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IntroductionEarly detection of Pneumocystis jirovecii as an opportunistic pathogen that may endanger predisposed persons, including COVID-19 patients, may help to choose the optimal management.MethodsIn this study, 585, including 530 COVID-19 patients, with clinical and radiological evidence of respiratory diseases, were investigated for P. jirovecii screening. Clinical specimens were examined by direct microscopy and PCR, and randomly selected positive PCR products were confirmed through DNA sequence analysis.ResultsThirty-one (5.3%) samples were positive in P. jirovecii-specific nested-PCR, while by direct microscopic tests, Pneumocystis was observed in 22 (3.76%) samples. Males (61.7%) and patients over 50 years old (75.6%) were more commonly affected than others, and malaise and fatigue (84%), and wheezing (75%) were the most common symptoms, followed by fever (40.48%) and dyspnea (39.51%). Among the Pneumocystis-positive patients, three cases had coinfection with Aspergillus fumigatus, A. flavus, and A. niger (each n = 1), as documented by direct microscopy, culture, and species identification by PCR-sequencing.ConclusionPneumocystis pneumonia is still a diagnostic challenge; therefore, additional large-scale studies are needed to clarify the epidemiology of the disease in immunocompromised or COVID-19 patients.

Candida meningitis is a rare life-threatening yeast infection mostly involving immunocompromised or paediatric patients undergoing neurosurgical procedures or shunt placement. Due to difficulties in diagnosis because of diverse clinical manifestations, the number of patients affected is most likely underestimated. Therefore, the correct diagnosis may be delayed for months, and accurate species identification is highly recommended for administering appropriate antifungal therapy. We report the first case of fluconazole-resistant Candida auris meningitis in a paediatric patient in Iran. This strain was probably imported, as it genotypically belonged to Clade I from South Asia. Furthermore, we include a literature review of C auris meningitis cases, as the number of cases with C auris meningitis has increased with reports from the United Kingdom, India and Iran. This problem might increase further in the era of COVID-19 due to attrition of experienced healthcare personnel and a high workload of hospital healthcare workers. To understand the precise prevalence of this emerging multidrug resistance pathogen, epidemiological surveillance studies are urgently warranted.

A fatal case of COVID-19-associated mucormycosis (CAM) affected a 40-year-old woman who was initially admitted to our hospital due to a SARS-CoV-2 infection. Her clinical condition worsened, and she finally died because of respiratory failure, hemodynamic instability, and mucormycosis with invasion into the orbit and probably the brain. According to DNA sequence analysis of the fungus isolated from the patient, Apophysomyces variabilis was involved. This is the first published case of CAM and the third case of mucormycosis due to this mold.

Nowadays apparently harmless Candida species especially rare ones, cause a rising number of infections. We report the first case of conjunctivitis due to Candida metapsilosis obtained from the eye discharge of 40-day-old girl with congenital heart disease admitted in the ICU. The isolate was resistant to fluconazole, anidulafungin and micafungin.

Aim: This study aimed to investigate the prevalence of coronavirus disease 2019 (COVID-19) among healthcare workers (HCWs) in Isfahan, Iran. Materials & methods: HCWs in COVID-19 wards of three referral COVID-19 hospitals in Isfahan were screened and tested for COVID-19 infection. Results: In total, 102 HCWs were screened whose median age was 43 years old. Moreover, 21 (20.5%) of them had a history of suspected infection with SARS-CoV2, mostly (66.6%) without any symptoms while six (28.5%) of them suffered from relatively mild diseases and one (4.7%) was diagnosed with pulmonary embolism. Conclusion: It was found that HCWs were prone to be asymptomatic carriers while their computed tomography images were normal. Therefore, it is recommended that reverse-transcriptase real-time-PCR be essential for the diagnosis of infections.

The incidence of invasive candidiasis in pediatric patients is increasing and is associated with significant morbidity and mortality. C. pelliculosa has been rarely reported as a human pathogen, however, it has been associated with serious nosocomial infections and clonal outbreaks with poor clinical outcomes in immunocompromised children were reported. Here, we describe the first case of candidemia due to Candida pelliculosa in a 5-year-old immunocompromised male suffered from Griscelli syndrome with hemophagocytic syndrome hospitalized in the pediatric intensive care unit (PICU), Tehran, Iran. In addition, the history of reported cases or case-series due to C. pelliculosa is reviewed.

Background: There is an increasing incidence of life-threatening systemic mycoses, specifically fulminant infections by the Candida species in hospitalised patients and in those who are immunocompromised. Management of the limited number of antifungal drugs currently available requires the identification of infections containing drug-resistant isolates. Objectives: The aim of this study was to identify the non-albicans Candida species as azole-resistant fungi, isolated from sputum and bronchoalveolar lavage specimens of hospitalised cases. Methods: The subjects included hospital-acquired infection (HAI) cases, with a primary diagnosis using a direct microscopic examination, performed for the detection of probable fungi. The molecular tests of PCR-restriction fragment length polymorphism (RFLP) and real-time PCR were performed to confirm the identity and molecular typing of the Candida isolates. Antifungal susceptibility testing (AFST), by the Clinical and Laboratory Standards Institute (CLSI) broth microdilution (BMD) minimum inhibitory concentration (MIC) (M27-A2) method, was performed on the hospital-isolated Candida species. Results: During 24 months, from August 2014 to September 2016, a total of 198 samples were obtained from cases with proven HAI. The results of experimental studies on the specimens showed 93 (47%) positive cases for a fungal or bacterial infection, of which 54 (58%) had a fungal infection. It was hypothesised that all of the isolated organisms were causative agents of the HAI. Conclusions: The results showed that the medium CHROMagar™ Candida is an accessible and easy-to-use method for the identification of infection, but not as accurate and reliable as PCR-RFLP and real-time PCR methods. Results also showed decreasing susceptibility to azoles (itraconazole in this study) of the Candida species.

Background Candida species cause a wide spectrum of disease entities. Candida africana and Candida dubliniensis–members of Candida albicans complex-are currently gaining both clinical and epidemiologic significance. Materials and methods Totally, 150 pediatric isolates that had previously been identified as C. albicans species complex based on a positive germ tube test were included. The isolates were cultured on CHROMagar Candida medium to ensure their purity and the results of germ tube test. For definitive speciation, PCR amplification and size polymorphism of the hyphal wall protein 1 (HWP1) gene was used. The results of HWP1-PCR were confirmed by sequencing the amplified fragments for randomly selected isolates of C. africana and C. dubliniensis. Results All 150 isolates included in this study were reconfirmed as C. albicans complex on chromogenic media. Based on the HWP1 gene size polymorphism, 141 (94%) isolates were identified as C. albicans, 2 (1.33%) as C. africana, and 1 (0.67%) as C. dubliniensis. The remaining 6 (4%) C. albicans complex isolates were a mix of C. albicans and C. africana. All isolates of C. africana and C. dubliniensis have been recovered from cases of candiduria. Conclusion C. africana, either alone or mixed with C. albicans, could be a cause of candiduria among pediatric patients and should not be ignored.

Multiple yeast species can cause human disease, involving superficial to deep-seated infections. Treatment of these infections depends on the accurate identification of causative agents; however, reliable methods are not available in many laboratories, especially not in resource-limited settings. Here, a new multiplex assay for rapid and low-cost identification of pathogenic yeasts is described.A two-step multiplex assay named YEAST PLEX that comprises of four tubes and identifies 17 clinically important common to rare yeasts was designed and evaluated. The set also provides PCR amplicon of unidentified species for direct sequencing. The specificity of YEAST PLEX was tested using 28 reference strains belonging to 17 species and 101 DNA samples of clinically important non-target bacteria, parasites, and fungi as well as human genomic DNA. The method was further analyzed using 203 previously identified and 89 unknown clinical yeast isolates. Moreover, the method was tested for its ability to identify mixed yeast colonies by using 18 mixed suspensions of two or three species.YEAST PLEX was able to identify all the target species without any non-specific PCR products. When compared to PCR-sequencing/MALDI-TOF, the results of YEAST PLEX were in 100% agreement. Regarding the 89 unknown clinical isolates, random isolates were selected and subjected to PCR-sequencing. The results of sequencing were in agreement with those of YEAST PLEX. Furthermore, this method was able to correctly identify all yeasts in mixed suspensions.YEAST PLEX is an accurate, low-cost, and rapid method for identification of yeasts, with applicability, especially in developing countries.

Background: Tracheoesophageal voice prostheses (TVPs) have been the gold standard in rehabilitation, after laryngectomy, producing faster and premier voicing towards esophageal speech. Fungal colonization shortens the device’s lifetime and leads to prosthesis dysfunction, leakage, and subsequent respiratory infection. Therefore, in the current study, we aimed to investigate the fungal colonization patterns and to propose prophylactic measures that shall increase the longevity of voice prosthesis. Methods: Failed TVPs were removed - due to leakage and/or aspiration - from 66 post laryngectomy patients and examined. They were referred to Amiralam and Rasoul Hospital, the main centers of Ear, Nose, and Throat in Tehran, Iran from April 2018 to January 2020. Fungal colonization patterns were assessed using DNA sequencing techniques. Furthermore, the susceptibility to fluconazole, amphotericin B, nystatin, and white vinegar was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Results: Resident fungal species from the upper airways colonized all the 66 TVPs (100%). Diabetes (31%) and smoking (98%) were the predominant underlying disease and predisposing factors, respectively. Among the 79 fungal agents isolated from the 66 TVPs, Candida glabrata (n=25, 31.7%) was the most common. A significant reduction in minimum inhibitory concentration (MIC) values were observed for white vinegar when used alone (P

Background and Purpose: The most common etiological agents of human dermatophytosis in various parts of the world are Trichophyton rubrum, Trichophyton interdigitale, and Epidermophyton floccosum. The main aim of this study was to design and evaluate a simple and straightforward multiplex polymerase chain reaction (PCR)assay for reliable identification/differentiation of these species in clinical isolates. Materials and Methods: The reliable sequences of several molecular targets of dermatophytes species were used to design a multiplex PCR for the identification of common pathogenic dermatophytes. The isolates and clinical specimens examined in this study included seven standard strains of dermatophytes, 101 isolates of dermatophytes and non-dermatophyte molds/yeasts which had already been identified by sequencing or PCR-restriction fragment length polymorphism (RFLP), and 155 clinical samples from patients suspected of cutaneous mycoses. Results: Species-specific primer pairs for T. rubrum and T. interdigitale/T. mentagrophytes were designed based on the sequence data of the translation elongation factor 1-alpha gene, and the primers for E. floccosum targeted the specific sequence of the internal transcribed spacer region (ITS). The multiplex PCR successfully detected T.rubrum, T. interdigitale/T. mentagrophytes, and E. floccosum strains that were identified by sequencing or PCR-RFLP. However, the primer pairs selected for T. interdigitale/T. mentagrophytes cross-reacted with Trichophyton tonsurans. In testing the PCR system directly for clinical samples, the proportion of positive multiplex PCR was higher than positive culture (68.1% vs. 55.4%, respectively). Conclusion: The multiplex assay could detect three common agents out of several causal agents of dermatophytosis, namely T. rubrum, T. interdigitale, and E. floccosum.Therefore, by adding pan-dermatophyte primers it can be used as a comprehensive detection/identification test.

Background and Purpose: Taxonomy of Candida is controversial and has changed due to the investigation of the novel species. Candida africana and Candida dubliniensis are new members of the C. albicans complex that are currently gaining both clinical and epidemiologic significance. This study aimed to report the prevalence of C. africana among the strains isolated from patients using hyphal wall protein 1 (HWP1) gene size polymorphism. Materials and Methods: In total, 235 yeasts confirmed as C. albicans complex based on chromogenic media and internal transcribed spacers sequencing isolated from various clinical forms of invasive and non-invasive candidiasis mainly candidemia were re-identified using HWP1 gene polymorphisms. The HWP1-polymerase chain reaction amplicons were re-confirmed by sequencing and BLAST analysis. Results: Based on the HWP1 gene size polymorphism, 223 strains were identified as C. albicans (94.89%) from which 7 isolates produced two DNA fragments (850 and 941 bp). The C. dubliniensis (n=4, 1.7%), C. africana (n=1, 0.42%), and mix of C. albicans and C. africana (n=7, 2.97%) were also identified. Conclusion: It can be said that C. albicans remains the most common Candida species, while C. dubliniensis and C. africana are rarely found among the patient isolates. Due to limited information on the molecular epidemiology of this novel yeast, more studies using molecular methods are recommended.

Purpose To elucidate the clinical and microbial epidemiology of otomycosis in Isfahan, Iran. Methodology From January 2016 to January 2017 all patients clinically suspected of otomycosis at Al-Zahra Hospital, Isfahan, Iran were recruited. Specimens were taken using sterile swabs by an otorhinolaryngologist and subjected to culture and microscopy using potassium hydroxide and Giemsa stain. Isolated fungi were identified based on morphological and molecular characteristics. Results Otomycosis was confirmed in 97/120 patients (80.8 %). Females (72.2 %) and patients aged 30-39 years (33 %) were more commonly affected than others. Manipulation of ear canal (62.9 %) was the most common predisposing factor. Pruritus was observed in 84.54 % of the patients followed by hearing impairment (81.4 %), and most episodes were detected over the summer (50.5 %). Culture was positive for 81 (83.5 %) of confirmed cases and molds were the most prevalent causative agents (n=51, 63 %) followed by yeasts (n=19, 23.4 %) and yeast/mold mixes (n=11, 13.6 %). For the 16 remaining patients, no growth was seen in culture despite a positive result on direct examination. In total, 92 isolates (63 molds and 29 yeasts) were recovered in culture. Application of molecular methods showed 18 fungal species and the vast majority of them belonged to Aspergillus (n=53, 57.6 %) and Candida genus. Among the species involved, Candida parapsilosis (n=22, 22.7 %) and Aspergillus tubingensis (n=15, 15.5 %) were the most encountered species. Conclusion Outcomes from this study showed a different picture of prevalence, where C. parapsilosis and A. tubingensis but not Aspergillus niger were the most species encountered from patients suffering from otomycosis.

Early and cost-effective diagnosis and monitoring of the infection caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are critically important to anticipate and control the disease. We aimed to set up a SYBR Green-based one-step real-time polymerase chain reaction (PCR) as a lower-cost alternative method to detect the virus.An in-house SYBR Green-based PCR assay targeting the envelope (E) and RNA-dependent RNA polymerase (RdRp) genes, was set up to diagnose the infection, and was compared with the reference probe-based PCR method.When the commercial probe-based assay was considered as the reference method, SYBR Green-based PCR had a slightly lower sensitivity (81.98% and 86.25% for E and RdRp targets, respectively) and a good specificity (100% and 94.44% for E and RdRp targets, respectively). For both gene targets, three different melting temperature (Tm) patterns were found in the PCRs of the nasopharyngeal/oropharyngeal swab samples, but no size polymorphism was seen in agarose gel electrophoresis.Further studies to improvement of the assay are needed to make it an inexpensive and reliable tool for the diagnosis of COVID-19.

Introduction: Fusarium species are hyaline saprophytic fungi that are frequently found in the soil, air, and water. They can cause severe systemic infections in immunocompromised patients. Clinical manifestations depend on the way of entry of the mold and host immune system status. The main ways of entrance are the airways, skin, and mucosal membranes. Disseminated fusariosis often occurs in patients with hematological disorders, patients with cancer, and solid organ transplant recipients. Case Presentation: Herein we report a case of Fusarium fungemia in a patient with adrenocortical carcinoma from Isfahan, Iran. The patient was a 41-year-old female with stage III adrenal cortical carcinoma. Despite antifungal therapy with liposomal amphotericin B, the patient passed away 6 days after admission. Internal transcribed spacer region sequencing applied for species identification and its sequence deposited in the GenBank (accession number: MK880379). Conclusions: Since the ideal strategies against invasive fungal infections remain uncertain and the mortality rate is high, we recommend primary prophylaxis with a broad-spectrum antifungal agent for vulnerable patients particularly those admitted to high-risk units such as oncology, hematology, and transplant units.

Otomycosis is a common finding in otorhinolaryngology clinics and is usually caused by species of Candida and Aspergillus, particularly black aspergilli. Meanwhile, other fungi can give rise to this infection, and the identification of these requires accurate methods. Here, we report three cases of otomycosis due to rare fungal pathogens. All the patients were young females, and manipulation of the ear canal was identified as a common potentially predisposing factor. In direct examination, filamentous fungal elements (in one case) and yeast cells (in two other cases) were seen. Culture was positive in all cases. Based on PCR-sequencing of internal transcribed spacers and β-tubulin (for mold isolate), the isolated fungi were identified as Talaromyces purpurogenus, Naganishia albida and Filobasidium magnum. By susceptibility testing of the isolates to fluconazole, itraconazole, voriconazole and amphotericin B, the lowest minimum inhibitory concentration values were observed for amphotericin B followed by voriconazole. Patients were successfully treated by a combination of antifungals and corticosteroids with no relapse over the next year, except for the case due to F. magnum, in which, despite partial recovery, a course of relapse was reported in the 1-year follow-up call.

Aspergillus infection is a well-known complication of severe influenza and severe acute respiratory syndrome coronavirus (SARS-CoV), and these infections have been related with significant morbidity and mortality even when appropriately diagnosed and treated. Recent studies have indicated that SARS-CoV-2 might increase the risk of invasive pulmonary aspergillosis (IPA). Here, we report the first case of Aspergillus ochraceus in a SARS-CoV-2 positive immunocompetent patient, which is complicated by pulmonary and brain infections. Proven IPA is supported by the positive Galactomannan test, culture-positive, and histopathological evidence. The patient did not respond to voriconazole, and liposomal amphotericin B was added to his anti-fungal regimen. Further studies are needed to evaluate the prevalence of IPA in immunocompetent patients infected with SARS-CoV-2. Consequently, testing for the incidence of Aspergillus species in lower respiratory secretions and Galactomannan test of COVID-19 patients with appropriate therapy and targeted anti-fungal therapy based on the primary clinical suspicion of IPA are highly recommended.