to select polymorphic patterns through qualitative and quantitative analyses to differentiate the species A. flavus, A. fumigatus, A. niger and A. tubingensis. Twenty-seven Aspergillus isolates from different species were typified using phenotypic (macro- and micromorphology) and genotypic (partial BenA gene sequencing) methods. Thirty-four primers were used to obtain polymorphic patterns, and with these a qualitative analysis was performed to select the primers that presented species-specific patterns to distinguish each species. For the quantitative selection, a database was built from the polymorphic patterns and used for the construction of logistic regression models; later, the model that presented the highest value of sensitivity against specificity was evaluated through ROC curves. The qualitative selection showed that the primers OPA-19, P54, 1253 and OPA-02 could differentiate the species. A quantitative analysis was carried out through logistic regression, whereby a species-specific correlation of sensitivity and specificity greater than 90% was obtained for the primers: OPC-06 with a 96.32% match to A. flavus; OPF-01 with a 100% match to A. fumigatus; OPG-13 with a 98.01% match to A. tubingensis; and OPF-07 with a 99.71% match to A. niger. The primer OPF-01 discriminated the four species as well as closely related species. The quantitative methods using the selected primers allowed discrimination between species and showed their usefulness for genotyping some of the species of medical relevance belonging to the genus Aspergillus. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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Review

Jump to: Research, Other

12 pages, 991 KiB

Open AccessReview

Molecular Markers: An Overview of Data Published for Fungi over the Last Ten Years

by Manuela Oliveira and Luísa Azevedo

J. Fungi 2022, 8(8), 803; https://doi.org/10.3390/jof8080803 - 29 Jul 2022

Cited by 15 | Viewed by 3289

Abstract

Fungi are amongst the most abundant and diverse organisms. Despite being widely known for their adverse role in food spoilage or as pathogens for humans, animals, or plants, they also present several beneficial effects. Fungi contribute to human well-being due to their role [...] Read more.

Fungi are amongst the most abundant and diverse organisms. Despite being widely known for their adverse role in food spoilage or as pathogens for humans, animals, or plants, they also present several beneficial effects. Fungi contribute to human well-being due to their role as decomposers, degrading decay matter into smaller molecules which can be easily used by other ecosystem members. These organisms can produce medicinal compounds or modulate protective immune responses in human intestine. Fungi intervene in diverse food processes or act as a food supply. Due to fungal diversity, the unequivocal identification of these organisms is crucial to increasing their practical applications and decreasing their adverse effects. The process of identification could be achieved through the integral sequencing of fungi genomes. However, this procedure would be time-consuming and rather cost-inefficient. Therefore, several molecular markers have been developed to overcome these limitations. The chronology of DNA-based molecular markers development can be divided into three main steps: (1) prior to the development of the PCR technique (RFLP); (2) after the development of the PCR technique (RAPD, AFLP, ISSR, VNTR, SNP, InDels, and DNA barcoding); (3) after the development of the massive parallel sequencing technique (Metabarcoding and WGS). Therefore, the present review covers an overview of the most recently developed molecular markers used for fungal detection and identification. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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Other

Jump to: Research, Review

21 pages, 3375 KiB

Open AccessSystematic Review

Nucleic Acid-Based Detection of Pythium insidiosum: A Systematic Review

by Thanawat Sridapan and Theerapong Krajaejun

J. Fungi 2023, 9(1), 27; https://doi.org/10.3390/jof9010027 - 23 Dec 2022

Cited by 1 | Viewed by 1671

Abstract

Pythiosis, a life-threatening infectious condition caused by Pythium insidiosum, has been increasingly reported in humans and animals worldwide. Antifungal drugs usually fail to control the pathogen. The surgical removal of an infected organ is the treatment of choice. Many affected patients die [...] Read more.

Pythiosis, a life-threatening infectious condition caused by Pythium insidiosum, has been increasingly reported in humans and animals worldwide. Antifungal drugs usually fail to control the pathogen. The surgical removal of an infected organ is the treatment of choice. Many affected patients die due to advanced infection. A timely and accurate diagnosis could lead to a better prognosis in pythiosis patients and save their lives. Although a standard culture method is available in microbiological laboratories, it is time-consuming, laborious, and insensitive for P. insidiosum identification. Immunological assays have been developed to improve the diagnosis of pythiosis. However, immunological methods are commercially unavailable and primarily detect anti-P. insidiosum antibodies, which constitute indirect evidence of pythiosis, making it challenging to differentiate a past from a recent infection. Moreover, such immunological tests cannot diagnose patients with a local infection, such as in the eye. Nucleic acid-based tests (NATs) are efficient for the direct and rapid detection of P. insidiosum DNA in trace-amount or culture-negative specimens. The reagents and equipment required for NATs are usually available in molecular diagnostic laboratories. Herein, we provide a systematic review to comprehensively present the principal and clinical usages, advantages, and limitations of such NATs in the detection of P. insidiosum. Various NATs have been established to detect P. insidiosum, which can be classified into amplification-based (i.e., PCR assays, isothermal tests, and next-generation sequencing methods) and non-amplification-based (i.e., DNA hybridization) techniques. This concise review on NATs constitutes an up-to-date reference with which healthcare professionals can learn about and decide upon which detection method is suitable for their respective laboratory environments. Full article

(This article belongs to the Special Issue Molecular Markers for Fungal Detection and Identification)

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