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Knowledge about the clinical characteristics and prognostic factors of Talaromyces marneffei infection in children is limited, especially in HIV-positive children. We performed a retrospective study of all HIV-positive pediatric inpatients with T. marneffei infection in a tertiary hospital in Southern China between 2014 and 2019 and analyzed the related risk factors of poor prognosis using logistic regression. Overall, 28 cases were enrolled and the prevalence of talaromycosis in AIDS children was 15.3% (28/183). The median age of the onset was 8 years (range: 1-14 years). The typical manifestation of skin lesion with central umbilication was not common (21.4%). All the children had very low CD4

Aim: This study aims to evaluate lumbar drainage (LD) for controlling refractory intracranial hypertension among non-HIV cryptococcal meningitis patients. Patients & methods: A case–control study was designed to compare LD (case) with repeated lumbar puncture (control). Results: Both LD and repeated lumbar puncture can efficiently control refractory intracranial hypertension. LD group showed better clinical symptom remission, such as lower rate of headache, vision disorders, signs of meningeal irritation and conscious disturbance, than control group. LD group was reported with higher intracranial pressure reduction (173.75 ± 17.72 mmH2O) than those among control group (113.50 ± 14.94 mmH2O; p

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As a novel risk factor, COVID-19 has led to an increase in the incidence of candidemia and an elevated mortality rate. Despite being of clinical importance, there is a lack of data regarding COVID-19-associated candidemia (CAC) among Iranian patients. Therefore, in this retrospective study, we assessed CAC epidemiology in the intensive care units (ICUs) of two COVID-19 centers in Mashhad, Iran, from early November 2020 to late January 2021. Yeast isolates from patients’ blood were identified by 21-plex polymerase chain reaction (PCR) and sequencing, then subjected to antifungal susceptibility testing according to the CLSI M27-A3 protocol. Among 1988 patients with COVID-19 admitted to ICUs, seven had fungemia (7/1988, 0.03%), among whom six had CAC. The mortality of the limited CAC cases was high and greatly exceeded that of patients with COVID-19 but without candidemia (100% (6/6) vs. 22.7% (452/1988)). In total, nine yeast isolates were collected from patients with fungemia: five Candida albicans, three C. glabrata, and one Rhodotorula mucilaginosa. Half of the patients infected with C. albicans (2/4) were refractory to both azoles and echinocandins. The high mortality of patients with CAC, despite antifungal therapy, reflects the severity of the disease in these patients and underscores the importance of rapid diagnosis and timely initiation of antifungal treatment.

Clonal outbreaks due to azole-resistant Candida parapsilosis (ARCP) isolates have been reported in numerous studies, but the environmental niche of such isolates has yet to be defined. Herein, we aimed to identify the environmental niche of ARCP isolates causing unremitting clonal outbreaks in an adult ICU from a Brazilian cancer referral center. C. parapsilosis sensu stricto isolates recovered from blood cultures, pericatheter skins, healthcare workers (HCW), and nosocomial surfaces were genotyped by multilocus microsatellite typing (MLMT). Antifungal susceptibility testing was performed by the EUCAST (European Committee for Antimicrobial Susceptibility Testing) broth microdilution reference method and ERG11 was sequenced to determine the azole resistance mechanism. Approximately 68% of isolates were fluconazole-resistant (76/112), including pericatheter skins (3/3, 100%), blood cultures (63/70, 90%), nosocomial surfaces (6/11, 54.5%), and HCW’s hands (4/28, 14.2%). MLMT revealed five clusters: the major cluster contained 88.2% of ARCP isolates (67/76) collected from blood (57/70), bed (2/2), pericatheter skin (2/3), from carts (3/7), and HCW’s hands (3/27). ARCP isolates were associated with a higher 30 day crude mortality rate (63.8%) than non-ARCP ones (20%, p = 0.008), and resisted two environmental decontamination attempts using quaternary ammonium. This study for the first time identified ARCP isolates harboring the Erg11-Y132F mutation from nosocomial surfaces and HCW’s hands, which were genetically identical to ARCP blood isolates. Therefore, it is likely that persisting clonal outbreak due to ARCP isolates was fueled by environmental sources. The resistance of Y132F ARCP isolates to disinfectants, and their potential association with a high mortality rate, warrant vigilant source control using effective environmental decontamination.

Background: Echinocandin resistance rarely occurs in clinical Candida parapsilosis isolates and the underlying mechanism is unknown. Objectives: To determine the prevalence of echinocandin resistance and the underlying mechanism for a Large collection of C. parapsilosis blood isolates and to determine whether the echinocandin-resistant isolates were clonally related. Methods: C. parapsilosis blood isolates (n = 213) were subjected to antifungal susceptibility testing (CLSI M27), for micafungin, anidulafungin, amphotericin B and, if appropriate, caspofungin. Hotspot (HS) 1 and HS2 of FKS1 were sequenced for all isolates (n = 213) and microsatellite typing was performed for echinocandin-resistant isolates. Results: ALL isolates were susceptible to amphotericin B and two isolates were intermediate to anidulafungin (MIC-4 mg/L), while micafungin resistance was noted in four isolates (MIC >8 mg/L); three of which were also fluconazole resistant and therefore were MDR. Interestingly, micafungin-resistant isolates, but not those intermediate to anidulafungin, carried novel mutation R658G in HS1 of Fks1p; three of which also harboured Y132F+K143R in Erg11. The first isolate (MICR1) was recovered in November 2017 from a patient admitted to paediatric gastroenterology who showed therapeutic failure under caspofungin treatment. MICR2-MICR4 were collected during 2018-19 and were recovered from three echinocandin-naive paediatric-surgery patients; the isolates shared the same genotype. Conclusions: Herein, for the first time (to the best of our knowledge), we identified micafungin-resistant C. parapsilosis blood isolates harbouring a novel mutation in HS1 of FKS1, which was Likely attributable to in vitro micafungin resistance and in vivo caspofungin therapeutic failure. The acquisition of micafungin-resistant C. parapsilosis isolates in echinocandin-naive patients Likely implicates clonal expansion, as supported by the close genetic relatedness of MICR2-MICR4., Major National R&D Projects of the National Health Department [2018ZX10101003]; National Natural Science Foundation of China [31770161]; Shanghai Science and Technology Committee [17DZ2272900, 14495800500]; Shanghai Municipal Commission of Health and Family Planning [2017ZZ01024-001]; Shanghai Sailing Program [19YF1448000]; Chinese Academy of Engineering [2019-XY-33], This work was supported by the Major National R&D Projects of the National Health Department (2018ZX10101003), the National Natural Science Foundation of China (31770161), the Shanghai Science and Technology Committee (17DZ2272900 and 14495800500), the Shanghai Municipal Commission of Health and Family Planning (2017ZZ01024-001), the Shanghai Sailing Program (19YF1448000) and the Chinese Academy of Engineering (2019-XY-33).

Coronavirus disease 2019 (COVID-19)–associated mucormycosis (CAM) has recently been increasingly reported, particularly among patients with uncontrolled diabetes. Patients with diabetes and hyperglycemia often display an inflammatory state that may be potentiated by the activation of antiviral immunity to SARS-CoV-2, and thus may favor secondary infections. We analyze 80 published and unpublished cases of CAM, with a predominance (42/80) of cases from India. Uncontrolled diabetes mellitus as well as systemic corticosteroid treatment represented major comorbid predisposing factors and rhino-orbital cerebral mucormycosis was the most frequent presentation of disease. Mortality was high at 49%, driven particularly by those with pulmonary or disseminated mucormycosis and those with cerebral involvement. Furthermore, a significant proportion of surviving patients suffered life-changing morbidities (loss of vision in 46% of survivors). Our review indicates that CAM may be a relevant complication of severe COVID-19, particularly in those with uncontrolled diabetes. Funding: Martin Hoenigl received funding from Astellas for two investigator initiated studies (ISR005824 and ISR005838), and was supported by the National Institutes of Health, Grant UL1TR001442. Agostinho Carvalho was supported by the Fundacao para a Ciencia e a Tecnologia (FCT) (UIDB/50026/2020 and UIDP/50026/2020), the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000039), the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 847507, and the “la Caixa” Foundation (ID 100010434) and FCT under the agreement LCF/PR/HR17/52190003. Declaration of Interest: MH received research funding from Gilead, Pfizer, Astellas, Scynexis and NIH. JPG received speaker and expert advice fees from Pfizer and Gilead. NK has received research grants or honoraria as a speaker or advisor from Astellas, Gilead, MSD, and Pfizer, outside the submitted work. KL received consultancy fees from SMB Laboratoires Brussels, MSD and Gilead, travel support from Pfizer, speaker fees from FUJIFILM WAKO, Pfizer and Gilead, a service fee from Thermo fisher Scientific. OAC is supported by the German Federal Ministry of Research and Education, is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) under Germany's Excellence Strategy – CECAD, EXC 2030 – 390661388 and has received research grants from, is an advisor to, or received lecture honoraria from Actelion, Allecra Therapeutics, Al-Jazeera Pharmaceuticals, Amplyx, Astellas, Basilea, Biosys, Cidara, Da Volterra, Entasis, F2G, Gilead, Grupo Biotoscana, Immunic, IQVIA, Janssen, Matinas, Medicines Company, MedPace, Melinta Therapeutics, Menarini, Merck/MSD, Mylan, Nabriva, Noxxon, Octapharma, Paratek, Pfizer, PSI, Roche Diagnostics, Scynexis, and Shionogi. PLW performed diagnostic evaluations and received meeting sponsorship from Bruker, Dynamiker, and Launch Diagnostics; Speakers fees, expert advice fees and meeting sponsorship from Gilead; and speaker and expert advice fees 489 from F2G and speaker fees MSD and Pfizer. Is a founding member of the European Aspergillus PCR Initiative. ACh received funding support from educational grant of Pfizer, MSD Pharmaceutical Ltd, and Gilead. All other authors no conflicts. Ethical Approval: MISSING

[Motivation]: Sequence analyses oriented to investigate specific features, patterns and functions of protein and DNA/RNA sequences usually require tools based on graphic interfaces whose main characteristic is their intuitiveness and interactivity with the user’s expertise, especially when curation or primer design tasks are required. However, interface-based tools usually pose certain computational limitations when managing large sequences or complex datasets, such as genome and transcriptome assemblies. Having these requirments in mind we have developed SeqEditor an interactive software tool for nucleotide and protein sequences’ analysis., [Result]: SeqEditor is a cross-platform desktop application for the analysis of nucleotide and protein sequences. It is managed through a Graphical User Interface and can work either as a graphical sequence browser or as a fasta task manager for multi-fasta files. SeqEditor has been optimized for the management of large sequences, such as contigs, scaffolds or even chromosomes, and includes a GTF/GFF viewer to visualize and manage annotation files. In turn, this allows for content mining from reference genomes and transcriptomes with similar efficiency to that of command line tools. SeqEditor also incorporates a set of tools for singleplex and multiplex PCR primer design and pooling that uses a newly optimized and validated search strategy for target and species-specific primers. All these features make SeqEditor a flexible application that can be used to analyses complex sequences, design primers in PCR assays oriented for diagnosis, and/or manage, edit and personalize reference sequence datasets., This work was supported by the European Union’s Horizon 2020 Research and Innovation programme under the Marie Sklodowska-Curie grant agreement [642095] of the OPATHY consortium; by the pre-doctoral research fellowship from Industrial Doctorates of MINECO [DI-17-09134]; and by the State Plan for Scientific and Technical Research and Innovation 2017-2020 of the Secretary of State for Digital Advancement of Ministry of Economic Affairs and Digital Transformation, Spain [TSI-100903-2019-11].

The high clinical mortality and economic burden posed by invasive fungal infections (IFIs), along with significant agricultural crop loss caused by various fungal species, has resulted in the widespread use of antifungal agents. Selective drug pressure, fungal attributes, and host- and drug-related factors have counteracted the efficacy of the limited systemic antifungal drugs and changed the epidemiological landscape of IFIs. Species belonging to Candida, Aspergillus, Cryptococcus, and Pneumocystis are among the fungal pathogens showing notable rates of antifungal resistance. Drug-resistant fungi from the environment are increasingly identified in clinical settings. Furthermore, we have a limited understanding of drug class-specific resistance mechanisms in emerging Candida species. The establishment of antifungal stewardship programs in both clinical and agricultural fields and the inclusion of species identification, antifungal susceptibility testing, and therapeutic drug monitoring practices in the clinic can minimize the emergence of drug-resistant fungi. New antifungal drugs featuring promising therapeutic profiles have great promise to treat drug-resistant fungi in the clinical setting. Mitigating antifungal tolerance, a prelude to the emergence of resistance, also requires the development of effective and fungal-specific adjuvants to be used in combination with systemic antifungals.

Dermatophytosis is a common superficial mycotic infection affecting individual's quality of life worldwide. The present study aimed to perform species-level identification and evaluate the antifungal susceptibility patterns of dermatophytes isolated in Shiraz, Iran. This cross-sectional study was conducted on clinical samples collected during 2017-2019 from 307 patients suspected of having dermatophytosis. The isolates were identified by direct microscopy, culture and internal transcribed spacer ribosomal DNA sequencing, and their antifungal susceptibility patterns were determined by the microdilution method. Among 307 patients, dermatophytosis was diagnosed by microscopy in 190 (61.8%) subjects and confirmed in 130 (42.3%) cases by both microscopy and culture. It was found out tinea pedis was the most common clinical manifestation, and Trichophyton mentagrophytes was the most prevalent species (28.4%), followed by T tonsurans (23.8%), Microsporum canis (11.5%), T interdigitale (10%), T verrucosum (6.9%), T rubrum (6.9%), T benhamiae (4.6%), T violaceum (3%), T simii (3%), Epidermophyton floccosum (0.7%) and M ferrugineum (0.7%). Moreover, it was revealed that luliconazole with a geometric mean (GM) minimum inhibitory concentration (MIC) of 0.03 μg ml-1 was the most effective agent against all tested isolates. Regardless of species, 30% of isolates responded to high MICs of griseofulvin (MIC90 > 2 μg ml-1 ). The increasing prevalence of nonindigenous species of T simii, T benhamiae and M ferrugineum in Shiraz, Iran, was a notable finding. In addition, infections due to zoophilic species showed an increasing trend. These epidemiological data, along with antifungal susceptibility patterns, may have implications for clinical decision-making and successful treatment.

Clonal outbreaks of fluconazole-resistant (FLZR) Candida parapsilosis isolates have been reported in several countries. Despite its being the second leading cause of candidemia, the azole resistance mechanisms and the clonal expansion of FLZR C. parapsilosis blood isolates have not been reported in Turkey. In this study, we consecutively collected C. parapsilosis blood isolates (n = 225) from the fifth largest hospital in Turkey (2007 to 2019), assessed their azole susceptibility pattern using CLSI M27-A3/S4, and sequenced ERG11 for all and MRR1, TAC1, and UPC2 for a selected number of C. parapsilosis isolates. The typing resolution of two widely used techniques, amplified fragment length polymorphism typing (AFLP) and microsatellite typing (MST), and the biofilm production of FLZR isolates with and without Y132F were compared. Approximately 27% of isolates were FLZR (60/225), among which 90% (54/60) harbored known mutations in Erg11, including Y132F (24/60) and Y132F+K143R (19/60). Several mutations specific to FLZR isolates were found in MRR1, TAC1, and UPC2. AFLP grouped isolates into two clusters, while MST revealed several clusters. The majority of Y132F/Y132F+K143R isolates grouped in clonal clusters, which significantly expanded throughout 2007 to 2019 in neonatal wards. Candida parapsilosis isolates carrying Y132F were associated with significantly higher mortality and less biofilm production than other FLZR isolates. Collectively, we documented the first outbreak of FLZR C. parapsilosis blood isolates in Turkey. The MRR1, TAC1, and UPC2 mutations exclusively found in FLZR isolates establishes a basis for future studies, which will potentially broaden our knowledge of FLZR mechanisms in C. parapsilosis. MST should be a preferred method for clonal analysis of C. parapsilosis isolates in outbreak scenarios. Copyright © 2020 American Society for Microbiology. All Rights Reserved., Shanghai Municipal Health and Family Planning Commission 2018ZX10101003 Science and Technology Commission of Shanghai Municipality, STCSM: 17DZ2272900, 14495800500 National Natural Science Foundation of China, NSFC: 31770161, This work was supported by the Major National R&D Projects of the National Health Department (2018ZX10101003), National Natural Science Foundation of China (31770161), Shanghai Science and Technology Committee (17DZ2272900 and 14495800500), Shanghai Municipal Commission of Health and Family Planning

Candida tropicalis is the fourth leading cause of candidemia in Turkey. Although C. tropicalis isolates from 1997 to 2017 were characterized as fully susceptible to antifungals, the increasing global prevalence of azole-non-susceptible (ANS) C. tropicalis and the association between high fluconazole tolerance (HFT) and fluconazole therapeutic failure (FTF) prompted us to re-evaluate azole susceptibility of C. tropicalis in Turkey. in this study, 161 C. tropicalis blood isolates from seven clinical centers were identified by ITS rDNA sequencing, genotyped by multilocus microsatellite typing, and tested for susceptibility to five azoles, two echinocandins, and amphotericin B (AMB); antifungal resistance mechanisms were assessed by sequencing of ERG11 and FKS1 genes. the results indicated that C. tropicalis isolates, which belonged to 125 genotypes grouped into 11 clusters, were fully susceptible to echinocandins and AMB; however, 18.6% of them had the ANS phenotype but only two carried the ANS-conferring mutation (Y132F). HFT was recorded in 52 isolates, 10 of which were also ANS. Large proportions of patients infected with ANS and HFT isolates (89 and 40.7%, respectively) showed FTF. Patients infected with azole-susceptible or ANS isolates did not differ in mortality, which, however, was significantly lower for those infected with HFT isolates (P = 0.007). There were significant differences in mortality (P = 0.02), ANS (P = 0.012), and HFT (P = 0.007) among genotype clusters. the alarming increase in the prevalence of C. tropicalis blood isolates with ANS and HFT in Turkey and the notable FTF rate should be a matter of public health concern., Major National R&D Projects of the National Health Department [2018ZX10101003]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31770161]; Shanghai Science and Technology CommitteeShanghai Science & Technology Committee [17DZ2272900, 14495800500]; Shanghai Municipal Commission of Health and Family Planning [2017ZZ01024-001]; Shanghai Sailing Program [19YF1448000]; Chinese Academy of Engineering [2019-XY-33], This work was supported by the Major National R&D Projects of the National Health Department (2018ZX10101003), National Natural Science Foundation of China (31770161), Shanghai Science and Technology Committee (17DZ2272900 and 14495800500), Shanghai Municipal Commission of Health and Family Planning (2017ZZ01024-001), Shanghai Sailing Program (19YF1448000), and the Chinese Academy of Engineering (2019-XY-33).

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.

Human fungal pathogens are attributable to a significant economic burden and mortality worldwide. Antifungal treatments, although limited in number, play a pivotal role in decreasing mortality and morbidities posed by invasive fungal infections (IFIs). However, the recent emergence of multidrug-resistant Candida auris and Candida glabrata and acquiring invasive infections due to azole-resistant C. parapsilosis, C. tropicalis, and Aspergillus spp. in azole-naïve patients pose a serious health threat considering the limited number of systemic antifungals available to treat IFIs. Although advancing for major fungal pathogens, the understanding of fungal attributes contributing to antifungal resistance is just emerging for several clinically important MDR fungal pathogens. Further complicating the matter are the distinct differences in antifungal resistance mechanisms among various fungal species in which one or more mechanisms may contribute to the resistance phenotype. In this review, we attempt to summarize the burden of antifungal resistance for selected non-albicansCandida and clinically important Aspergillus species together with their phylogenetic placement on the tree of life. Moreover, we highlight the different molecular mechanisms between antifungal tolerance and resistance, and comprehensively discuss the molecular mechanisms of antifungal resistance in a species level. D.S.P. was funded by grant AI109025 from the National Institutes of Health, as well as from Astellas for the Perlin lab’s Reference Center for Antifungal Drug Resistance.

Systematic candidemia studies, especially in southern Iran, are scarce. In the current prospective study, we investigated candidemia in three major healthcare centers of Shiraz, the largest city in southern Iran. Yeast isolates from blood and other sterile body fluids were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and subjected to antifungal susceptibility testing (AFST) using the broth microdilution method. Clinical data were retrieved from patients' medical records. In total, 113 yeast isolates were recovered from 109 patients, over 60% of whom received fluconazole. Antifungal drugs were prescribed without considering species identification or AFST. The all-cause mortality rate was 28%. Almost 30% of the patients were from intensive care units (ICUs). Candida albicans (56/113; 49.5%) was the most prevalent species followed by C. glabrata (26/113; 23%), C. parapsilosis (13/113; 11.5%), C. tropicalis (7/113; 6.2%), and C. dubliniensis (5/113; 4.4%). Only five isolates showed antifungal resistance or decreased susceptibility to fluconazole: one C. orthopsilosis isolate from an azole-naïve patient and two C. glabrata, one C. albicans, and one C. dubliniensis isolates from patients treated with azoles, who developed therapeutic failure against azoles later. Our results revealed a low level of antifungal resistance but a notable rate of azole therapeutic failure among patients with candidemia due to non-albicans Candida species, which threaten the efficacy of fluconazole, the most widely used antifungal in southern regions of Iran. Candidemia studies should not be confined to ICUs and treatment should be administered based on species identification and AFST resultsLay summary: Landscape of candidemia is blurred in Iran, and only two studies from Tehran have extensively explored the epidemiology of candidemia. However, candidemia data from the other regions are notoriously scarce, which precludes from reaching a consensus regarding species distribution, the burden of antifungal resistance, and the clinical features of infected patients. Therefore, we conducted the current prospective candidemia study in Shiraz, one of the largest cities located in the south of Iran, from April 2016 to April 2018. More than 63% of the candidemia infections were treated by fluconazole and species identification and antifungal susceptibility testing were not used for decision making regarding the choice of antifungal treatment. Approximately 70% of the candidemia cases occurred in the wards outside of the ICUs. Candida albicans, C. glabrata, C. parapsilosis, C. tropicalis, and C. dubliniensis were the five leading causative agents of candidemia. Antifungal resistance was rare and fluconazole resistance and/or non-wild type phenotypes were noticed in five isolates, only one was C. albicans and the rest were non-albicans Candida (NAC) species, including C. glabrata, C. dubliniensis, and C. orthopsilosis. Except for C. orthopsilosis, which was isolated from an azole-naïve patient, the rest of isolates were recovered from patients treated with azoles and all showed therapeutic failure to azoles. Collectively, our data will complete the candidemia picture in Iran and show that, although the level of resistance was rare, the therapeutic failure was notable among NAC species, which threatens the efficacy of fluconazole, the most widely used antifungal in Southern regions of Iran. Moreover, we showed that candidemia is poorly managed in Iran since species identification tools along with antifungal susceptibility testing were not used to select appropriate antifungal treatment.

Like severe influenza, coronavirus disease-19 (COVID-19) resulting in acute respiratory distress syndrome (ARDS) has emerged as an important disease that predisposes patients to secondary pulmonary aspergillosis, with 35 cases of COVID-19 associated pulmonary aspergillosis (CAPA) published until June 2020. The release of danger-associated molecular patterns during severe COVID-19 results in both pulmonary epithelial damage and inflammatory disease, which are predisposing risk factors for pulmonary aspergillosis. Moreover, collateral effects of host recognition pathways required for the activation of antiviral immunity may, paradoxically, contribute to a highly permissive inflammatory environment that favors fungal pathogenesis. Diagnosis of CAPA remains challenging, mainly because bronchoalveolar lavage fluid galactomannan testing and culture, which represent the most sensitive diagnostic tests for aspergillosis in the ICU, are hindered by the fact that bronchoscopies are rarely performed in COVID-19 patients due to the risk of disease transmission. Similarly, autopsies are rarely performed, which may result in an underestimation of the prevalence of CAPA. Finally, the treatment of CAPA is complicated by drug–drug interactions associated with broad spectrum azoles, renal tropism and damage caused by SARS-CoV-2, which may challenge the use of liposomal amphotericin B, as well as the emergence of azole-resistance. This clinical reality creates an urgency for new antifungal drugs currently in advanced clinical development with more promising pharmacokinetic and pharmacodynamic profiles., AC was supported by the Fundação para a Ciência e a Tecnologia (FCT) (CEECIND/03628/2017, UIDB/50026/2020 and UIDP/50026/2020), and the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023). This research received no other external funding.

Diabetic foot ulcer infections are the main causes of hospitalization in diabetics. The present study aimed to develop vancomycin and imipenem/cilastatin loaded core-shell nanofibers to facilitate the treatment of diabetic foot ulcers. Therefore, novel core-shell nanofibers composed of polyethylene oxide, chitosan, and vancomycin in shell and polyvinylpyrrolidone, gelatin, and imipenem/cilastatin in core compartments were prepared using the electrospinning technique. The nanofibers were characterized using scanning electron microscopy, transmission electron microscopy, Fourier transform infrared spectroscopy, tensile test, and drug release. The antibacterial activity of drug-loaded nanofibers in different drugs concentrations was evaluated against Methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, and Pseudomonas aeruginosa by disk diffusion method. Furthermore, the cytotoxicity of fibers was investigated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide assay. The obtained results showed that the prepared nanofibers were smooth having a core-shell structure with almost no cytotoxicity. The nanofibrous mats exhibited significant antibacterial activity against S. aureus and MRSA with the inhibition zones of 2.9 and 2.5 cm and gram-negative bacteria species of E. coli and P. aeruginosa with the inhibition zones of 1.9 and 2.8 cm, respectively. With respect to the significant antibacterial activities of these nanofibrous mats, they might be used as suitable drug delivery devices not only for diabetic foot ulcer infections but also for other chronic wounds.

BackgroundDespite being associated with a high mortality and economic burden, data regarding candidemia are scant in Algeria. The aim of this study was to unveil the epidemiology of candidemia in Algeria, evaluate the antifungal susceptibility pattern of causative agents and understand the molecular mechanisms of antifungal resistance where applicable. Furthermore, by performing environmental screening and microsatellite typing we sought to identify the source of infection.MethodsWe performed a retrospective epidemiological-based surveillance study and collected available blood yeast isolates recovered from the seven hospitals in Algiers. To identify the source of infection, we performed environmental screening from the hands of healthcare workers (HCWs) and high touch areas. Species identification was performed by API Auxa-Color and MALDI-TOF MS and ITS sequencing was performed for species not reliably identified by MALDI-TOF MS. Antifungal susceptibility testing followed CLSI M27-A3/S4 and included all blood and environmental yeast isolates.ERG11sequencing was performed for azole-resistantCandidaisolates. Microsatellite typing was performed for blood and environmentalCandidaspecies, where applicable.ResultsCandida tropicalis(19/66) was the main cause of candidemia in these seven hospitals, followed byCandida parapsilosis(18/66), Candida albicans(18/66), andCandida glabrata(7/66). The overall mortality rate was 68.6% (35/51) and was 81.2% forC. tropicalis-infected patients (13/16). Fluconazole was the main antifungal drug used (12/51); 41% of the patients (21/51) did not receive any systemic treatment.Candida parapsilosiswas isolated mainly from the hands of HCWs (7/28), and various yeasts were collected from high-touch areas (11/47), includingNaganishia albida, C. parapsilosisandC. glabrata. Typing data revealed interhospital transmission on two occasions forC. parapsilosisandC. glabrata, and the same clone ofC. parapsilosisinfected two patients within the same hospital. Resistance was only noted forC. tropicalisagainst azoles (6/19) and fluconazole-resistantC. tropicalisisolates (≥8 μg/ml) (6/19) contained a novel P56S (5/6) amino acid substitution and a previously reported one (V234F; 1/6) in Erg11p.ConclusionsCollectively, our data suggest an urgent need for antifungal stewardship and infection control strategies to improve the clinical outcome of Algerian patients with candidemia. The high prevalence ofC. tropicalisjoined by fluconazole-resistance may hamper the therapeutic efficacy of fluconazole, the frontline antifungal drug used in Algeria.

ObjectivesDevelopment and validation of a single-step and accurate reverse transcriptase loop-mediated isothermal amplification technique (RT-LAMP) for rapid identification of SARS COV-2 relative to commercial quantitative reverse transcriptase real-time PCR (qRT-PCR) assays to allow prompt initiation of proper medical care and containment of virus spread.MethodsPrimers showing optimal in-silico features were subjected to analytical sensitivity and specificity to assess the limit of detection (LOD) and cross-reaction with closely- and distantly-related viral species, and clinically prominent bacterial and fungal species. In order to evaluate the clinical utility, our RT-LAMP was subjected to a large number of clinical samples, including 213 negative and 47 positive patients, relative to two commercial quantitative RT-PCR assays.ResultsThe analytical specificity and sensitivity of our assay was 100% and 500 copies/ml when serial dilution performed in both water and sputum. Subjecting our RT-LAMP assay to clinical samples showed a high degree of specificity (99.5%), sensitivity (91.4%), positive predictive value (97.7%), and negative predictive value (98.1%) when used relative to qRT-PCR. Our RT-LAMP assay was two times faster than qRT-PCR and is storable at room temperature. A suspected case that later became positive tested positive using both our RT-LAMP and the two qRT-PCR assays, which shows the capability of our assay for screening purposes.ConclusionsWe present a rapid RT-LAMP assay that could extend the capacity of laboratories to process two times more clinical samples relative to qRT-PCR and potentially could be used for high-throughput screening purposes when demand is increasing at critical situations.

Background Candida glabratais the third leading cause of candidaemia in Turkey; however, the data regarding antifungal resistance mechanisms and genotypic diversity in association with their clinical implication are limited. Objectives To assess genotypic diversity, antifungal susceptibility and mechanisms of drug resistance ofCglabratablood isolates and their association with patients' outcome in a retrospective multicentre study. Patients/Methods Isolates from 107 patients were identified by ITS sequencing and analysed by multilocus microsatellite typing, antifungal susceptibility testing, and sequencing ofPDR1andFKS1/2hotspots (HSs). Results Candida glabrataprevalence in Ege University Hospital was twofold higher in 2014-2019 than in 2005-2014. Six of the analysed isolates had fluconazole MICs >= 32 mu g/mL; of them, five harboured uniquePDR1mutations. Although echinocandin resistance was not detected, three isolates had mutations in HS1-Fks1 (S629T, n = 1) and HS1-Fks2 (S663P, n = 2); one of the latter was also fluconazole-resistant. All patients infected with isolates carrying HS-FKSmutations and/or demonstrating fluconazole MIC >= 32 mu g/mL (except one without clinical data) showed therapeutic failure (TF) with echinocandin and fluconazole; seven such isolates were collected in Ege (n = 4) and Gulhane (n = 3) hospitals and six detected recently. Among 34 identified genotypes, none were associated with mortality or enriched for fluconazole-resistant isolates. Conclusion Antifungal susceptibility testing should be supplemented with HS-FKSsequencing to predict TF for echinocandins, whereas fluconazole MIC >= 32 mu g/mL may predict TF. Recent emergence ofC glabrataisolates associated with antifungal TF warrants future comprehensive prospective studies in Turkey., Major National R&D Projects of the National Health Department [2018ZX10101003]; National Natural Science Foundation of ChinaNational Natural Science Foundation of China (NSFC) [31770161]; Shanghai Science and Technology CommitteeShanghai Science & Technology Committee [17DZ2272900, 14495800500]; Shanghai Municipal Commission of Health and Family Planning [2017ZZ01024-001]; Shanghai Sailing Program [19YF1448000]; Chinese Academy of Engineering [2019-XY-33]; NIHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [AI10902], This work was supported by the Major National R&D Projects of the National Health Department [2018ZX10101003], National Natural Science Foundation of China [31770161], Shanghai Science and Technology Committee [17DZ2272900 and 14495800500], Shanghai Municipal Commission of Health and Family Planning [2017ZZ01024-001], Shanghai Sailing Program [19YF1448000], and the Chinese Academy of Engineering [2019-XY-33]. DSP was supported by NIH grant #AI10902.

The recent global pandemic of COVID-19 has predisposed a relatively high number of patients to acute respiratory distress syndrome (ARDS), which carries a risk of developing super-infections. Candida species are major constituents of the human mycobiome and the main cause of invasive fungal infections, with a high mortality rate. Invasive yeast infections (IYIs) are increasingly recognized as s complication of severe COVID-19. Despite the marked immune dysregulation in COVID-19, no prominent defects have been reported in immune cells that are critically required for immunity to Candida. This suggests that relevant clinical factors, including prolonged ICU stays, central venous catheters, and broad-spectrum antibiotic use, may be key factors causing COVID-19 patients to develop IYIs. Although data on the comparative performance of diagnostic tools are often lacking in COVID-19 patients, a combination of serological and molecular techniques may present a promising option for the identification of IYIs. Clinical awareness and screening are needed, as IYIs are difficult to diagnose, particularly in the setting of severe COVID-19. Echinocandins and azoles are the primary antifungal used to treat IYIs, yet the therapeutic failures exerted by multidrug-resistant Candida spp. such as C. auris and C. glabrata call for the development of new antifungal drugs with novel mechanisms of action., M.H. received research funding by Gilead and Pfizer. D.S.P. receives research support and/or serves on advisory boards for Amplyx, Cidara, Scynexis, N8 Medical, Merck, Regeneron, and Pfizer. He also has a patent covering the detection of fungal species and drug resistance, as well as a pending patent on COVID-19 detection licensed to T2 Biosystems. A.C. was supported by the Fundação para a Ciência e a Tecnologia (FCT) (CEECIND/03628/2017 and PTDC/MED-GEN/28778/2017). Additional support was provided by FCT (UIDB/50026/2020 and UIDP/50026/2020), the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (ERDF) (NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023), the European Union’s Horizon 2020 research and innovation program under grant agreement no. 847507, and the “la Caixa” Foundation (ID 100010434) and FCT under the agreement LCF/PR/HP17/52190003.

The genus Madurella comprising four species, M. fahalii, M. mycetomatis, M. pseudomycetomatis, and M. tropicana, represents the prevalent cause of eumycetoma worldwide. The four species are phenotypically similar and cause an invariable clinical picture, but differ markedly in their susceptibility to antifungal drugs, and epidemiological pattern. Therefore, specific identification is required for optimal management of Madurella infection and to reveal proper epidemiology of the species. In this study, a novel multiplex real-time PCR targeting the four Madurella species was developed and standardized. Evaluation of the assay using reference strains of the target and non-target species resulted in 100% specificity, high analytical reproducibility (R2 values >0.99) and a lowest detection limit of 3 pg target DNA. The accuracy of the real-time PCR was further assessed using biopsies from eumycetoma suspected patients. Unlike culture and DNA sequencing as gold standard diagnostic methods, the real-time PCR yielded accurate diagnosis with specific identification of the causative species in three hours compared to one or two weeks required for culture. The novel method reduces turnaround time as well as labor intensity and high costs associated with current reference methods., Author summary Mycetoma, a progressive and disfiguring disease, is one of the neglected tropical diseases, caused by both bacteria and fungi. Eumycetoma is the fungal type and mainly caused by species of the genus Madurella. Madurella mycetomatis is the most prevalent species worldwide. However, other species such as M. fahalii, M. pseudomycetomatis, and M. tropicana can also cause mycetoma and have a different susceptibility towards the drug used for treating mycetoma patients. Currently, we lack a rapid and non-culture-based technique that can readily identify these four species from clinical samples. Due to its sensitivity, and specificity, real-time PCR is re-recognized by European Organization for Research and Treatment of Cancer (EORTC) to directly identify fungal agents from clinical samples. We developed and validated a multiplex real-time PCR-based technique using the least expensive chemistry to identify Madurella species within 3–4 hours. Development of such a technique will allow rapid diagnosis of eumycetoma and timely initiation of appropriate antifungal therapy.

This study was conducted to find the etiology of acute diarrhea in Iranian children and determine the antimicrobial resistance patterns. The pathogenic bacteria were recovered from 110/269 (40.9%) diarrheal fecal samples with the following profiles: the most predominant pathogen was diarrheagenic Escherichia coli (DEC) (43.6%), comprising enteroaggregative E. coli (23.6%), enteropathogenic E. coli (10.9%), enteroinvasive E. coli (5.5%), and enterotoxigenic E. coli (3.6%); Shigella spp. (37.3%), Salmonella spp. (12.7%) and Campylobacter jejuni (6.4%) were ranked second and fourth in terms of prevalence, respectively. The rates of extended-spectrum beta-lactamase (ESBL) production were 66.7% and 53.7% in DEC and Shigella, respectively. Resistance to ampicillin (AMP) (95.1%), trimethoprim/sulfamethoxazole (SXT) (73.2%), azithromycin (ATH) (21.9%), and ciprofloxacin (CIP) (14.6%) was observed among Shigella isolates. Multidrug resistance phenotype was observed in 24.4% (10/41) of Shigella isolates, with the most common pattern of resistance to cefotaxime, ceftriaxone, ceftazidime, AMP, SXT, and ATH. This study indicates an alarming increase in the ESBL production of DEC and Shigella spp. and identifies them as the two most prevalent diarrhea-causing enteropathogens in the region. The results show that CIP could be an alternative to third-generation cephalosporins against these two pathogens. Therefore, it is proposed that further investigation be done in the pursuit of alternative antibiotics that are effective against the resistant cases. For instance, one study could look into the comparative clinical effectiveness of third-generation cephalosporins versus CIP, the latter not being presently the drug of choice for the treatment of acute diarrhea in children in Iran.

Objective: To perform a laboratory investigation to identify and characterize the causative pathogens of a diarrheal outbreak in the south of Iran in July 2015. Methods: Laboratory investigation was done through standard cultures and molecular methods for causative agent and possible source identification. Antibiotic resistant patterns, extended-spectrum β-lactamase (ESBL) production ability and plasmid profiling were used to characterize the isolated pathogens. Results: Out of 16 stool samples received in the lab, 14 were positive for enteroaggregative E. coli (EAEC), enteroinvasive E. coli (EIEC) and non-O157 enterohaemorrhagic E. coli (EHEC). Of 5 EIEC isolates, 3 were similar in terms of plasmid patterns and ESBL production ability and virulence genes (ipaH+ and virF+). The EAEC isolates were positive for at least one of two virulence genes, agg and aap. Out of 7 EAEC isolates, 2 had the same patterns of plasmid profiles, antibiotic resistance and virulence gene (aap+). Of the 7 EAEC isolates, four had ESBL production ability. The two non-O157 EHEC isolates were positive for stx2 virulent gene and were also susceptible to all tested antibiotics. Conclusions: Laboratory investigation showed that the outbreak was caused by mixed diarrheagenic E. coli pathogroups, possibly due to waste contamination of drinking water.

Extensive advances in technology offer a vast variety of diagnostic methods that save time and costs, but identification of fungal species causing human infections remains challenging in developing countries. Since the echinocandins, antifungals widely used to treat invasive mycoses, are still unavailable in developing countries where a considerable number of problematic fungal species are present, rapid and reliable identification is of paramount importance. Unaffordability, large footprints, lack of skilled personnel, and high costs associated with maintenance and infrastructure are the main factors precluding the establishment of high-precision technologies that can replace inexpensive yet time-consuming and inaccurate phenotypic methods. In addition, point-of-care lateral flow assay tests are available for the diagnosis of Aspergillus and Cryptococcus and are highly relevant for developing countries. An Aspergillus galactomannan lateral flow assay is also now available. Real-time PCR remains difficult to standardize and is not widespread in countries with limited resources. Isothermal and conventional PCR-based amplification assays may be alternative solutions. The combination of real-time PCR and serological assays can significantly increase diagnostic efficiency. However, this approach is too expensive for medical institutions in developing countries. Further advances in next-generation sequencing and other innovative technologies such as clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostic tools may lead to efficient, alternate methods that can be used in point-of-care assays, which may supplement or replace some of the current technologies and improve the diagnostics of fungal infections in developing countries.

Although Cyberlindnera fabinaii is a rare opportunist yeast species, its ability to cause septicemia, produce biofilm, and rapid acquisition of resistance to fluconazole and voriconazole, reinforced the urge for its identification from its closely related species. Widely used biochemical assays mainly identify Cyberlindnera fabinaii as Cyberlindnera jadinii and Wickerhamomyces anomalus, resulting in underestimation of this yeast in clinical settings. Moreover, the urge for a reliable molecular means of identification remains unsolved for 28 years. In order to unequivocally differentiate Cy. fabianii, Cy. mississipiensis, Cy. jadinii, and W. anomalus, we designed a dual-function multiplex polymerase chain reaction (PCR) assay. Challenging our dual-function multiplex PCR assay with 30 most clinically important yeast species, proved its specificity. Although conventional PCR could differentiate four target species, the real-time PCR counterpart due to Tm overlap misidentified Cy. mississipiensis as Cy. jadinii. Alongside of presenting a comprehensive literature review of published cases of Cy. fabianii from 1990 to 2018, we collected various clinical isolates from Tehran, Shiraz, and Fasa (July 1, 2017, to December 31, 2017) to find a passive relative distribution of these closely-related species in Iran. Subjecting our Iranian collection of yeast isolates to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS and LSU and ITS rDNA sequencng revealed six isolates of Cy. fabianii (central venous catheter n = 2 and vaginal swabs n = 4) and one isolate of Cy. jadinii (vaginal swabs). Due to the use of biochemical assays in global ARTEMIS study, we encourage reidentification of clinical isolates of Cy. jadinii and Cy. jadinii using MALDI-TOF or Sanger sequencing that might lead to correcting the distribution of this fungus.

Establishing an effective empirical antifungal therapy requires that national surveillance studies be conducted. Herein, we report the clinical outcome of infections with and the microbiological features of Iranian isolates of Candida glabrata derived from patients suffering from candidemia. C. glabrata isolates were retrospectively collected from four major cities in Iran; identified by a 21-plex PCR, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and large subunit of ribosomal DNA sequencing; and genotyped by amplified fragment length polymorphism (AFLP). Mutations in PDR1, ERG11, and hot spot 1 (HS1) of FKS1 and FKS2 were investigated, and antifungal susceptibility testing (AFST) was performed (by the CLSI M27-A3 and M27-S4 methods). Seventy isolates of C. glabrata were collected from 65 patients with a median age of 58 years. Fluconazole was the most widely used (29.23%) and least effective antifungal agent. The overall crude mortality rate was 35.4%. Only one strain was resistant to fluconazole, and 57.7% and 37.5% of the isolates were non-wild type (non-WT) for susceptibility to caspofungin and voriconazole, respectively. All isolates showed the WT phenotype for amphotericin B, posaconazole, and itraconazole. HS1 of FKS1 and FKS2 did not harbor any mutations, while numerous missense mutations were observed in PDR1 and ERG11. AFLP clustered our isolates into nine genotypes; among them, genotypes 1 and 2 were significantly associated with a higher mortality rate (P=0.034 and P= 0.022, a PDR1, T745A, died despite treatment with fluconazole or caspofungin. Overall, Iranian isolates of C. glabrata were susceptible to the major antifungal drugs. Application of genotyping techniques and sequencing of a specific gene (PDR1) might have prognostic implications.

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=100; 46.9%), vaginal swabs (n=20; 9.4%), bronchoalveolar lavage (n=10; 4.7%) and sputum (n=12; 5.6%) from 68 patients 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.

Echinocandins are the recommended first-line antifungals for treatment of invasive candidiasis. The increasing number of Candida glabrata strains resistant against echinocandins is an emerging health care concern. The rapid detection of resistant C. glabrata isolates is an urgent requirement for clinical laboratories. In this study, we developed the MALDI Biotyper antibiotic (antifungal) susceptibility test rapid assay (MBT ASTRA) for the rapid detection of anidulafungin-resistant C. glabrata isolates directly from positive blood cultures. Of 100 C. glabrata strains, MBT ASTRA classified 69 as susceptible and 29 as resistant. Microdilution assays performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, used as a standard reference, identified 65 susceptible, 9 intermediate, and 26 resistant isolates. Sequencing of hot spot 1 and hot spot 2 regions of the FKS1 and FKS2 genes classified 86 susceptible and 14 resistant isolates. The MBT ASTRA had sensitivity and specificity of 80% and 95%, respectively, compared to the microdilution method. Positive and negative agreement of MBT ASTRA was calculated at 100% and 80%, respectively, compared with the molecular sequencing approach. Together, these results revealed a high accuracy of MBT ASTRA compared to microdilution according to the CLSI and PCR analysis, resulting in a categorical agreement of 90% and 83%, respectively. The validity of MBT ASTRA was 98%. Importantly, MBT ASTRA provided antifungal susceptibility testing (AFST) within 6 h that was both accurate and reliable compared to the other two approaches, which require at least 24 h or are costly. Therefore, this method has the potential to facilitate clinical AFST rapidly at low sample costs for clinical labs already equipped with matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS).

Aim: Presenting the first clinical case of Wickerhamomyces myanmarensis. Patients & methods: Yeast cells were isolated from blood and central venous catheter of a 5.5-year-old male subject. API 20C AUX, MALDI-TOF MS, ITS and LSU rDNA sequencing, and our qPCR assay were used for identification and the MIC values were determined by CLSI M27-A3. Results: ITS and LSU rDNA sequencing identified both isolates as W. myanmarensis, while API 20C AUX and MALDI-TOF MS did not identify them correctly. Our qPCR specifically distinguished W. myanmarensis from W. anomalus. Isolate obtained from blood showed a higher MIC value for fluconazole, voriconazole and posaconazole. Conclusion: Utilization of reliable identification tools might reveal the genuine spectrum of opportunistic yeast species.

Background: Oral candidiasis (OC) has a profound effect on the life quality of immunocompromised patients, such as those undergoing chemotherapy. Objective: Systematic investigation of clinical outcome and microbiological features of yeast isolates recovered from the oral cavity of 150 Iranian patients with hematological malignancies. Design: MALDI-TOF MS, 21-plex PCR, and rDNA sequencing were used for identification. Antifungal susceptibility testing (broth microdilution, CLSI M27-A3/S4) and genotypic diversity of yeast isolates (amplified fragment length polymorphism) were assessed. Results: Nystatin treatment resulted in 70% therapeutic failure and administration of 150 mg fluconazole (FLZ) + nystatin for patients with OC relapse showed 70% clinical failure. Previous history of OC was significantly correlated with FLZ treatment requirement and nystatin failure (P = 0.005, α Candida albicans (80.3%) and Kluyveromyces marxianus (C. kefyr) (12.7%) were the two most prevalent yeast species isolated. FLZ and AMB exhibited the highest geometric mean values. 21-PCR showed 98.9% agreement with MALDI-TOF MS. K. marxianus isolates had the same genotype, while C. albicans isolates grouped in 15 genotypes. Conclusions: Marked rate of therapeutic failure of nystatin necessitated OC treatment with systemic antifungals. K. marxianus was the second most prevalent yeast and 21-plex PCR could be considered as an inexpensive identification tool.

The multidrug-resistant opportunistic yeast species of Candida auris, Candida haemulonii, Candida duobushaemulonii and Candida pseudohaemulonii continue to endanger the healthcare settings around the globe. Due to the lack of a specific qPCR assay for detection of these species from clinical samples, we developed a multiplex qPCR assay. Analytical specificity and sensitivity showed 100% specificity and the sensitivity of up to ten genomes of target species with a high value of reproducibility (R2 >0.99). Subsequently, from spiked serum samples, our qPCR specifically could detect up to ten genomes of C. auris and one genome of C. haemulonii, C. duobushaemulonii and C. pseudohaemulonii (R2 >0.98). Lack of cross reaction with the human DNA, a high degree of specificity and sensitivity, showed the potential of our multiplex PCR for direct detection of C. auris and closely related species from serum samples of suspected patients. Future studies are warranted to assure its applicability in clinical settings.

Identification of opportunistic yeasts in developing countries is mainly performed by phenotypic assays, which are time-consuming and prone to errors. Wrong species identification may result in suboptimal treatment and inaccurate epidemiological data. To improve rapidity and accuracy of species identification, a diagnostic strategy using a stepwise "YEAST PANEL multiplex PCR assays" targeting 21 clinically important yeast species of Candida, Trichosporon, Rhodotorula, Cryptococcus, and Geotrichum was designed. Four hundred CBS reference strains were used for optimization and specificity testing. Eight hundred clinical species were prepared in blinded sets for multiplex polymerase chain reaction (PCR) and matrix-assisted laser desorption time of flight mass spectrophotometry (MALDI-TOF MS) investigation. Results obtained from YEAST PANEL multiplex PCR assay were 100% consistent with those of MALDI-TOF MS. Utilization of pure colony testing showed distinct amplicons for each species, thus eliminating the need for DNA extraction. The targeted yeast species of this assay are responsible for 95% of the yeast infections. In conclusion, due to the high accuracy and coverage of a broad range of yeasts, this assay could be useful for identification in routine laboratories and epidemiological studies.

While being the third leading cause of candidemia worldwide, numerous studies have shown severe clonal outbreaks due to fluconazole-resistant (FLCR) Candida parapsilosis isolates associated with fluconazole therapeutic failure (FTF) with enhanced mortality. More recently, multidrug resistant (MDR) C. parapsilosis blood isolates have also been identified that are resistant to both azole and echinocandin drugs. Amphotericin B (AMB) resistance is rarely reported among C. parapsilosis isolates and proper management of bloodstream infections due to FLZR and MDR isolates requires prompt action at the time of outbreak. Therefore, using a well-established Galleria mellonella model, we assessed whether (a) laboratory-based findings on azole or echinocandin (micafungin) resistance in C. parapsilosis lead to therapeutic failure, (b) LAMB could serve as an efficient salvage treatment option, and (c) distinct mutations in ERG11 impact mortality. Our in vivo data confirm fluconazole inefficacy against FLCR C. parapsilosis isolates carrying Y132F, Y132F + K143R, Y132F + G307A, and G307A + G458S in Erg11p, while LAMB proved to be an efficacious accessible option against both FLCR and MDR C. parapsilosis isolates. Moreover, positive correlation of in vitro and in vivo data further highlights the utility of G. melonella as a reliable model to investigate azole and polyene drug efficacy. © 2020 by the authors. Licensee MDPI, Basel, Switzerland., Pfizer Gilead Sciences Merck Sharp and Dohme, MSD Austrian Science Fund, FWF Astellas Pharma Christian Doppler Forschungsgesellschaft, CDG, Funding: This work was financially supported by Christian DopplerForschungsgesellschaft (CD-Labor Invasive Pilzinfektionen) to C.L.-F., Conflicts of Interest: In the past five years, C. Lass-Flörl has received grant support from the Austrian Science Fund (FWF), MFF Tirol, Astellas Pharma, Gilead Sciences, Pfizer, Schering Plough, and Merck Sharp & Dohme. She has been an advisor/consultant to Gilead Sciences, Merck Sharp & Dohme, Pfizer, and Schering Plough. She has received travel/accommodation expenses from Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas, and Schering Plough and has been paid for talks on behalf of Gilead Sciences, Merck Sharp & Dohme, Pfizer, Astellas, and Schering Plough. All other authors have no conflict of interest to declare., This work was financially supported by Christian Doppler Forschungsgesellschaft (CD-Labor Invasive Pilzinfektionen) to C.L.-F.

Establishing an effective empirical antifungal therapy requires that national surveillance studies be conducted. Herein, we report the clinical outcome of infections with and the microbiological features of Iranian isolates of Candida glabrata derived from patients suffering from candidemia., Establishing an effective empirical antifungal therapy requires that national surveillance studies be conducted. Herein, we report the clinical outcome of infections with and the microbiological features of Iranian isolates of Candida glabrata derived from patients suffering from candidemia. C. glabrata isolates were retrospectively collected from four major cities in Iran; identified by a 21-plex PCR, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and large subunit of ribosomal DNA sequencing; and genotyped by amplified fragment length polymorphism (AFLP). Mutations in PDR1, ERG11, and hot spot 1 (HS1) of FKS1 and FKS2 were investigated, and antifungal susceptibility testing (AFST) was performed (by the CLSI M27-A3 and M27-S4 methods). Seventy isolates of C. glabrata were collected from 65 patients with a median age of 58 years. Fluconazole was the most widely used (29.23%) and least effective antifungal agent. The overall crude mortality rate was 35.4%. Only one strain was resistant to fluconazole, and 57.7% and 37.5% of the isolates were non-wild type (non-WT) for susceptibility to caspofungin and voriconazole, respectively. All isolates showed the WT phenotype for amphotericin B, posaconazole, and itraconazole. HS1 of FKS1 and FKS2 did not harbor any mutations, while numerous missense mutations were observed in PDR1 and ERG11. AFLP clustered our isolates into nine genotypes; among them, genotypes 1 and 2 were significantly associated with a higher mortality rate (P = 0.034 and P = 0.022, α

Background: Candida albicans, Candida glabrata, and Candida parapsilosis are three prevalent causes of candidiasis, worldwide. These species are considered as nine medically important complex species. Limited knowledge about these newly recognized species prompted us to develop a one-step, multiplex PCR to detect and identify them in clinical settings.Methods: Primers targeting Hyphal Wall Protein I gene for the C. albicans, C. dubliniensis, C. africana, Intergenic Spacer for the C. glabrata, C. nivariensis, C. bracarensis, and Intein and ITS rDNA for the C. parapsilosis, C. orthopsilosis, and C. metapsilosis were designed. Using 168 CBS reference strains and 280 clinical isolates, the specificity and reproducibility of the developed assay were evaluated.Results: Our developed assay successfully identified and distinguished all the nine species. No cross-reaction with closely- and distantly-related yeast species, Aspergillus species and human DNA was observed, resulting in 100% specificity. The ambiguous results obtained by MALDI-TOF for C. albicans and C. africana were corrected by our 9-plex PCR assay. This assay identified all the cryptic complex species from two test sets from Iran and China, correctly.Conclusions: Our developed multiplex assay is accurate, specific, cost/time-saving, and works without the tedious DNA extraction steps. It could be integrated into routine clinical laboratories and as a reliable identification tool and has the potential to be implemented into epidemiological studies to broaden the limited knowledge of cryptic species complexes.

Background: Acute diarrhea, a leading cause of morbidity and mortality worldwide, still remains a major global health problem, especially among children in the developing countries. Diarrheagenic E. coli represent one of the most common etiological causes of diarrhea in children worldwide. Objectives: This study was conducted in order to determine the rate of Enteroaggregative E. coli (EAEC) among 50 E. coli isolates as well as its antimicrobial resistance patterns. Methods: A total of 50 Escherichia coli strains had been isolated among children under 5 years of age during 75 reported outbreaks in various provinces of Iran from October 2013 to May 2014. PCR was employed for the identification of different groups of diarrheagenic E. coli. Antimicrobial susceptibility testing was performed using disc diffusion methods. In addition, extended spectrum beta-lactamase (ESBL) production ability was checked by way of combination disc methods of CLSI. Minimum inhibitory concentration of cefotaxime, ceftriaxone, and ceftazidime in EAEC with the ability of ESBL production was determined using the micro-broth dilution method of CLSI. Results: Out of the 50 E. coli isolates, 17 were identified as Enteroaggregative E. coli (EAEC) in that they were positive for at least 1 of the 2 tested virulent genes: agg and aap. ESBL production ability was observed in 4/17 (23.5%) EAEC isolates. MIC of cefotaxime and ceftriaxone in ESBL positive EAEC varied between 8 - 32 µg/mL and 8 - 64 µg/mL, respectively. Resistance to ampicillin and nalidixic-acid (47.1%), trimethoprim/sulfamethoxazole (41.2%), and ciprofloxacin (11.8%) was observed among the EAEC isolates. No evidence of resistance to gentamycin and meropenem was detected. Conclusions: This research has revealed that the most common type of diarrheagenic E. coli among children, who were affected in the diarrheal outbreak in different cities of the country, is Enteroaggregative E. coli (34%). The rate of ESBL positive cases in EAEC isolates were 23.5 %.

Item does not contain fulltext Aspergillus terreus infections are difficult to treat because of the intrinsic resistance to amphotericin B, and higher mortality compared to infections caused by other Aspergillus species. The aim of the present study was to determine the in vitro antifungal activity of amphotericin B and 11 comparators against clinical (n = 36) and environmental (n = 45) A. terreus isolates. In vitro antifungal susceptibility was performed using the CLSI M38-A2 procedure. Amphotericin B exhibited the highest MICs (MIC range, 0.125-4 mug/mL; MIC90 , 2 mug/mL), followed by terbinafine (MIC range, 0.002-1 mug/mL; MIC90 , 1 mug/mL). Only one isolate (1/81) showed amphotericin B MIC above the epidemiologic cut-off value (ECV; 4 mug/mL). None of the isolates had a MIC of >/= ECV for voriconazole, itraconazole and posaconazole. The reasons for the difference in amphotericin B susceptibility patterns between studies remain unknown. The genetic and species diversity, clinical, environmental and ecological factors in Terrei section on various amphotericin B susceptibility profiles in different countries should be considered more as the main reasons associated with these differences.

Although Cyberlindnera fabinaii is a rare opportunist yeast species, its ability to cause septicemia, produce biofilm, and rapid acquisition of resistance to fluconazole and voriconazole, reinforced the urge for its identification from its closely related species. Widely used biochemical assays mainly identify Cyberlindnera fabinaii as Cyberlindnera jadinii and Wickerhamomyces anomalus, resulting in underestimation of this yeast in clinical settings. Moreover, the urge for a reliable molecular means of identification remains unsolved for 28 years. In order to unequivocally differentiate Cy. fabianii, Cy. mississipiensis, Cy. jadinii, and W. anomalus, we designed a dual-function multiplex polymerase chain reaction (PCR) assay. Challenging our dual-function multiplex PCR assay with 30 most clinically important yeast species, proved its specificity. Although conventional PCR could differentiate four target species, the real-time PCR counterpart due to Tm overlap misidentified Cy. mississipiensis as Cy. jadinii. Alongside of presenting a comprehensive literature review of published cases of Cy. fabianii from 1990 to 2018, we collected various clinical isolates from Tehran, Shiraz, and Fasa (July 1, 2017, to December 31, 2017) to find a passive relative distribution of these closely-related species in Iran. Subjecting our Iranian collection of yeast isolates to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS and LSU and ITS rDNA sequencng revealed six isolates of Cy. fabianii (central venous catheter n = 2 and vaginal swabs n = 4) and one isolate of Cy. jadinii (vaginal swabs). Due to the use of biochemical assays in global ARTEMIS study, we encourage reidentification of clinical isolates of Cy. jadinii and Cy. jadinii using MALDI-TOF or Sanger sequencing that might lead to correcting the distribution of this fungus.

Occurrence of non-Candida albicans Candida (NCAC) species that are associated with elevated MIC values and therapeutic failures are increasing. As a result, timely and accurate means of identification to the species level is becoming an essential part of diagnostic practices in clinical settings. In this study, 301 clinically isolated yeast strains recovered from various anatomical sites [Blood (n = 145), other sites (n = 156)] were used to assess the accuracy and practicality of API 20C AUX and 21-plex PCR compared to MALDI-TOF MS and large subunit rDNA (LSU rDNA). MALDI-TOF MS correctly identified 98.33% of yeast isolates, 100% of top five Candida species, 95.7% of rare yeast species, while 1.3% of isolates were misidentified. API 20C AUX correctly identified 83.7% of yeast isolates, 97.2% of top five Candida species, 61.8% of rare yeast species, while 16.2% of yeast isolates were misidentified. The 21-plex PCR, accurately identified 87.3% of yeast isolates, 100% of top five Candida species, 72% of rare yeast species, but it misidentified 1.3% of rare yeast species while 9.9% of whole yeast isolates were not identified. The combination of rapidity of 21-plex PCR and comprehensiveness of API 20C AUX, led to correct identification of 92% of included yeast isolates. Due to expensiveness of MALDI-TOF MS and sequencing, this combination strategy could be the most accurate and inexpensive alternative identification strategy for developing countries. Moreover, by the advent and development of cost-effective, reliable, and rapid PCR machines that cost 130 US dollars, 21-plex could be integrated in routine laboratories of developing and resource-limited countries to specifically identify 95% causative agents of yeast-related infections in human. Databases of MALDI-TOF MS, API 20C AUX, and the number of target species identified by 21-plex require further improvement to keep up with the diverse spectrum of yeast species.