Yeast species in the respiratory samples of COVID-19 patients; molecular tracking of Candida auris.

Introduction: Although the existence of Candida species in the respiratory tract is often considered commensal, it is crucial to recognize the significance of Candida colonization in immunocompromised or COVID-19 patients. The emergence of Candida auris as an emerging pathogen further emphasizes the importance of monitoring yeast infection/colonization, particularly in COVID-19 patients. Methods: In this study, respiratory samples mainly from COVID-19 patients, primarily those suspected of having a fungal infection, were cultured on Sabouraud dextrose agar plates and the yeast colonies were identified using a two-step multiplex PCR method. The samples suspected of C. auris underwent specific nested PCR followed by sequence analysis. Results: A total of 199 respiratory samples were collected from 73 women and 126 men, ranging in age from 1.6 to 88 years. Among the patients, 141 had COVID-19, 32 had cancer, 5 were hospitalized in ICU, 2 had chronic obstructive pulmonary disease)COPD(, and others were patients with combination diseases. From these samples, a total of 334 yeast strains were identified. C. albicans (n=132, 39.52%) was the most common species, followed by C. tropicalis (n=67, 20%), C. glabrata (n=56, 16.76%), C. krusei (n=18, 5.4%), C. parapsilosis (n=17, 5.08%), Saccharomyces cerevisiae (n=10, 3%), C. kefyr (n=9, 2.6%), C. dubliniensis (n=7, 2.1%), C. lusitaniae (n=5, 1.5%), C. auris (n=3, 0.9%), C. guilliermondii (n=2, 0.6%), C. rugosa (n=1, 0.3%), C. intermedia (n=1, 0.3%), and Trichosporon spp. (n=1, 0.3%). C. auris was detected in a patient in ICU and two COVID-19 patients. While its presence was confirmed through sequence analysis, our extensive efforts to isolate C. auris were unsuccessful. Conclusion: While C. albicans colonization remains prevalent, our study found no evidence of Candida lung infection. Since the role of Candida colonization in airway secretions remains ambiguous due to limited research, further studies are imperative to shed light on this matter.

COVID-19 associated mucormycosis (CAM) in Kashan, Iran: clinical presentations, risk factors, management, and outcomes.

BACKGROUND: This study aimed to estimate the disease burden and describe the clinical presentation, risk factors, and outcome of CAM in a single centre in Iran. METHODS: A case of mucormycosis was defined as one that had clinical and radiological features consistent with mucormycosis along with demonstration of the fungus in tissue via KOH mount/culture/histopathological and molecular examination. RESULTS: We report 30 cases of COVID-19 associated mucormycosis (CAM). The results of this study showed the affected age group in the range of 40-79 years (median = 65.5; IQR = 5) with women (16/30, 53%) affected more than men (14/30, 47%). Among the fungi recovered, Rhizopus oryzae had the highest frequency (79%). Out of the 30 patients, 28 (93%) patients were diabetic with 24 (80%) patients having other co-morbidities. Headache followed by retro-orbital pain, proptosis/ptosis and rapid diminution of vision was a common sequence of symptoms reported by the majority of cases. Use of mechanical ventilation (58% vs. 6%, p = 0.003), O2 required (92% vs. 50%, p = 0.024), and development of renal dysfunction during hospital stay (17% vs. 0%, p = 0.041) was significantly higher in non-survivors than survivors. Temperature (C°), PR (pulse rate), mean levels of serum creatinine, BUN, troponin, and neutrophils were significantly higher in non-survivors (p < 0.05). Besides, Albumin and PO2 were also significantly higher in survivors than non-survivors. CONCLUSION: Despite medical and surgical treatment, the mortality rate among CAM patients is still high. Thus, concerted efforts of revamping surveillance, diagnosis and management, along with public awareness and patient education, are the requisites for managing COVID-19 and mucormycosis.

Molecular and Antigen Detection, Phylogenetics, and Immunoinformatics Study of the Zoonotic Coronavirus in Iranian Diarrheic Calves.

Background: Bovine coronaviruses (BCoVs) are zoonotic diseases that result in substantial economic losses due to mortality, impaired growth, and increased medication expenses in large animals. These viruses pose a risk to children who live beside infected animal, as they can cause diarrhea. This study was dedicated to molecular and antigen detections and phylogenetic and immunoinformatics analysis of zoonotic coronavirus (CoV) in Iran. Materials and Methods: A total of 77 diarrheic samples were collected from Holstein dairy herds in selected provinces of Iran. Samples were tested by capture antibody enzyme-linked immunosorbent assay (ELISA) to detect CoV and reverse transcriptase-polymerase chain reaction (RT-PCR) for verification of detection and also genotyping of spike glycoprotein in CoV-positive samples. After statistical analysis, nucleotide sequence alignment, and nucleotide and protein phylogenetic tree construction, the centralized sequence for vaccine strains was obtained using computationally optimized broadly reactive antigen (COBRA)'s center-of-the-tree (COT) method. Results: Twenty-two (28.5%) and eight (10.3%) of 77 samples were positive according to RT-PCR and ELISA, respectively. (Basic Local Alignment Search Tool) BLAST and phylogenetic analysis revealed that most similar sequences to the Iranian CoV sequence were for European countries. Furthermore, there were strong correlations to other CoVs in humans and wild and domesticated animals. As CoV has variable COT, the most recent strains and COBRA vaccine strains were obtained. Conclusion: Based on the high prevalence of this viral disease in calves and its economic impact on the breeding industry, as well as the potential transmission to humans and correlation with World Health Organization (WHO) One Health approach guidelines, the study emphasizes the importance of implementing preventive strategies such as animal vaccination.

Candida palmioleophila candidemia and bacterial co-infection in a 3-month-old infant with biliary atresia.

Candidemia caused by rare and uncommon Candida species is becoming more prevalent in pediatric healthcare settings, resulting in significant morbidity and mortality. One such species, Candida palmioleophila, is resistant to fluconazole but highly susceptible to echinocandins. Here, we report the first documented case of C. palmioleophila candidemia in Iran that occurred in a male infant with biliary atresia who had been hospitalized for 2 months. The patient's blood and urine cultures were positive for both yeast and bacterial species. Through DNA sequence analysis, the yeast isolate was identified as C. palmioleophila. In vitro antifungal susceptibility testing of the isolate against amphotericin B, fluconazole, itraconazole, voriconazole, isavuconazole, posaconazole, and nystatin revealed MIC values of 2, 16, 0.25, 0.0625, 0.125, 0.25, and 4 µg/mL, respectively, and minimum effective concentration for caspofungin was 0.031 µg/mL. Despite receiving antibacterial and antifungal therapies, the patient unfortunately expired due to bradycardia and hypoxemia. Proper identification and epidemiological surveillance studies are needed to understand the exact prevalence of these emerging yeast pathogens. Previously reported cases of C. palmioleophila infection, primarily associated with bloodstream infections and catheter-related candidemia, were reviewed.

Sensitive detection of specific cell-free DNA in serum samples from sheep with cystic echinococcosis.

BACKGROUND: Developing more sensitive methods for the diagnosis of echinococcosis is essential. In this study PCR assay for sensitive detection of specific cell-free DNA (cfDNA) of Echinococcus granulosus sensu lato in the sera of the sheep naturally infected with echinococcosis was investigated. METHODS: To extract cfDNA from 35 infected sheep, the modified phenol-chloroform method was used for two different volumes (0.5 and 2 ml) of serum samples. From each extracted sample, two DNA volumes (5 and 10 µl) were amplified using both standard PCR and semi-nested PCR targeting NADH dehydrogenase subunit I. RESULTS: Standard and semi-nested PCR on 0.5 ml of serum samples detected Echinococcus DNA in 8 and 12 out of 35 sheep, respectively; however, using 2 ml of serum samples, they detected 24 and 27 samples. By increasing the volume of template DNA, the PCRs could detect 29 and 33 out of 35 samples. The results were confirmed by sequencing of randomly selected PCR amplicons and comparing them with GenBank databases. CONCLUSIONS: Larger volumes of serum for DNA extraction, greater volumes of DNA template for PCR, and employing a semi-nested PCR protocol, increased the sensitivity of PCR to 95%. This approach can also be applied to the diagnosis of echinococcosis in humans.

Invasive Fusarium rhinosinusitis in COVID-19 patients: report of three cases with successful management.

Invasive fungal rhinosinusitis (IFRS) is a life-threatening infection that can occur in immunocompromised patients, including those with COVID-19. Although Mucorales and Aspergillus species are the most common causes of IFRS, infections caused by other fungi such as Fusarium are rare. In this report, we present three cases of proven rhinosinusitis fusariosis that occurred during or after COVID-19 infection. The diagnosis was confirmed through microscopy, pathology, and culture, and species identification of the isolates was performed by DNA sequencing the entire ITS1-5.8 rRNA-ITS2 region and translation elongation factor 1-alpha (TEF-1α). Antifungal susceptibility testing was conducted according to CLSI guidelines. The causative agents were identified as Fusarium proliferatum, F. oxysporum + Aspergillus flavus, and F. solani/falciforme. Treatment involved the administration of antifungal medication and endoscopic sinus surgery to remove the affected mucosa, leading to the successful resolution of the infections. However, one patient experienced a recurrence of IFRS caused by A. flavus 15 months later. Early diagnosis and timely medical and surgical treatment are crucial in reducing mortality rates associated with invasive fusariosis. Additionally, the cautious use of corticosteroids in COVID-19 patients is highly recommended.

Diagnosis of mucormycosis using a simple duplex PCR assay: Analysis of 160 clinical samples from COVID-19 patients.

Early diagnosis of mucormycosis, a severe and potentially fatal complication in immunocompromised and COVID-19 patients, is crucial for initiating timely antifungal therapy and reducing infection mortality. In this study, the diagnostic performance of a duplex polymerase chain reaction (PCR) assay was evaluated to detect Mucorales-specific and Rhizopus oryzae-specific targets in 160 clinical samples collected from 112 COVID-19 patients suspected of invasive fungal rhinosinusitis (IFRS). During potassium hydroxide (KOH) direct microscopy, non-septate hyphae were observed in 73 out of 160 samples (45.63%); however, using duplex PCR, 82 out of 160 specimens (51.25%) tested positive. Among the positive PCR samples, 67 (81.71%) exhibited a double band (both 175 and 450 base pairs [bp]) indicating the presence of R. oryzae, and 15 (18.29%) showed only a single band (175 bp), suggesting the presence of non-R. oryzae Mucorales. DNAs from 10 microscopically negative samples and 4 samples with septate hyphae in microscopy were successfully amplified in PCR. Considering Calcofluor white fluorescence microscopy as the gold standard for laboratory diagnosis of mucormycosis, the duplex PCR assay utilized in this study exhibited a sensitivity of 93.88%, a specificity of 100%, a negative predictive value of 91.18%, and a positive predictive value of 100% for detecting mucormycosis in IFRS specimens. The duplex PCR assay demonstrated higher sensitivity compared to direct examination with KOH (82 vs. 73) and culture (82 vs. 41), enabling rapid detection/identification of Mucorales even in samples with negative culture or in biopsies with only a few hyphal elements.

Early diagnosis of mucormycosis, a severe complication in COVID-19 patients, is critical for reducing the mortality of the infection. In this study, a sensitive and rapid PCR assay to detect all Mucorales and delineate Rhizopus oryzae was developed and assessed to improve the diagnosis of mucormycosis.

Molecular strategy for the direct detection and identification of the most common fungal community in cerumen specimens by multiplex PCR.

Introduction. Otomycosis is a superficial fungal infection that is responsible for approximately 9-27 % of otitis externa. However, fungal communities in otomycosis are varied, but Aspergillus spp. and Candida spp. are the most common causes of this infection.Hypothesis Statement. The multiplex PCR assay is postulated to be able to directly detect more than one fungal genus in cerumen specimens.Aim. This study aimed to develop and evaluate the role of the multiplex PCR assay in detecting the most common genus of fungi that cause otomycosis directly from the cerumen specimens.Methodology. To detect Candida and Aspergillus/Penicillium genera, three pairs of primers, including pan-fungal, pan-Candida, and pan-Aspergillus/Penicillium, were used in a multiplex PCR. In order to evaluate the performance and reproducibility of the multiplex PCR. the cerumen of 140 patients suspected of otomycosis were investigated.Results. Pan-Candida and pan-Aspergillus/Penicillium primers were designed to amplify the ITS1-5.8S-ITS2 region and the ß-tubulin gene, respectively. In the multiplex PCR assay, 64 (47.40 %) and 118 (87.40 %) specimens were positive with pan-Candida and pan-Aspergillus/Penicillium primers, respectively. Double amplicon bands of Candida and Aspergillus were obtained in 51 (37.77 %) specimens. In the culture method, yeast (n=18, 13.33 %) and mould (n=117, 86.66 %) were isolated from 135 cerumen specimens. The sensitivity, specificity, and positive and negative predictive values of the multiplex PCR assays using culture method results as the gold standard were determined to be 94, 33, 97, and 22 %, respectively.Conclusion. In our study, multiplex PCR assays enabled simultaneous detection of two common genera of the causative agent of otomycosis in a cerumen specimen. Regarding the high sensitivity of the first step of the multiplex PCR assay, this assay may be used for the direct detection of Candida and Aspergillus genera in other clinical specimens.

A case of COVID-19-associated mucormycosis due to Lichtheimia ramosa.

BACKGROUND: Mucormycosis is a life-threatening invasive fungal infection in immunocompromised and COVID-19 patients. CASE REPORT: Here, we report a fatal rhino-orbito-cerebral mucormycosis caused by Lichtheimia ramosa, in a 79-year-old diabetic female. She was initially admitted to the hospital for COVID-19 infection and received broad-spectrum antibiotics and corticosteroids. After 1 month, she was admitted again because of persistent headaches and decreased right eye movement when the computed tomography scan showed mucosal thickening and opacification of paranasal sinuses. Microbiological investigations, including culture and direct microscopy, and histopathological findings confirmed the diagnosis of proven mucormycosis. The isolated causal agent was identified as Lichtheimia ramosa by sequencing the entire ITS region of nuclear ribosomal DNA. Despite surgical debridement and administration of liposomal amphotericin B 5 mg/kg/day, the patient's level of consciousness suddenly deteriorated; she was intubated and mechanically ventilated in the ICU and died on the same day. CONCLUSION: To our knowledge, this is the first worldwide case of COVID-19-associated rhino-orbito-cerebral mucormycosis due to Lichtheimia ramosa.

Molecular and microscopy detection of Pneumocystis jirovecii in hospitalized patients during the COVID-19 pandemic.

Introduction: Early detection of Pneumocystis jirovecii as an opportunistic pathogen that may endanger predisposed persons, including COVID-19 patients, may help to choose the optimal management. Methods: In 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. Results: Thirty-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. Conclusion: Pneumocystis 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.

Case report: COVID-19-associated mucormycosis co-infection with Lomentospora prolificans: The first case and review on multiple fungal co-infections during COVID-19 pandemic.

Along with the pandemic COVID-19 spreads, new clinical challenges have emerged in the health care settings, among which there is a high risk of secondary invasive fungal infections with significant mortality. Here, we report a case of invasive fungal rhino orbital sinusitis due to the simultaneous co-infection by Rhizopus oryzae and Lomentospora prolificans, both identified by sequencing, in a 70-year-old Afghanistanian female with COVID-19. The patient was subjected to surgical debridement as well as taking liposomal amphotericin B, voriconazole, and on discharge, her condition was good. As far as we know, this is the first case of co-infection of COVID-19-associated mucormycosis (CAM) and Lomentospora prolificans infection. Multiple fungal co-infections in COVID-19 patients are reviewed.

Microbial and clinical epidemiology of invasive fungal rhinosinusitis in hospitalized COVID-19 patients, the divergent causative agents.

Since COVID-19 spread worldwide, invasive fungal rhinosinusitis (IFRS) has emerged in immunocompromised patients as a new clinical challenge. In this study, clinical specimens of 89 COVID-19 patients who presented clinical and radiological evidence suggestive of IFRS were examined by direct microscopy, histopathology, and culture, and the isolated colonies were identified through DNA sequence analysis. Fungal elements were microscopically observed in 84.27% of the patients. Males (53.9%) and patients over 40 (95.5%) were more commonly affected than others. Headache (94.4%) and retro-orbital pain (87.6%) were the most common symptoms, followed by ptosis/proptosis/eyelid swelling (52.8%), and 74 patients underwent surgery and debridement. The most common predisposing factors were steroid therapy (n = 83, 93.3%), diabetes mellitus (n = 63, 70.8%), and hypertension (n = 42, 47.2%). The culture was positive for 60.67% of the confirmed cases, and Mucorales were the most prevalent (48.14%) causative fungal agents. Different species of Aspergillus (29.63%) and Fusarium (3.7%) and a mix of two filamentous fungi (16.67%) were other causative agents. For 21 patients, no growth was seen in culture despite a positive result on microscopic examinations. In PCR-sequencing of 53 isolates, divergent fungal taxons, including 8 genera and 17 species, were identified as followed: Rhizopus oryzae (n = 22), Aspergillus flavus (n = 10), A. fumigatus (n = 4), A. niger (n = 3), R. microsporus (n = 2), Mucor circinelloides, Lichtheimia ramosa, Apophysomyces variabilis, A. tubingensis, A. alliaceus, A. nidulans, A. calidoustus, Fusarium fujikuroi/proliferatum, F. oxysporum, F. solani, Lomentospora prolificans, and Candida albicans (each n = 1). In conclusion, a diverse set of species involved in COVID-19-associated IFRS was observed in this study. Our data encourage specialist physicians to consider the possibility of involving various species in IFRS in immunocompromised and COVID-19 patients. In light of utilizing molecular identification approaches, the current knowledge of microbial epidemiology of invasive fungal infections, especially IFRS, may change dramatically.

Invasive fungal rhinosinusitis (IFRS) may infect people with diabetes, cancer, or COVID-19. In this study, various types of fungi were identified from COVID-19-associated-IFRS, encouraging physicians to consider specific treatments.

A case of Candida metapsilosis conjunctivitis in a neonate admitted to the cardiac heart intensive care unit.

Harmless commensal Candida species, especially uncommon and rare ones may rarely cause a serious infection. Candida metapsilosis is a recently described yeast that is phenotypically indistinguishable from Candida parapsilosis and molecular methods are essential for its identification. We report the first case of Candida conjunctivitis due to C. metapsilosis obtained from the eye discharge of a 40-day-old girl with congenital heart disease admitted to the cardiac intensive care unit (CICU). The yeast isolate was identified by sequencing the entire ITS1-5.8 rRNA-ITS2 region. Antifungal susceptibility testing performed according to the CLSI M27-A3 showed that the isolate was susceptible to amphotericin B, fluconazole, itraconazole, voriconazole, clotrimazole, nystatin, terbinafine, 5-fluorocytosine, and caspofungin. Differentiation of the fungal new species allows us the accurate diagnosis and treatment, and a better understanding the microbial epidemiology.

The first case of Wickerhamomyces anomalus fungemia in Iran in an immuneodeficient child, a review on the literature.

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.

A case of COVID-19-associated rhino-orbito-cerebral mucormycosis caused by Apophysomyces variabilis with a review of the literature.

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.

Melting Curve-based Assay as an Alternative Technique for the Accurate Detection of SARS-CoV-2.

Background: 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. Materials and Methods: 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. Results: 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. Conclusion: Further studies to improvement of the assay are needed to make it an inexpensive and reliable tool for the diagnosis of COVID-19.

Multiplex size marker (YEAST PLEX) for rapid and accurate identification of pathogenic yeasts.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: YEAST PLEX is an accurate, low-cost, and rapid method for identification of yeasts, with applicability, especially in developing countries.

Molecular Characterization of Fungal Colonization on the Provox™ Tracheoesophageal Voice Prosthesis in Post Laryngectomy Patients.

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<0.05). CONCLUSION: White vinegar at a very low concentration could decrease the amount of fungal colonization on TVPs without any adverse effects; its wide accessibility and affordability ensure a decrease in the overall health cost.

South Asian (Clade I) Candida auris meningitis in a paediatric patient in Iran with a review of the literature.

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.

Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR.

Background: Considering the importance of differential diagnosis of infectious otitis externa (OE), a stepwise PCR-based assay using universal and genus- or species-specific primers for the detection/identification of the most prevalent bacterial and fungal OE was developed and evaluated on the ear aspiration specimens of clinically suspected patients. Methods and Materials: A total of 120 ear aspiration specimens with otomycosis suspicion were subjected to manual DNA extraction using phenol-chloroform extraction after tissue digestion with a lysis buffer. The multiplex PCR was initially performed using pan-fungal and bacterial homemade primers. Pseudomonas and Staphylococcus specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents, Candida species-specific multiplex primers targeting the most clinically important Candida species causing OE (i.e., C. albicans, C. parapsilosis, and C. auris), as well as Aspergillus related multiplex PCR identifying the most prevalent Aspergillus species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size. Results: The overall multiplex PCR-based detection rate of bacterial (n = 88; 73.3%) and fungal (n = 97; 81%) OE was documented to be 100% along with and complete consistency with the results of direct examination and Giemsa staining. Double amplicon bands of bacterial and fungal pathogens were evidenced in 76 specimens (63.3%). Moreover, the positivity rate of pan-fungal PCR was higher than that of the culture result. Out of 88 pan-bacterial positive PCR specimens, 66 and 47 ones were positive for Staphylococcus and Pseudomonas, respectively. In addition, 30 samples exhibited mixed infection of both, and five specimens remained negative. Out of 97 pan-fungal positive PCR specimens, 67 and 51 ones contained Candida and Aspergillus species, respectively. It should be noted that dual amplicon bands of Candida and Aspergillus-related multiplex PCR were yielded in 30 specimens. Conclusion: The stepwise multiplex PCR assay proved to be more sensitive, more rapid, as well as less cumbersome in detection and identification of fungal and bacterial OE, compared to culture.