An investigation on non-invasive fungal sinusitis; Molecular identification of etiologic agents.

BACKGROUND: Fungal sinusitis is increasing worldwide in the past two decades. It is divided into two types including invasive and noninvasive. Noninvasive types contain allergic fungal sinusitis (AFS) and fungus ball. AFS is a hypersensitivity reaction to fungal allergens in the mucosa of the sinonasal tract in atopic individuals. The fungus ball is a different type of noninvasive fungal rhinosinusitis which is delineated as an accumulation of debris and fungal elements inside a paranasal sinus. Fungal sinusitis caused by various fungi such as Aspergillus species, Penicillium, Mucor, Rhizopus, and phaeohyphomycetes. The aim of the present study is to identify fungal species isolated from noninvasive fungal sinusitis by molecular methods. MATERIALS AND METHODS: During 2015-2016, a total of 100 suspected patients were examined for fungal sinusitis. Functional endoscopic sinus surgery was performed using the Messerklinger technique. Clinical samples were identified by phenotypic and molecular methods. Polymerase chain reaction (PCR) sequencing of ITS1-5.8S-ITS2 region and PCR-restriction fragment length polymorphism with MspI restriction enzyme was performed for molecular identification of molds and yeasts, respectively. RESULTS: Twenty-seven out of 100 suspected cases (27%) had fungal sinusitis. Nasal congestion (59%) and headache (19%) were the most common clinical signs among patients. Fifteen patients (55.5%) were male and 12 patients (44.5%) were female. Aspergillus flavus was the most prevalent fungal species (26%), followed by Penicillium chrysogenum (18.5%) and Candida glabrata species complex (15%). CONCLUSION: Since clinical manifestations, computed tomography scan, endoscopy, and histopathological findings are very nonspecific in AFS and fungus ball; therefore, molecular investigations are compulsory for precise identification of etiologic agents and appropriate management of these fungal infections.

Detection of Opportunistic Fungi from the Bronchoalveolar Lavage Specimens of Patients with Pulmonary Diseases.

Background: Opportunistic fungi are a constantly evolving group of pathogens that become active when the immune system is compromised, begin to multiply, and soon overwhelm the weakened immune system. This study was performed to evaluate the number of opportunistic fungi in bronchoalveolar lavage (BAL) samples of patients with pulmonary diseases. Materials and Methods: After receiving patients' consent and demographic forms, a total of 120 BAL samples were taken by a pulmonary physician. The etiologic agents were identified by standard morphological and molecular methods. Yeast cells were counted on culture media, and direct smears were precisely examined for the presence of yeasts elements, Pneumocystis, and filamentous fungi. Results: In this study, 29 (24.1%) patients showed positive direct smears for yeast elements in their BAL samples. The mean colony count of yeasts was 42,000 (CFU/mL) on culture media. Six (5%) species of filamentous fungi, including three (2.5%) isolates of Penicillium species (P. variabile, P. glabrum, and P. thomii), two (1.67%) Aspergillus species (A. flavus and A. fumigatus), 1 case (0.83%) Pseudallescheria boydii were detected. Seven cases (5.83%) of Pneumocystis cysts were observed in the direct smears stained with Giemsa. Identification of all fungi confirmed by molecular or sequencing methods. Conclusions: Due to the presence of a large number of fungi in the BAL samples and possible physical interference with the selected drugs for treatment, we draw the attention of pulmonologists to this important issue. Rapid diagnosis of fungal infections is essential to optimize treatments and outcomes.

Isolation and molecular characterization of clinical and environmental dematiaceous fungi and relatives from Iran.

Background and Purpose: The frequency and genetic diversity of black fungi in environmental and clinical settings have not been fully studied in Iran. This study aimed to identify and evaluate intra- and inter-species DNA sequence variation and also understand the phylogenetic relationships of melanized fungi and relatives isolated from different geographical regions of Iran. Materials and Methods: In total, 111 clinical and environmental strains of dematiaceous fungi were isolated, and their internal transcribed spacer ribosomal DNA (rDNA) regions were sequenced and analyzed. Results: An inter-species nucleotide sequence diversity rate of 1 to 464 nucleotides was observed between the species. Intra-species differences were found in the strains of Alternaria alternata, Cladosporium cladosporioides, Alternaria tenuissima, Curvularia spicifera, Aureobasidium pullulans, Curvularia hawaiiensis, Neoscytalidium dimidiatum, Alternaria terricola, Alternaria chlamydospora, Didymella glomerata, and Drechslera dematioidea by 0-59, 0-22, 0-4, 0-4, 0-3, 0-2, 0-2, 0-2, 0-2, 0-1, and 0-1 nucleotide, respectively. Conclusion: The internal transcribed spacer rDNA is useful for the discrimination of several taxa of dematiaceous fungi. However, a better understanding of the taxonomy of species of Alternaria requires a larger rDNA region or a library of other gene sequences.

Identification of Candida albicans and Candida dubliniensis Species Isolated from Bronchoalveolar Lavage Samples Using Genotypic and Phenotypic Methods.

BACKGROUND: Candida dubliniensis is a newly diagnosed species very similar to Candida albicans phenotypically and first discovered in the mouth of people with AIDS in 1995. Among the different phenotypic and genotypic methods, a cost-effective method should be selected which makes it possible to differentiate these similar species. MATERIALS AND METHODS: Polymerase chain reaction (PCR)-restriction fragment length polymorphism with MspI enzyme and the Duplex-PCR method were done by DNA extraction using boiling. The sequencing of the amplified ribosomal region was used to confirm the C. dubliniensis species. Direct examination and colony count of the yeasts were applied for bronchoalveolar lavage (BAL) samples and the growth rate of the yeasts were studied at 45°C. To understand the ability formation of chlamydoconidia in yeast isolates, they were separately cultured on the sunflower seed agar, wheat flour agar, and corn meal agar media. RESULTS: Fifty-nine (49.2%) yeast colonies were identified from the total of 120 BAL specimens. Twenty-nine isolated yeasts; including 17 (58.6%) of C. albicans/dubliniensis complex and 12 (41.4%) of nonalbicans isolates produced pseudohypha or blastoconidia in direct smear with a mean colony count of 42000 CFU/mL. C. albicans with the frequency of 15 (42.9%) were the most common isolated yeasts, whereas C. dubliniensis was identified in two nonHIV patients. CONCLUSION: Sequencing of the replicated gene fragment is the best method for identifying the yeasts, but the determination of the species by phenotypic methods such as the creation of chlamydoconidia in sunflower seeds agar and wheat flour agar media can be cost-effective, have sensitivity and acceptable quality.

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

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