Clinical epidemiology of pulmonary aspergillosis in hospitalized patients and contribution of Cyp51A, Yap1, and Cdr1B mutations to voriconazole resistance in etiologic Aspergillus species.

Pulmonary aspergillosis is a life-threatening fungal infection with worldwide distribution. In the present study, clinical epidemiology of pulmonary aspergillosis and antifungal susceptibility of etiologic Aspergillus species were evaluated in one-hundred fifty patients with special focus on the frequency of voriconazole resistance. All the cases were confirmed by the clinical pictures, laboratory findings, and isolation of etiologic Aspergillus species which belonged to two major species, i.e., A. flavus and A. fumigatus. Seventeen isolates displayed voriconazole MIC greater than or equal to the epidemiological cutoff value. Expression of cyp51A, Cdr1B, and Yap1 genes was analyzed in voriconazole-intermediate/resistant isolates. In A. flavus, Cyp51A protein sequencing showed the substitutions T335A and D282E. In the Yap1 gene, A78C replacement led to Q26H amino acid substitution that was not reported previously in A. flavus resistant to voriconazole. No mutations associated with voriconazole resistance were found in the three genes of A. fumigatus. The expression of Yap1 was higher than that of two other genes in both A. flavus and A. fumigatus. Overall, voriconazole-resistant strains of both A. fumigatus and A. flavus demonstrated overexpression of Cdr1B, Cyp51A, and Yap1 genes compared to voriconazole-susceptible strains. Although there are still ambiguous points about the mechanisms of azole resistance, our results showed that mutations were not present in majority of resistant and intermediate isolates, while all of these isolates showed overexpression in all three genes studied. As a conclusion, it seems that the main reason of the emergence of mutation in voriconazole-resistant isolates of A. flavus and A. fumigatus is previous or prolonged exposure to azoles.

Local Administration of Sodium Bicarbonate for Preventing COVID-19 Associated Mucormycosis.

Background: One important complication of the coronavirus disease 2019 (COVID-19) is COVID Associated Mucormycosis (CAM), especially in patients with conditions such as diabetes and in immunosuppressed patients. Systemic acidosis, hyperglycemia, and other biochemical factors such as free iron and ß-hydroxybutyrate (BHB) can play a role in this complication. Materials and Methods: Rhizopus oryzae was isolated from a patient at Masih Daneshvari Hospital microbiology laboratory and sub-cultured on the Potato Dextrose Agar (PDA) for 48 hours at 37 °C. Subsequently, Roswell Park Memorial Institute (RPMI) 1640 Broth medium buffered to pH 7.0 with 3-N-morpholino-propane sulfonic acid. Macrodilution and microdilution methods were performed with 8.4% sodium bicarbonate. After 24 hours of incubation at 35°C, the minimum inhibitory concentration (MIC) and the minimum fungicidal concentrations (MFC) were evaluated. Results: We found that the minimum inhibitory and fungicidal concentrations are at 1.05 % and 2.1 % respectively. Therefore, the minimum concentration is 2% sodium bicarbonate, which requires achieving the desired environmental pH for fungal inhibition and fungicidal effects. Conclusion: Regulation of systemic acidosis by sodium bicarbonate could be used to decrease the chance of mucormycosis. In addition, According to our study and some others, an alkaline environment can prevent fungal growth. We found that a minimum concentration of 2% sodium bicarbonate is required to achieve the desired mucosal pH to inhibit the fungus. Therefore, sodium bicarbonate inhalation, as a cost-effective and well-tolerated medicine, is a good candidate for the prevention of mucormycosis. In this regard, extensive clinical and laboratory research is needed to achieve more accurate doses and appropriate administration intervals.

Coinfection by Aspergillus and Mucoraceae Species in Two Cases of Acute Rhinosinusitis as a Complication of COVID-19.

Acute invasive fungal rhinosinusitis (AIFR) is a life-threatening infection often found in immunocompromised patients. In the COVID-19 era, reports of AIFR have emerged, with high mortality and morbidity rate. This paper presents two cases of COVID-19 associated AIFR with the combined proven fungal etiology of Aspergillus flavus and Rhizopus arrhizus in case 1 and Aspergillus fumigatus and Rhizopus arrhizus in case 2. Both patients received liposomal amphotericin B then posaconazole combined with aggressive surgical debridement of necrotic tissues with a favorable clinical outcome. Mixed etiology AIFR can influence the outcome; hence, further studies are required upon this new threat.

Delayed post-pneumonectomy empyema necessitans caused by Aspergillus flavus: An unusual report.

Post-pneumonectomy empyema (PPE), with or without bronchopleural fistula, is a challenging and serious entity with significant mortality and morbidity. PPE is usually caused by bacteria such as staphylococci, streptococci and also gram-negative rods. Among fungal pathogens, Aspergillus species is a very rare cause of this entity. Herein, we describe an unusual case of delayed post-pneumonectomy empyema necessitans caused by Aspergillus flavus in a 65-year-old man with favourable clinical outcome by combined surgical and antifungal therapy.

Mixed etiology COVID-19 associated acute rhinosinusitis caused by two Aspergillus species.

INTRODUCTION: Acute invasive fungal rhino-sinusitis (AIFR) is a life-threatening infection that is mostly found in immunocompromised patients with serious morbidity and mortality. Recently, reports of AIFR have also emerged among SARS-CoV-2 infected patients. CASE PRESENTATION: A 50-year-old diabetic woman, previously diagnosed with COVID-19 pneumonia, was presented to the hospital with left facial pain on day 12 after discharge. Paranasal sinuses computed tomography was performed and according to the mucosal thickening in both maxillary sinuses and ethmoidal air cells, the patient underwent functional endoscopic sinus surgery (FESS) and necrosis were observed. The histopathologic examination revealed mycelium with septation suspected to Aspergillus and the culture was consistent with Aspergillus flavus and also Aspergillus niger . We reported a case of COVID-19 associated AIFR with two combined Aspergillus species from Iran. The patient received liposomal amphotericin B, which then switched to voriconazole combined with aggressive surgical debridement of necrotic tissues with a clinically favorable outcome. CONCLUSION: Mixed etiology AIFR can influence the outcome. However, further investigation is required upon this new threat.

Phylogenetic relationship of Fusarium species isolated from keratitis using TEF1 and RPB2 gene sequences.

Background and Objectives: Fusarium species are known to be one of the common causes of keratitis. This study was conducted to identify Fusarium spp. causing keratitis and to investigate their genetic diversity using TEF1 and RPB2 gene sequences. Materials and Methods: Twenty-four clinical isolates of Fusarium were isolated from the patient with keratitis. Phylogenetic analysis of two-locus of the 24 clinical isolates and three reference strains was carried out using the maximum parsimony and RAxML methods. Results: Based on gene sequences of the 24 clinical isolates, 17, 4, and 3 isolates were identified as Fusarium solani species complex (FSSC), Fusarium fujikuroi species complex (FFSC), and Fusarium oxysporum, respectively. FFSC include F. proliferatum (n=1), F. globosum (n=1), F. verticillioides (n=1), and F. brevicatenulatum (n=1), respectively. Conclusion: Given that sequence of a sole gene can be challenging and on the other hand, due to the high resistance to antifungal drugs, identification of Fusarium species is of substantial significance. In this study, by designing a novel set of primers for the RPB2 area and using TEF1 primer, we were able to differentiate 24 Fusarium spp. isolated from patients with keratitis.

Molecular discrimination and antifungal susceptibility profile of cryptic Candida albicans complex species isolated from patients in Iran.

Background and Objectives: Candida albicans complex species are well known as the main cause of candidiasis, particularly among susceptible individuals. In this study, we report the genetic diversity of Candida spp. and the antifungal susceptibility pattern of the cryptic C. albicans complex isolates in Kerman, Iran. Materials and Methods: A total of 112 yeast isolates were obtained from different clinical samples, and molecular identification was performed. All C. albicans complex isolates were tested for susceptibility of them to amphotericin B, fluconazole, and itraconazole. Results: The majority of clinical isolates were C. albicans complex (n=48) followed by C. glabrata complex (n=34), C. parapsilosis complex (n=21), and C. krusei (n=9). Among C. albicans complex, 45 isolates were C. albicans (94%), 2 isolates were C. dubliniensis (4%), and 1 isolate was C. africana (2%). Amphotericin B was the most active antifungal, whereas 8.9% and 6.7% of the isolates were resistant to fluconazole and itraconazole, respectively. Conclusion: Regarding the high incidence of Candida infections particularly in susceptible populations and the emergence of an infrequent yeast species with elevated MICs, which is indistinguishable with conventional methods, developing accurate molecular methods for laboratory diagnosis should be considered in the clinical setting.

Case Report: COVID-19-associated Rhinosinusitis Mucormycosis Caused by Rhizopus arrhizus: A Rare but Potentially Fatal Infection Occurring After Treatment with Corticosteroids.

COVID-19 first emerged in Wuhan, China, in December 2019. Since that time, the frequency of bacterial and fungal coinfections has been continuously increasing. Although invasive pulmonary aspergillosis is being increasingly recognized in association with COVID-19, there is limited information regarding COVID-19-associated mucormycosis. We describe a 50-year-old woman with uncontrolled diabetes who received systemic corticosteroids and remdesevir during her admission for COVID-19. A few days after discharge, the patient was readmitted because of facial swelling and numbness, and a diagnosis of COVID-19-associated rhinosinusitis mucormycosis caused by Rhizopus arrhizus (formerly called Rhizopus oryzae) was confirmed with sequencing of the internal transcribed spacer region of the ribosomal DNA. This report aimed to address the importance of short-term follow-up for COVID-19 patients who have received systemic corticosteroids, particularly those with predisposing conditions, because early detection and prompt, aggressive treatment are essential for the management of invasive fungal infections.

Fatal invasive pulmonary aspergillosis in an immunocompetent patient with COVID-19 due to Aspergillus terreus: A case study.

Case reports of CAPA emerged. In most of the reports, the predominant species is Aspergillus fumigatus. Uncommon species are less reported. Due to poor clinical outcome with Aspergillus terreus, the increasing reports with this agent require attention.

Diagnosis of allergic bronchopulmonary aspergillosis in patients with persistent allergic asthma using three different diagnostic algorithms.

BACKGROUND: Allergic bronchopulmonary aspergillosis (ABPA) has been reported in various degrees among patients with persistent allergic asthma (PAA). Currently, there is no gold standard approach for diagnosis of ABPA. OBJECTIVES: In the current study, we aimed the evaluation of three different mainly used algorithms as Rosenberg & Patterson (A), ISHAM Working Group (B) and Greenberger (C) for diagnosis of ABPA in 200 patients with underlying PAA. METHODS: All patients were evaluated using Aspergillus skin prick test (SPTAf), Aspergillus-specific IgE (sIgEAf) and IgG (sIgGAf), total IgE (tIgE), pulmonary function tests, radiological findings and peripheral blood eosinophil count. The prevalence rate of ABPA in PAA patients was estimated by three diagnostic criteria. We used Latent Class Analysis for the evaluation of different diagnostic parameters in different applied ABPA diagnostic algorithms. RESULTS: Aspergillus sensitisation was observed in 30 (15.0%) patients. According to algorithms A, B and C, nine (4.5%), six (3.0%) and 11 (5.5%) of patients were diagnosed with ABPA, respectively. The sensitivity and specificity of criteria B and C were (55.6% and 99.5%) and (100.0% and 98.9%) respectively. sIgEAf and sIgGAf showed the high significant sensitivity. The performance of algorithm A, in terms of sensitivity and specificity, was somewhat better than algorithm B. CONCLUSION: Our study demonstrated that the sensitivity of different diagnostic algorithms could change the prevalence rate of ABPA. We also found that all of three criteria resulted an adequate specificity for ABPA diagnosis. A consensus patterns combining elements of all three criteria may warrant a better diagnostic algorithm.

Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens.

INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

Relationship between antifungal susceptibility profile and virulence factors in Candida albicans isolated from nail specimens

Abstract INTRODUCTION: The aim of this study was to evaluate some virulence factors in Candida albicans isolates from patients with onychomycosis and determine the correlation between these factors and the antifungal resistance profile. METHODS: Seventy species of C. albicans were confirmed using polymerase chain reaction amplification of the HWP1 gene. According to the Clinical & Laboratory Standards Institute guidelines, the susceptibility profile of four antifungal agents was investigated, and the production of aspartyl protease, phospholipase, haemolysin, and biofilm was determined. The correlation between these profiles was also investigated. RESULTS: The isolates indicated different levels of resistance and production of virulence factors. Significant correlations were observed between the minimum inhibitory concentration (MIC) of fluconazole/itraconazole and biofilm production, between phospholipase production and fluconazole/itraconazole MIC, and between fluconazole MIC and hemolytic activity in C. albicans isolates. The results also showed significant correlations between phospholipase activity and biofilm production. CONCLUSIONS: Our findings will contribute to a better understanding of the pathogenesis of C. albicans and characterize the relationship between virulence factors and antifungal resistance, which may suggest new therapeutic strategies considering the possible involvement of the virulence mechanism in the effectiveness of treatment.

Genetic diversity and antifungal susceptibility of Candida albicans isolated from Iranian patients.

A total of 105 independent Candida albicans strains isolated from patients in Iran were investigated. According to CLSI documents M27-A3 and M27-S4, the 24 h geometric mean MICs of caspofungin, itraconazole, and fluconazole were 0.27, 3.19, and 11.91 µg/ml, respectively. Microsatellites analysis of CEF3, CAIII, LOC4 Loci identified 93 different allelic genotypes clustered apart into six different clades. Antifungal susceptibility was not linked with the source of isolation and the corresponding genotype of C. albicans strains.

Genetic Diversity and Antifungal Susceptibility of Candida parapsilosis Sensu Stricto Isolated from Bloodstream Infections in Turkish Patients.

Candida parapsilosis sensu stricto is an emerging cause of hospital-acquired Candida infections, predominantly in southern Europe, South America, and Asia. We investigated the genetic diversity and antifungal susceptibility profile of 170 independent C. parapsilosis sensu stricto strains obtained from patients with candidemia who were treated at the Ege University Hospital in Izmir, Turkey, between 2006 and 2014. The identity of each strain was confirmed via PCR amplification and digestion of the secondary alcohol dehydrogenase-encoding gene. The 24-h geometric mean minimum inhibitory concentrations of the antifungal agents, in increasing order, were as follows: posaconazole, 0.10 µg/mL; voriconazole, 0.21 µg/mL; caspofungin, 0.38 µg/mL; amphotericin B, 0.61 µg/mL; anidulafungin, 0.68 µg/mL; and fluconazole, 2.95 µg/mL. Microsatellite genotyping of the isolates (using fluorescently labeled primers and a panel of four different short-nucleotide repeat fragments) identified 25, 17, 17, and 8 different allelic genotypes at the CP6, B5, CP4, and CP1 locus, respectively. Posaconazole, caspofungin, and amphotericin B showed the greatest in vitro activity of the tested systemic azole, echinocandin, and polyene agents, respectively, and the observed antifungal susceptibility of the isolates was shown to be independent of their isolation source. We obtained a combined discriminatory power of 0.99 with a total of 130 genotypes for 170 isolates tested. Finally, microsatellite profiling analysis confirmed the presence of identical genotype between separate isolates, supporting that effective surveillance and infection-prevention programs are essential to limit the impact of C. parapsilosis sensu stricto on hospitalized patients' health.

Prevalence of specific immunoglobulin E and G against Aspergillus fumigatus in patients with asthma.

BACKGROUND AND PURPOSE: Aspergillus fumigatus as a ubiquitous fungus can be found in the respiratory tract of the asthmatic and healthy people. The inhalation of Aspergillus spores leads to an immune response in individuals with asthma and results in the aggravation of the clinical symptoms. The present study aimed to investigate the prevalence of specific immunoglobulin E and G (IgE and IgG) against A.fumigatus in asthmatic patients. MATERIALS AND METHODS: This study was conducted on 200 consecutive patients with moderate to severe asthma referring to Masih Daneshvari hospital Tehran, Iran, from January 2016 to February 2018. Skin prick test (SPT) was performed in all subjects with Aspergillus allergens. Moreover, all patients underwent specific IgE testing for Aspergillus using Hycor method. Enzyme immune assay was applied to measure total IgE and Aspergillus-specific IgG. RESULTS: According to the results, the mean age of the patients was 45.8 years (age range: 18-78 years). The mean levels of total IgE and Aspergillus specific IgE in asthmatic patients were obtained as 316.3 (range: 6-1300 IU/ml) and 1.5 (range: 0.1-61.3 IU/ml), respectively. Out of 200 patients, 27 (13.5%), 65 (32.5%), 22 (11.0%), and 86 (43.0%) cases had positive Aspergillus SPT, total IgE of > 417 IU/ml, Aspergillus -specific IgE, and IgG, respectively. The level of these variables in patients with severe asthma were 16 (16.5%), 36 (37.1%), 15 (15.5%), and 46 (47.4%), respectively. CONCLUSION: As the findings indicated, reactivity to Aspergillus is a remarkable phenomenon in asthmatic patients. It is also emphasised that the climatic condition may affect the positive rate of hypersensitivity to Aspergillus.

Multi-Gene Expression in Anthracosis of the Lungs as One of the Risk Factors for Non-Small Cell Lung Cancer

Background: Anthracosis of the lung occurs due to the deposition of carbon and silica in the mucosa and submucosa, manifested as black lesions. The association of anthracosis with lung cancer has remained to be clearly elucidated The current study aimed to assess the P16, CDH1 and LUNX genes expression level to evaluate the association of anthracotic lesions in the lungs with the occurrence of non-small cell lung cancer. Methods: Forty biopsy samples were taken from the center and 40 from the margins of black anthracotic lesions in the lungs; RNA was extracted from the samples and cDNA was synthesized. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was performed to assess the expression of P16, CDH1 and LUNX genes. All steps were performed in triplicate. Results: A significant reduction in P16 gene expression was noted at the center compared to the margins of the lesions (P<0.001). expression level of CDH1 at the center of lesions was significantly lower than margins (P<0.001). However, LUNX gene had significantly higher expressionlevel at the center compared to margins (P<0.001). Conclusion: Decreased expression of P16 and CDH1 and increased expression of LUNX tumor genes were noted at the center of anthracotic lesions. Significant increase in expression of LUNX gene in NSCLC indicates an association between anthracosis and NSCLC, according to which, anthracotic patients may carry a high risk for NSCLC.

Morphological changes and induction of antifungal resistance in Aspergillus fumigatus due to different CO2 levels.

BACKGROUND AND PURPOSE: Aspergillosis is one of the most common opportunistic fungal infections in immunocompromised and neutropenic patients. Aspergillus fumigatus (A. fumigatus) is the most common causative agent of this infection. Due to variable CO2 concentrations that pathogens are exposed to during the infection process and to understand the role of CO2, we examined the effects of various CO2 concentrations as one of the environmental factors on morphological changes and induction of antifungal resistance in A. fumigatus. MATERIALS AND METHODS: A. fumigatus strains were cultured and incubated under 1%, 3%, 5%, and 12% CO2 atmospheres, each time for one, two, and four weeks. The control culture was maintained for one week without CO2 atmosphere. Morphological changes were investigated and antifungal susceptibility test was performed according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) M38-A2 document. The results of different CO2 atmospheres were compared with that of the control sample. RESULTS: We found that 1%, 3%, 5%, and 12% CO2 atmospheres were associated with morphological colony changes. Macroscopically, the colonies were shallow dark green, smooth, crisp to powdery with reduced growth; microscopic examination revealed the absence of conidiation. The induction of antifungal resistance in the susceptible strains to itraconazole, voriconazole, and amphotericin B increased after exposure to 12% CO2 atmosphere and four weeks of incubation. The MIC values for itraconazole, voriconazole, and amphotericin B were 16 g/ml, 1 g/ml, and 16 g/ml, respectively. These values for the control group were 0.125 g/ml, 0.125 g/ml, and 2 g/ml, respectively. CONCLUSION: Exposure to different CO2 atmospheres induced morphological changes in A. fumigatus, it seems to increase the MIC values, as well. In parallel, resistance to both itraconazole and voriconazole was also observed.

Molecular identification and antifungal susceptibility profile of Candida species isolated from patients with vulvovaginitis in Tehran, Iran.

BACKGROUND: Rapid and accurate identification and evaluation of antifungal susceptibility pattern of Candida isolates are crucial to determine suitable antifungal drugs for the treatment of patients with vulvovaginitis candidiasis. MATERIALS AND METHODS: Vaginal samples were collected from 150 women with suspicious vaginal candidiasis, and then cultured on Sabouraoud's Dextrose Agar with chloramphenicol to isolate Candida species. After identification of Candida isolates using polymerase chain reaction-restriction fragment length polymorphism technique, antifungal susceptibility testing of four azolic antifungal drugs was carried out using broth microdilution method according to the CLSI M27-A3. RESULTS: Candida species were isolated from eighty suspected patients (61.79%). The most common pathogen was Candida albicans (63.75%). Resistance to fluconazole and ketoconazole was observed in 27.5% and 23.75% of Candida isolates, respectively, and only 2% of Candida isolates were resistant to miconazole. Interestingly, resistance to fluconazole in C. albicans was more than other Candida species. CONCLUSION: The results indicated that therapy should be selected according to the antifungal susceptibility tests for the prevention of treatment failure and miconazole therapy can be considered as the best therapeutic choice in the management of vulvovaginitis.

Effects of Pistacia atlantica subsp. kurdica on Growth and Aflatoxin Production by Aspergillus parasiticus.

BACKGROUND: Aflatoxins are highly toxic secondary metabolites mainly produced by Aspergillus parasiticus. This species can contaminate a wide range of agricultural commodities, including cereals, peanuts, and crops in the field. In recent years, research on medicinal herbs, such as Pistacia atlantica subsp. kurdica, have led to reduced microbial growth, and these herbs also have a particular effect on the production of aflatoxins as carcinogenic compounds. OBJECTIVES: In this study, we to examine P. atlantica subsp. kurdica as a natural compound used to inhibit the growth of A. parasiticus and to act as an anti-mycotoxin. MATERIALS AND METHODS: In vitro antifungal susceptibility testing of P. atlantica subsp. kurdica for A. parasiticus was performed according to CLSI document M38-A2. The rate of aflatoxin production was determined using the HPLC technique after exposure to different concentrations (62.5 - 125 mg/mL) of the gum. The changes in expression levels of the aflR gene were analyzed with a quantitative real-time PCR assay. RESULTS: The results showed that P. atlantica subsp. kurdica can inhibit A. parasiticus growth at a concentration of 125 mg/mL. HPLC results revealed a significant decrease in aflatoxin production with 125 mg/mL of P. atlantica subsp. kurdica, and AFL-B1 production was entirely inhibited. Based on quantitative real-time PCR results, the rate of aflR gene expression was significantly decreased after treatment with P. atlantica subsp. kurdica. CONCLUSIONS: Pistacia atlantica subsp. kurdica has anti-toxic properties in addition to an inhibitory effect on A. parasiticus growth, and is able to decrease aflatoxin production effectively in a dose-dependent manner. Therefore, this herbal extract maybe considered a potential anti-mycotoxin agent in medicine or industrial agriculture.