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.

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.

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.

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.

Candidemia among Hospitalized Pediatric Patients Caused by Several Clonal Lineages of Candida parapsilosis.

Candida parapsilosis is the second most common cause of candidemia in some geographical areas and in children in particular. Yet, the proportion among children varies, for example, from 10.4% in Denmark to 24.7% in Tehran, Iran. As this species is also known to cause hospital outbreaks, we explored if the relatively high number of C. parapsilosis pediatric cases in Tehran could in part be explained by undiscovered clonal outbreaks. Among 56 C. parapsilosis complex isolates, 50 C. parapsilosis were genotyped by Amplified Fragment Length Polymorphism (AFLP) fingerprinting and microsatellite typing and analyzed for nucleotide polymorphisms by FKS1 and ERG11 sequencing. AFLP fingerprinting grouped Iranian isolates in two main clusters. Microsatellite typing separated the isolates into five clonal lineages, of which four were shared with Danish isolates, and with no correlation to the AFLP patterns. ERG11 and FKS1 sequencing revealed few polymorphisms in ERG11 leading to amino-acid substitutions (D133Y, Q250K, I302T, and R398I), with no influence on azole-susceptibilities. Collectively, this study demonstrated that there were no clonal outbreaks at the Iranian pediatric ward. Although possible transmission of a diverse C. parapsilosis community within the hospital cannot be ruled out, the study also emphasizes the necessity of applying appropriately discriminatory methods for outbreak investigation.

Differentiation of Candida albicans complex species isolated from invasive and non-invasive infections using HWP1 gene size polymorphism.

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.

Species distribution and antifungal susceptibility profile of Candida isolates from blood and other normally sterile foci from pediatric ICU patients in Tehran, Iran.

As data on pediatric invasive candidiasis (IC) and the antifungal susceptibility pattern of associated isolates are scarce in Iran, this study aimed to determine species distribution and antifungal susceptibility profile of Candida species isolated from pediatric patients with suspected or documented IC. A total of 235 yeast strains recovered from normally sterile body fluids of patients admitted at the intensive care units of Children's Medical Centre, Tehran, Iran, were identified using CHROMagar Candida, molecular methods (ITS PCR-RFLP and sequencing), and MALDI-TOF. Susceptibility to amphotericin B, fluconazole, voriconazole, micafungin, and anidulafungin was determined according to the European on Antimicrobial Susceptibility testing reference microdilution method (EUCAST E.Def 7.3.1). Candida albicans (53.6%), C. parapsilosis (24.7%), and C. tropicalis (8.5%) were the most common species, followed by C. lusitaniae (4.3%), C. glabrata (3.0%), C. guilliermondii and C. orthopsilosis (each 1.7%), C. kefyr (1.3%), C. dubliniensis (0.8%), and C. intermedia (0.4%). Amphotericin B MICs were ≤1 mg/l for all Candida isolates. C. albicans isolates were susceptible to all five antifungal agents. All C. parapsilosis isolates categorised as intermediate to micafungin and anidulafungin, except two isolates that had the MICs >2 mg/l for micafungin. MIC50, MIC90, and MIC range for fluconazole were 0.25 mg/l, 1 mg/l, and 0.125 - ≥32 mg/l, respectively. Fluconazole and voriconazole showed 100% activity against the most prevalent Candida species. The low resistance rate, favorable safety profile and low cost of fluconazole make it a reasonable choice for treatment of candidemia/invasive candidemia in Iran.

Antifungal susceptibility, genotyping, resistance mechanism, and clinical profile of Candida tropicalis blood isolates.

Candida tropicalis is one of the major candidaemia agents, associated with the highest mortality rates among Candida species, and developing resistance to azoles. Little is known about the molecular mechanisms of azole resistance, genotypic diversity, and the clinical background of C. tropicalis infections. Consequently, this study was designed to address those questions. Sixty-four C. tropicalis bloodstream isolates from 62 patients from three cities in Iran (2014-2019) were analyzed. Strain identification, antifungal susceptibility testing, and genotypic diversity analysis were performed by MALDI-TOF MS, CLSI-M27 A3/S4 protocol, and amplified fragment length polymorphism (AFLP) fingerprinting, respectively. Genes related to drug resistance (ERG11, MRR1, TAC1, UPC2, and FKS1 hotspot9s) were sequenced. The overall mortality rate was 59.6% (37/62). Strains were resistant to micafungin [minimum inhibitory concentration (MIC) ≥1 µg/ml, 2/64], itraconazole (MIC > 0.5 µg/ml, 2/64), fluconazole (FLZ; MIC ≥ 8 µg/ml, 4/64), and voriconazole (MIC ≥ 1 µg/ml, 7/64). Pan-azole and FLZ + VRZ resistance were observed in one and two isolates, respectively, while none of the patients were exposed to azoles. MRR1 (T255P, 647S), TAC1 (N164I, R47Q), and UPC2 (T241A, Q340H, T381S) mutations were exclusively identified in FLZ-resistant isolates. AFLP fingerprinting revealed five major and seven minor genotypes; genotype G4 was predominant in all centers. The increasing number of FLZ-R C. tropicalis blood isolates and acquiring FLZ-R in FLZ-naive patients limit the efficiency of FLZ, especially in developing countries. The high mortality rate warrants reaching a consensus regarding the nosocomial mode of C. tropicalis transmission.

Molecular Identification, Genotypic Diversity, Antifungal Susceptibility, and Clinical Outcomes of Infections Caused by Clinically Underrated Yeasts, Candida orthopsilosis, and Candida metapsilosis: An Iranian Multicenter Study (2014-2019).

Despite the increasing occurrence of Candida orthopsilosis and Candida metapsilosis in clinical settings, little is known about their microbiological and clinical properties. Herein, we conducted a national retrospective study (2014-2019) from multiple centers in Iran. Among the 1,770 Candida isolates collected, we identified 600 Candida parapsilosis species complex isolates. Isolate identification was performed by 9-plex PCR, matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS), and rDNA sequencing, and antifungal susceptibility testing (AFST) followed CLSI M27-A3/S4; genotyping was performed by amplified fragment length polymorphism (AFLP) analysis; and clinical information was mined. Thirty-one isolates of C. orthopsilosis from various clinical sources, one mixed sample (blood) concurrently containing C. orthopsilosis and C. parapsilosis and one isolate of C. metapsilosis from a nail sample were identified. Although both 9-plex PCR and MALDI-TOF successfully identified all isolates, only 9-plex PCR could identify the agents in a mixed sample. For the C. orthopsilosis isolates, resistance (non-wild type) was noted only for itraconazole (n = 4; 12.5%). Anidulafungin and fluconazole showed the highest and voriconazole had the lowest geometric mean values. AFLP analysis showed three main and four minor genotypes. Interestingly, 90% of nail isolates clustered with 80% of the blood isolates within two clusters, and four blood isolates recovered from four patients admitted to a hospital clustered into two genotypes and showed a high degree of similarity (>99.2%), which suggests that C. orthopsilosis disseminates horizontally. Supported by our data and published case studies, C. orthopsilosis and C. metapsilosis can be linked to challenging clinical failures, and successful outcomes are not always mirrored by in vitro susceptibility. Accordingly, conducting nationwide studies may provide more comprehensive data, which is required for a better prognosis and clinical management of patients.

Molecular characterization and antifungal susceptibility testing of Candida nivariensis from blood samples - an Iranian multicentre study and a review of the literature.

PURPOSE: Identification of the emerging yeast species Candida nivariensis among presumptively identified Iranian Candida glabrata isolates. METHODOLOGY: Clinical C. glabrata species complex isolates from blood (n=71; 33.3 %), urine (n=100; 46.9 %), vaginal swabs (n=20;9.4 %), BAL (n=10; 4.7 %), and sputum (n=12; 5.6 %) from Iran were investigated. Isolates were characterized by CHROMagar, multiplex PCRs, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), amplified fragment length polymorphism (AFLP) fingerprinting, internal transcribed spacer (ITS)/large subunit (LSU) rDNA and FKS1/FKS2 sequencing, and the European Committee on Antimicrobial Susceptibility Testing broth microdilution method. A comprehensive literature review was conducted and all the relevant clinical and microbiological data were collected. RESULTS: Four C. nivariensis isolates were recovered from blood samples of three subjects and were all consistently identified by nine-plex PCR, Bruker MALDI-TOF MS, and LSU and ITS rDNA sequencing. AFLP genotyping clustered the isolates into two groups. Sequencing of the FKS1 and FKS2 hotspots showed no accountable amino acid substitutions. All isolates were susceptible to amphotericin B, fluconazole, itraconazole, posaconazole, voriconazole, anidulafungin and micafungin. CONCLUSION: In total, 4 out of 213 clinical C. glabrata species complex candidemia isolates were C. nivariensis. Improvement of the BioMerieux Vitek MS database is required to accurately identify C. nivariensis and it is advised to alternatively use CHROMagar and/or PCR-based techniques. As other species within the Nakaseomyces clade may cause infection and showed high MIC values for antifungals, inclusion of their spectra into the MALDI-TOF MS database seems relevant. Due to developing resistance to fluconazole and insufficient efficacy of caspofungin, the combination of catheter removal plus treatment with caspofungin, or voriconazole, or micafungin might be effective for patients.

Study on the relationship between the concentration and type of fungal bio-aerosols at indoor and outdoor air in the Children's Medical Center, Tehran, Iran.

Fungal bio-aerosols are of concern due to their adverse health effects, especially in indoor environments. The aim of this study was to evaluate the relationship between the concentration and type of fungal bio-aerosols in the indoor and outdoor of Children's Medical Center in Tehran, Iran. In the present descriptive-analytical study, the fungal bio-aerosols' concentrations in both indoor and outdoor of the hospital air were measured. The measurements were carried out by the Anderson method using a Quick Take 30 pump at 28.3 L min-1 and 2.5 min sampling that was placed on a Sabouraud dextrose agar with chloramphenicol. The average concentrations of total fungal bio-aerosols in the hospital indoor and outdoor air were 40.48 and 119.6 CFU/m3, respectively. Onco-hematology and bone marrow transplantation wards were the most and least contaminated units, respectively (11.09 CFU/m3 vs 1.47 CFU/m3). The most common fungi isolated from the indoor environment were Penicillium spp. (45.86%) which was followed by Cladosporium spp. (31.92%), Aspergillus section Nigri (6.26%), sterilized mycelia (5.05%), and Aspergillus section Flavi (2.83%). Cladosporium spp. (61.10 CFU/m3) and Penicillium spp. (18.56 CFU/m3) had the highest mean concentrations in outdoor and indoor air, respectively. The indoor-to-outdoor ratio of fungal aerosols was < 1 at most sampling sites, indicating that the indoor fungal bio-aerosols may have originated from the outdoor environment.