CLSI M27-A3 Research Articles

Evaluation of the Sensititre YeastOne and Etest in Comparison with CLSI M38-A2 for Antifungal Susceptibility Testing of Three Azoles, Amphotericin B, Caspofungin, and Anidulafungin, against Aspergillusfumigatus and Other Species, Using New Clinical Breakpoints and Epidemiological Cutoff Values.

Aspergillosis is an invasive fungal disease associated with high mortality. Antifungal susceptibility testing (AFST) is receiving increasing consideration for managing patients, as well as for surveilling emerging drug resistance, despite having time-consuming and technically complex reference methodologies. The Sensititre YeastOne (SYO) and Etest methods are widely utilized for yeasts but have not been extensively evaluated for Aspergillus isolates. We obtained Posaconazole (POS), Voriconazole (VCZ), Itraconazole (ITC), Amphotericin B (AMB), Caspofungin (CAS), and Anidulafungin (AND) minimum inhibitory concentrations (MICs) for both the Etest (n = 330) and SYO (n = 339) methods for 106 sequenced clinical strains. For 84 A. fumigatus, we analyzed the performance of both commercial methods in comparison with the CLSI-AFST, using available cutoff values. An excellent correlation could be demonstrated for Etest-AMB and Etest-VCZ (p < 0.01). SYO-MICs of AMB, VCZ, and POS resulted in excellent essential agreement (>93%), and >80% for AMB, VCZ, and ITC Etest-MICs. High categoric agreement was found for AMB, ITC, and CAS Etest-MICs (>85%) and AMB SYO-MICs (>90%). The considerable number of major/very major errors found using Etest and SYO, possibly related to the proposed cutoffs and associated with the less time-consuming processes, support the need for the improvement of commercial methods for Aspergillus strains.

Chemical composition of Prangos ferulacea (L.) Lindl., and Prangos uloptera DC. essential oils and their antifungal activities

Introduction: Candida albicans is an important opportunistic pathogen that is responsible for most fungal infections in humans. Secondary metabolites are known to be antimicrobial and antifungal agents. This study aimed to investigate the chemical composition of Prangos ferulacea and P. uloptera essential oils and evaluate the sensitivity of four genera of Candida. Methods: After collecting plant samples, their essential oils were extracted by the distillation method, and their components were analyzed using Gas chromatography–mass spectrometry to identify constituents. In total, 48 species of Candida isolated from clinical specimens were examined in this study. The antifungal activities of essential oils of P. ferulacea and P. uloptera were evaluated according to CLSI M27-A3 compared to fluconazole. Results: Out of the two tested plants, P. ferulacea had the lowest minimum inhibitory concentration (MIC) against Candida species. However, MIC of this plant against C. albicans isolates was higher than 0.121 μL/mL non-albicans species. Both plants were able to inhibit non- albicans species with MIC90 values of 0.0097 and 0.039 μL/mL. However, their MIC90 values were less than fluconazole against Candida isolates. Conclusion: The results of this study suggest that P. ferulacea and P. uloptera essential oils might be used as new antifungal agents.

P364 Quantitative monitoring of Aspergillus fumigatus mycelial growth by optical density

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectivesFilamentous fungi generally form hyphal pellets in liquid culture. This property prevents filamentous fungi from applying the growth monitoring methods used for unicellular organisms such as yeast and bacteria. We have analyzed the biological functions of cell wall polysaccharide α-1,3-glucan and extracellular polysaccharide galactosaminogalactan (GAG) in Aspergillus species, and revealed that both α-1,3-glucan and GAG contribute to the pellet formation. Here we constructed the double disruption mutant of α-1,3-glucan and GAG biosynthesis-related genes (Δags1Δgtb3) in Aspergillus fumigatus AfS35 strain, and used the mutant for quantitative monitoring of the mycelial growth by optical density.MethodsTo measure the optical density of conidia and mycelia in shake-flask culture, conidia (final concentration, 1.0 × 10⁷/ml) of AfS35 or Δags1Δgtb3 strain were inoculated into 50 ml of Aspergillus minimal medium (AMM) medium in a 200-ml Erlenmeyer flask and rotated at 160 rpm at 37°C. At each sampling point, the culture (2 ml) was withdrawn, and 100 μL of the culture was mixed with 100 μL of 4% paraformaldehyde solution in a 96-well plate. OD₆₀₀ was measured in a microplate reader. To evaluate the susceptibility of the Δags1Δgtb3 strain to antifungal agents, conidia (final concentration, 5.0 × 10⁶/ml) were inoculated into 500 μL of RPMI medium containing an antifungal agent (voriconazole, itraconazole, amphotericin B, flucytosine, or micafungin) in a 48-well plate and rotated at 300 rpm using a microplate mixer at 35°C for 15 h, and OD₆₀₀ was measured in triplicate.ResultsIn AMM flask culture, pellets became visible in AfS35 from 9 h, and their size increased with time, whereas the Δags1Δgtb3 hyphae were continuously dispersed (Fig. 1a). The OD₆₀₀ measurements of AfS35 suggested no correlation between apparent AfS35 growth and OD₆₀₀ (Fig. 1b). In the Δags1Δgtb3 strain, the first and third quartiles were 0.633 and 0.595 at 15 h (Fig. 1b), suggesting that the measurement of OD₆₀₀ is suitable for monitoring the growth of the mutant. As an application of the monitoring method, the Δags1Δgtb3 strain was grown in an RPMI medium containing the indicating antifungal agent for 15 h in a 48-well plate (Fig. 2). Growth was repressed in the presence of antifungal agents tested, except for micafungin (Fig. 2). Growth was completely inhibited at 2 μg/ml of voriconazole, 1 μg/ml of itraconazole, 0.5 μg/ml of amphotericin B, and 256 μg/ml of flucytosine (Fig. 2), which was in agreement with MICs determined by CLSI M38-A2.ConclusionWe established a convenient strategy to monitor A. fumigatus hyphal growth. Our method can be directly applied to screening for novel antifungals against Aspergillus species.

P476 Monitoring antifungal resistance in a global collection of Candida spp. surveillance isolates, including C. auris—analysis of resistance in antifungals (ARIA) 2020 study

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesAnalysis of resistance in antifungals (ARIA) is a recent longitudinal global surveillance initiative collecting yeast and fungal isolates from hospitals worldwide designed to determine susceptibility profiles and monitor the resistance trends among antifungal agents. ARIA reports the susceptibility patterns of data concerning echinocandins, second-generation triazoles, and fluconazole against clinical Candida spp., and filamentous fungal isolates from worldwide sources.Methods Candida spp. isolates (n = 662) were collected from hospitals worldwide during 2020 from 13 different sites—Argentina (n = 1), Australia (n = 2), Germany (n = 1), India (n = 2), Italy (n = 1), Panama (n = 1), Spain (n = 1), Turkey (n = 1), United Kingdom (n = 1), and United States (n = 2). These isolates were shipped to a central laboratory at IHMA Europe, Switzerland, and re-identified by MALDI-TOF or molecular methods. The MICs were performed by broth microdilution method in line with CLSI susceptibility testing standards—CLSI M27-A4 and M38-A2—methodologies and percentage susceptibility (%S) were calculated. Antifungals tested were amphotericin B (AMB), anidulafungin (ANID), fluconazole (FLU), isavuconazole (ISA), caspofungin (CASP), micafungin (MIC), posaconazole (POS), and voriconazole (VOR).ResultsTable 1: Summary MIC and susceptibility data of Candida spp. isolates for all countries combinedConclusionsThe data from the ARIA 2020 study indicate that overall antifungal resistance is low among the Candida spp. isolates except for C. glabrata and C. krusei where resistance to one or more antifungal agents was observed. However, there was no significant difference in susceptibility pattern was observed when susceptibility data of C. glabrata and C. krusei from different continents were compared. The emergence of resistance was evident among C. auris isolates as they have shown reduced susceptibility to azoles in this study.Antifungal resistance surveillance and investigation into resistance mechanisms are of paramount importance. The ongoing ARIA surveillance study will provide resources to monitor antifungal resistance trends, provide key information to caregivers and provide essential information with respect to the development of novel antifungal agents.

P051 A case of recalcitrant sporotrichosis by infection of Sporothrix globosa

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesSporotrichosis is the leading subcutaneous mycosis caused by the Sporothrix (S.) schenckii complex. S. globosa is the causative organism of fixed sporotrichosis in Korea. The preferred regimen of cutaneous sporotrichosis is itraconazole for 3-6 months, however, there were few studies for recalcitrant sporotrichosis.MethodsIn 2018, we performed a histological examination of a patient who suffered sporotrichosis for 3 years and cultured part of the specimen. Despite various regimens for years, improvement and exacerbation were repeated, so we took another skin biopsy and cultured it in 2021. Isolates from the 2018 and 2021 lesions were identified as S. globosa by ribosomal DNA ITS sequencing (GenBank accession number: MH499862 and MH499863). The in vitro antifungal sensitivity tests were performed by broth microdilution method according to CLSI M38-A2 guidelines or Sensititre YeastOne® manufacturer's instructions. They were incubated at 30°C in a non-CO2 incubator for 7 days.ResultsIn 2018, histologically, we observed chronic inflammatory granuloma comprising lymphocytes, histiocytes, and giant cells, and several spores with periodic acid-Schiff (PAS) staining. Microscopic findings and ITS sequences of rRNA gene were identical with S. globosa. The antifungal susceptibility profile in 2018 revealed sensitive to terbinafine (0.125 μg/ml), and moderate to high MIC values for amphotericin B (2 μg/ml), itraconazole (>16 μg/ml), voriconazole (>16 μg/ml), and echinocandins (>16 μg/ml). Treatment with terbinafine, itraconazole, or amphotericin B, the skin lesions were partially improved, but were not cured. In 2021, we took another skin biopsy and culture specimen. Histopathological and mycological examination results were the same as before. The antifungal susceptibility profile revealed sensitive to itraconazole (0.5/ml), and high MIC for others. Clinically, skin lesions were not improved with the use of itraconazole 200 mg/d. Itraconazole 400 mg/d with local heating induced moderate improvement. There was no evidence of immune deficiency.ConclusionWe experienced recalcitrant sporotrichosis that did not respond to itraconazole and terbinafine, and the sensitivity of antifungal was changed. In this case, the combination treatment including local heating, saturated KI may be considered, and frequent antifungal susceptibility tests are needed.

P107 Spectrum of Dermatophyte infections and drug susceptibility pattern of Dermatophytes in patients visiting to tertiary care hospital in Chhattisgarh state of India

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectives1. To isolate and identify various species of Dermatophytes from clinical specimens 2. To perform and analyze the antifungal susceptibility testing of isolated Dermatophytes for commonly used antifungal agents; terbinafine and itraconazole.MethodsA prospective study was conducted from December 2019 to October 2021. Clinical specimens (skin, hair, and nail) from suspected cases of dermatophytosis were received and processed in the department of microbiology. All the samples were subjected to microscopic examination and culture by standard techniques. Their clinico-demographic profile was obtained. Specimen were processed for KOH and fungal culture. Dermatophytes were identified by studying macroscopic and microscopic characteristics of the isolates. The conidium-forming dermatophyte isolates were processed for antifungal susceptibility testing for terbinafine and itraconazole by Microbroth dilution testing following the CLSI M-38A2 guidelines.ResultsTotal 248 patients with male predominance (68%) were noted in the above-mentioned study period. Predominance of study population belonged to rural area. Maximum numbers of cases were from the age group 21-30 years. Majority of patients belong to poor socioeconomic status. Out of 248 samples, 178 (72%) had a positive KOH mount amongst which 72% had positive culture results. Amongst 2 4881% were skin scraping, 17% were nail, and 1.6% hair samples were processed. Out of culture-positive samples 52% were Dermatophytes. The most clinical form of dermatophytosis was combination of both Tinea cruris and T. carporis (31%) followed by T. cruris (22%), and T. corporis (17%) for which skin scraping was processed. The most common isolate was Trichophyton tonsurans (73%) followed by T. mentagrophytes (10%), and T. verrucosum. Onychomycosis was diagnosed in 17% patients of which 59% were positive by KOH 49% were culture positive.11.5% isolates from nails were dermatophytes.Antifungal susceptibility testing was done by Microbroth dilution method and analyzed the range. The MIC range of major isolates, i.e., T. tonsurans showed MIC ranges against terbinafine <0.03-4 μg/ml and itraconazole 0.03-2 μg/ml. Trichophyton mentagrophyte for terbinafine <0.12-4 μg/ml and for itraconazole 0.12-2 μg/ml. Four isolates of T. tonsurans had higher MIC values for terbinafine and two isolates had higher MIC for itraconazole. One isolate of T. mentagrophytes had higher MIC values of itraconazole, and one another isolate had higher MIC for terbinafine.ConclusionThis study highlights the change in pattern of causative agents of dermatophytosis. The present study showed the predominance of T. tonsurans. More extensive studies are needed to evaluate the cut-off range of antifungal susceptibility testing of dermatophytes with clinical follow-up to see the response of respective antifungals and to guide the therapy.

P009 Investigation of in vitro antifungal susceptibility testing and genetic diversity of clinical isolates of Trichophyton benhamiae and Trichophyton eriotrephon in Iran

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMBackground Trichophyton benhamiae is a zoophilic dermatophyte, known as one of the causative agents of dermatophytosis.ObjectivesThe purpose of this study was to explore the genotypes of T. benhamiae strains isolated from geographically different areas of Iran and also to evaluate in vitro antifungal susceptibility profile of these strains against seven antifungal drugs.MethodsA total of 22 strains of T. benhamiae and 2 strains of T. eriotrephon were isolated from patients with distinct types of dermatophytosis. DNA extraction and amplification of rDNA regions using ITS1 and ITS4 primers were conducted on the isolates. The in vitro antifungal susceptibility of posaconazole (PSC), voriconazole (VRC), itraconazole (ITC), ketoconazole (KET), caspofungin (CAS), terbinafine (TRB) and griseofulvin (GRZ) was evaluated according to CLSI M38-A2 protocol.ResultsThe multiple alignments of the ITS-rDNA sequences of T. benhamiae indicated a mean similarity of 99.5%, with 0-3 interspecies nucleotide differences. The geometric mean (GM) values of minimum inhibitory concentrations (MICs) and minimum effective concentrations (MECs) across the all isolates were respectively: TRB: 0.025 mg/l, PSC: 0.032 mg/l, ITC: 0.050 mg/l, and VRC: 0.059 mg/l with lower values and CAS: 0.31 mg/l, KTZ: 0.56 mg/l, and GRZ: 0.76 mg/l with higher values.ConclusionDiverse ITS sequence types of T. benhamiae were shown in different geographical regions of Iran. The TRB, PSC, and ITC were the most effective drugs against T. benhamiae strains, respectively. Furthermore, in our study, two strains of T. eriotrephon as a scarce dermatophyte species were described.

P071 Isolate profiling and antifungal susceptibility determination for terbinafine and itraconazole among dermatophytes in a tertiary care hospital of western rajasthan

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectiveTo determine the species distribution of causative agents causing dermatophytosis, and their antifungal susceptibility pattern for terbinafine and itraconazole in trichophyton spp among samples collected in patients with dermatophytosis clinical suspicion during the period of December 1, 2020 to January 31, 2022.Materials and MethodsThis is a prospective study conducted in the Department of Microbiology of a tertiary care super specialty and referral Centre of western Rajasthan from December 1, 2020 to January 31, 2022.Skin scraping, nail clipping, and hair pluckings were collected in mycology lab from clinically suspected cases of dermatophytosis presenting to the department of dermatology for conventional identification, and antifungal susceptibility testing.The specimens were subjected to direct KOH and calcofluor white microscopy and conventional fungal culture on SDA at 25 ͦ C and 37 ͦ C.The cultures positive for dermatophytes were speciated by microslide culture lactophenol cotton blue mount, hair perforation test, and urease test.The isolates identified as Trichophyton spp were taken up for antifungal susceptibility testing against terbinafine and itraconazole by microbroth dilution according to CLSI-M38 A2 guidelines. Further terbinafine resistance gene evaluation for detection of C1191A and T1189C single nucleotide polymorphism in Squalene epoxide by Amplified Refractory Mutation System-Polymerase chain Reaction (ARMS-PCR) is undergoing for trichophyton spp.ResultsOver the 14-month study period, the laboratory processed total of 174 specimens: 134 skin scraping, 36 nail clipping, and 4 hair pluckings. Of them, 106 (61.62%) specimens were microscopy positive and 111 (63.79%) were culture positive. Out of the 111 culture-positive agents isolated, 94 (84.68%) were found to be dermatophytes. On isolate profiling of 94 dermatophytes T. mentagrophyte was found to be most common 45 (48%) followed by T. rubrum 27 (29%), T. tonsurans 20 (21%), T. verrucosum 1 (1.1%), and Microsporum spp 1(1.1%). Antifungal susceptibility of 93 Trichophyton spp against terbinafine showed resistance among 58.06% isolates with 83.33% isolates among terbinafine resistant cases showing ≥4 μg/ml minimum inhibitory concentration. There was no resistance detected for itraconazole with microbroth dilution.ConclusionA total of (54.02%) skin, hair, nail infections were found to be caused by dermatophytes.On isolate profiling, T. mentagrophyte, T. rubrum, and T. tonsurans were found to be predominant species among our isolates showing altered trend of local isolates from T. tonsurans being second most common spp isolated in past.On antifungal susceptibility >55% isolates showed resistance for Terbinafine with >80% having higher MIC of ≥4 μg/ml on the contrary there was no observed resistance for itraconazole.There is a need for encouraging dermatologists for prescribing routine fungal microscopy, culture, and AFST for dermatophytes in Western Rajasthan, to reduce the indiscriminate use of antifungals.

P053 Invasive Mucormycosis (Zygomycosis) -Pre-Covid Era and Covid Era: a retrospective study at a tertiary care centre, chennai

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PMObjectivesTo compare the prevalence and clinical presentation of Mucormycosis in pre-COVID era (January 2017-December 2019) and COVID era (January 2020-till date).To compare the AFST pattern of the zygomycetes causing Mucormycosis in pre-COVID era and COVID era.MethodsThis is a retrospective, hospital-based descriptive study. This study included patients admitted during the pre-COVID and COVID era at a tertiary care center, Chennai. The cases were categorized into two: (1) possible mucormycosis cases which included direct microscopy [Potassium hydroxide (KOH) and histopathological examination] positives and (2) confirmed cases which included direct microscopy and culture positives. Direct microscopic examinations like KOH wet mount and histopathological examination (H and E stain and special stains) were performed. Samples were cultured on Sabourauds dextrose agar and identification was done by analyzing the microscopic morphology using lactophenol cotton blue mount. AFST was performed for culture positive isolates with amphotericin B, itraconazole, posaconazole, voriconazole and isavuconazole by microbroth dilution method according to CLSI M38-A2.ResultsDuring the Pre-COVID era, out of the 365 samples received in the laboratory, 35 were possible mucormycosis cases. Only 17 were confirmed cases, out of which 16 grew Rhizopus oryzae and 1 grew Apophysomyces elegans. During the COVID era, among 886 samples received in the laboratory, 143 were possible mucormycosis cases, and 31 were confirmed cases that grew Rhizopus oryzae (26), Rhizomucor pusillus (2), Mucor sp (2), and Basidiobolus ranarum (1). Though the risk factors were common during the pre-COVID and COVID era, additional risk factors like steroid therapy (19.2%), and COVID infection (28.7%) were seen during the COVID era. Though clinical presentations were common during both pre-COVID and COVID era, additional complications like epistaxis (0.57%), orbital cellulitis (32.7%), and loss of smell (8.04%) were seen during COVID era. The prevalence of complications was more during COVID era compared to pre-COVID era. Treatment received during the pre-COVID era was only amphotericin B, whereas during the COVID era majority of the patients received posaconazole (74.5%) followed by liposomal amphotericin B (25.5%). The antifungal susceptibility test showed the following mean minimum inhibitory concentration (MIC) values: amphotericin B (1.8 μg/ml), itraconazole (3.6 μg/ml), posaconazole (0.31 μg/ml), and voriconazole (1.61 μg/ml) during the pre-COVID era while the mean MIC values during the COVID era had the following variations: amphotericin B (0.97 μg/mL), itraconazole (13.6 μg/ml), Posaconazole (13.4 μg/ml), voriconazole (14.5 μg/ml), and isavuconazole (1.10 μg/ml).ConclusionHigh incidence of Mucormycosis during the COVID-19 era may be related to common risk factors of COVID and mucormycosis. Though most of the risk factors and clinical presentations were similar during the pre-COVID and COVID era, serious complications like loss of vision and the percentage of complications were more during COVID era which may be attributed to the increased invasiveness of Zygomycetes during COVID infection. The high mean MIC value of amphotericin B during pre-COVID era and higher mean MIC value of posaconazole during COVID era may be contributed to the higher usage of these antifungals. Usage of the antifungal agents is the main contributor toward the resistance. Newer azole like isavuconazole which had a low mean MIC in our study, can be considered as a good therapeutic option for the future management of resistant infections. Hence timely management of the patients with an appropriate antifungal agent by performing AFST will help in the reduction of resistance in the future.

P441 Molecular identification, genotyping, and antifungal susceptibility of Trichosporon species isolated from clinical samples of patients at various parts of the Indian subcontinent

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives(1) To study mycological characteristics of strains belonging to Trichosporon and its related genera obtained from clinical samples of patients from India. (2) Molecular identification by intergenic spacer (IGS) region 1 sequencing of the rDNA locus. (3) Genotyping of the major causative agent, T. asahii, and its in vitro drug susceptibility testing.Materials and Methods: A total of 55 clinical isolates of Trichosporon species were collected from NCCPF (National culture collection of pathogenic fungi) PGIMER, Chandigarh along with different health institutions of India. These isolates were recovered from urine, blood, sputum, nail, tissue biopsy, pleural fluid, hair, BAL, and wound discharge over a period of 12 years (2006–2018). The isolates were molecularly characterized and genotyped using IGS-1 region sequencing. In vitro drug susceptibility testing of the isolates was performed against amphotericin-B, fluconazole, itraconazole, voriconazole, and posaconazole according to the CLSI M27-A3 guidelines (CLSI 2008).ResultsPredominant underlying risk factors identified were presence of an indwelling catheter, use of broad-spectrum antibiotics, and presence of comorbid conditions such as diabetes, hypertension, and anemia. A total of 47 (85%) of the 55 isolates were identified as T. asahii, 6 were T. inkin (11%), and 2 were Cutaneo Trichosporon dermatis (3.6%). Trichosporon asahii genotype III (22; 41%) was the most common type, followed by genotype IV (12; 22%), I (8; 15%), and VII (2; 4%). In addition to the 15 known T. asahii genotypes, one novel genotype was identified in this study. Indian T. asahii isolates showed high MIC ranges to amphotericin B (0.06-4 μg/l) and fluconazole (0.25-64 μg/l). Relatively low MIC ranges were found in the case of voriconazole (0.03-1 μg/l), Posaconazole (0.06-1 μg/l), and itraconazole (0.06-1 μg/l). Voriconazole appeared to be the most active drug in maximum T. asahii isolates. The MICs for all the drugs were comparatively lower in the case of non-T. asahii strains.Conclusion Trichosporon asahii remains the most common etiology of Trichosporonosis in India and presents a challenge for both diagnosis and treatment. With increasing drug resistance, therapeutic options are limited, and antifungal regimens with triazoles especially voriconazole appear to be the best. Accurate timely identification, removal of indwelling catheters/central venous lines, and voriconazole-based treatment along with control of underlying conditions were associated with favorable outcomes. Identification of the novel genotype has epidemiological implications and requires further work up.

Genotypic diversity and antifungal susceptibility of Scedosporium species from clinical settings in China.

Scedosporium species have drawn significant interest as inhabitants of polluted soil and water and as cause of high mortality in near-drowning patients. So far, most cases have been reported from Europe and Australia, while knowledge on their prevalence and genotypic diversity from Asia is scant. To increase the knowledge of the genetic diversity and in vitro antifungal susceptibility of Scedosporium species involved in human infections from China. Here, we applied the ISHAM-MLST consensus scheme for molecular typing of Scedosporium species and revealed both high species diversity and high genotypic diversity among 45 Chinese clinical Scedosporium isolates. Among the five species, Scedosporium boydii (n=22) was the most common, followed by S. apiospermum (n=18), S. aurantiacum (n=4) and S. dehoogii (n=1). S. aurantiacum was reported for the first time from clinical samples in China. The predominant sequence types (STs) were ST17 in S. apiospermum, ST4 in S. boydii and ST92 in S. aurantiacum, including four novel STs (ST40, ST41, ST42 and ST43) in S. apiospermum. Based on the CLSI-M38 A2 criterion, voriconazole was the only antifungal compound with low MIC values (MIC90 ≤ 1μg/ml) for all Scedosporium isolates in our study. The genetic diversity of clinical isolates of Scedosporium species from China is extremely high, with S. boydii being predominant and S. aurantiacum being firstly reported here. VOR was the only antifungal compound with low MIC values for all Scedosporium isolates in our study, which should be recommended as the firstline antifungal treatment against scedosporiosis in China.

Activities and mechanisms of oregano, marjoram and rosemary essential oils against Malassezia pachydermatitis isolates from canine and feline otitis against Malassezia pachydermatitis isolates from canine and feline otitis

Malassezia pachydermatis is an opportunistic yeast found in the ear canal of small animals; however, the current azole-based therapy applied to it has failed to achieve clinical success due to the antifungal resistance. This issue has encouraged the studies in natural products, such as Origanum vulgare (oregano), Origanum majorana (marjoram) and Rosmarinus officinalis (rosemary) essential oils, although their mechanism of action remains unclear. Malassezia pachydermatis specimens deriving from otitis cases in dogs (n = 22) and cats (n = 2) were subjected to CLSI M27-A3. Sorbitol protection and ergosterol effect were analyzed to investigate their mechanism of action. Fungistatic (MIC) and fungicidal (MFC) activities were observed for oregano (MIC$_{90}$/MFC$_{90}$: 0.625 mg/mL); marjoram (MIC$_{90}$/MFC$_{90}$: 2.5 mg/mL) and rosemary MIC$_{90}$/MFC$_{90}$ &gt; 2.5 mg/mL). Oregano showed superior antifungal effect even at lower MIC and MFC values. All three oils acted on cell wall and at complexation to fungal ergosterol. By gas chromatography (GC-FID), carvacrol was the major compound found in oregano (73.9%); 1,8-cineole was for marjoram and rosemary (20.9% and 49.4%, respectively). These findings support the potential use of these essential oils to treat canine and feline otitis caused by Malassezia pachydermatis.

Multicenter Study of Susceptibility of Aspergillus Species Isolated from Iranian University Hospitals to Seven Antifungal Agents.

ABSTRACTAspergillus species are a major cause of life-threatening invasive infections and noninvasive diseases. This study seeks to investigate the frequency of Aspergillus species among Iranian patients and their susceptibility to seven antifungals. In a cross-sectional study, 233 Aspergillus isolates were collected from 11 university hospitals in Iran between 2018 and 2021. Aspergillus isolates were identified based on colony morphology, microscopic characteristics, PCR-restriction fragment length polymorphism (RFLP), and sequencing of the beta-tubulin gene. The CLSI M38-A2 reference methodology was used for antifungal susceptibility testing of amphotericin B, voriconazole, posaconazole, itraconazole, luliconazole, isavuconazole, and caspofungin. Members of Aspergillus section Flavi (117/233, 50.2%), Aspergillus section Nigri (77/233, 33.1%), Aspergillus section Fumigati (21/233, 9%), Aspergillus section Terrei (14/233, 6%), Aspergillus pseudodeflectus (2/233, 0.85%), and Aspergillus melleus (2/233, 0.85%) were isolated from the samples. The lowest 0.25 MIC90 values for all isolates tested were for luliconazole (0.016 μg/mL) and isavuconazole (0.250 μg/mL), and the highest value was observed for itraconazole (≥ 8μg/mL). The 90% minimum effective concentration (MEC90) value for caspofungin was 0.125 μg/mL. MIC90 values for voriconazole, amphotericin B, and posaconazole were 1, 2, and 2 μg/mL, respectively. The non-wild-type species were presented for amphotericin B (3%), voriconazole (1.3%), posaconazole (2.6%), luliconazole (1.3%), isavuconazole (1.7%), and caspofungin (4.7%). Positive correlations in the MIC values of azole antifungals were observed, and using one azole increases the MIC value rates of other ones. None of the species were pan-azole resistant. Species of Aspergillus section Flavi were the most common Aspergillus species isolated from Iranian samples. Luliconazole, caspofungin, and isavuconazole present the most effective antifungal agents for treatment of infection due to Aspergillus species. Susceptibility tests should be performed frequently in each region for the best management of patients.IMPORTANCE Aspergillus species are the leading cause of invasive aspergillosis in immunocompromised hosts. The susceptibility of Aspergillus species to antifungal agents might be different. Azole-resistant species have emerged worldwide. Performing susceptibility testing in each region can help in the best management of patients. Here, we show the epidemiology and distribution of Aspergillus species in Iran and their susceptibility patterns for seven antifungal agents. The significant points of the present study are that species of Aspergillus section Flavi are the most prevalent Aspergillus species isolated from 11 university hospitals. Luliconazole, caspofungin, and isavuconazole were effective antifungal agents against all Aspergillus species.

Green synthesis of selenium nanoparticles and evaluate their effect on the expression of ERG3, ERG11 and FKS1 antifungal resistance genes in Candida albicans and Candida glabrata.

Drug resistance in Candida species has been considerably increased in the last decades. Given the opposition to antifungal agents, toxicity and interactions of the antimicrobial drugs, identifying new antifungal agents seems essential. This study assessed the antifungal effects of nanoparticles (NPs) on the standard strains of Candida albicans and Candida glabrata and determined the expression genes, including ERG3, ERG11 and FKS1. Selenium nanoparticles (Se-NPs) were biosynthesized with a standard strain of C. albicans and approved by several methods including, ultraviolet-visible spectrophotometer, X-ray diffraction technique, Fourier-transform infrared analysis, field-emission scanning electron microscopy and EDX diagram. The antifungal susceptibility testing performed the minimum inhibitory concentrations (MICs) using the CLSI M27-A3 and M27-S4 broth microdilution method. The expression of the desired genes was examined by the real-time PCR assay between untreated and treated by antifungal drugs and Se-NPs. The MICs of itraconazole, amphotericin B and anidulafungin against C. albicans and C. glabrata were 64, 16 and 4 µg ml-1 . In comparison, reduced the MIC values for samples treated with Se-NPs to 1 and 0·5 µg ml-1 . The results obtained from real-time PCR and analysis of the ∆∆Cq values showed that the expression of ERG3, ERG11 and FKS1 genes was significantly down-regulated in Se-NPs concentrations (P < 0·05). This study's evidence implies biosafety Se-NPs have favourable effects on the reducing expression of ERG3, ERG11 and FKS1 antifungal resistance genes in C. albicans and C. glabrata.

In vitro activity of immunosuppressive agents against Cryptococcus neoformans

IntroductionInfections caused by Cryptococcus neoformans are a major cause of fungal mortality in HIV-infected/AIDS patients and in those receiving organ transplants. We evaluated the in vitro activity of tacrolimus and cyclosporine in combination with amphotericin B and fluconazole against C. neoformans. MethodsMICs were determined against a total of 30 clinical isolates of C. neoformans by the microdilution method following the CLSI M27-A3 guidelines and by the checkerboard method. ResultsTacrolimus and cyclosporine A showed in vitro activity against cryptococcal isolates. The combination of amphotericin B with cyclosporine A or tacrolimus was synergistic against 90% and 30% of isolates, respectively. Synergism was also observed with the combination of fluconazole with cyclosporine A or tacrolimus, against 70% and 20% of isolates, respectively. ConclusionsThe synergistic interactions between the calcineurin inhibitors and antifungal drugs against C. neoformans isolates, could potentially have a role in devising novel therapeutic strategies for this opportunistic mycosis.

Anti-Pythium insidiosum activity of MSI-78, LL-37, and magainin-2 antimicrobial peptides.

We investigated the anti-Pythium insidiosum activity of the antimicrobial peptides (AMPs) MSI-78, LL-37, and magainin-2. To detect the minimum inhibitory concentration (MIC), fourteen clinical strains were incubated with the AMPs following the CLSI M38-A2 protocol. All three AMPs showed antimicrobial activity with an MIC range of 20-80mg/L against all strains. We concluded that the evaluated AMPs have great potential as anti-Pythium insidiosum agents, and their activity deserves to be more explored in further research. Antimicrobial peptides were tested against Pythium insidiosum, a microorganism that causes a difficult-to-treat disease in animals and humans. These peptides have been shown to be able to kill P. insidiosum and may be candidates for use in the treatment of this infection.

Antifungal susceptibility profile and biofilm production of Candida spp. isolated from catheterised patients with urinary tract infection

BACKGROUND: Candida species, which are present as normal flora in healthy individuals, are known to cause opportunistic infections with high rates of mortality, especially in immunocompromised individuals. Urinary tract infection (UTI) is the most common type of nosocomial infection, and about 10%-15% of them are due to Candida species. Catheter-associated infections due to Candida spp. are mainly due to biofilm formation.OBJECTIVE: This study aims to speciate Candida isolates from catheterised patients with UTI infection and to compare the antifungal susceptibility pattern with their biofilm production.MATERIALS AND METHODS: A total of 55 Candida species were isolated from the urine samples of catheterised patients with UTIs over a period of nine months. Patients' demographic details and risk factors were collected. All the isolates were identified and confirmed by routine phenotypic and genotypic techniques. The rare species that could not be identified by routine techniques were identified by sequencing the internal transcribed spacer region. Biofilm production was detected by tube method, and the antifungal susceptibility testing was done by broth microdilution method (CLSI M27-A3 guidelines).RESULTS: In our study, 55 Candida species were identified, among which the most predominant species was found to be Candida tropicalis 23 (42%). Among 48 biofilm producers (87.2%), only 2 (4.1%) isolates of Candida albicans and 3 (6.2%) isolates of C. tropicalis were resistant to Amphotericin B and Fluconazole. The MIC values for Amphotericin B, Fluconazole and Itraconazole were high in both Candida auris (2) and Candida lusitaniae (1), although one isolate of each species was a potent biofilm producer. In Candida catenulata (4), three isolates had high MIC value for Amphotericin B and two isolates had high MIC value for Itraconazole and three isolates produced biofilm.

Isolation of antifungal-resistant Candida from the blowholes of captive dolphins.

In this study, we isolated eight strains of Candida albicans from the blowhole air cultures of eight dolphins (one Pacific white-sided dolphin and seven bottlenose dolphins) housed at the Enoshima Aquarium. The minimum inhibitory concentrations of antifungals for these isolates were determined by conducting E-test and broth microdilution assays using the CLSI M27-A3 protocol antifungal susceptibility testing method. Only one of the eight dolphins from which Candida had been isolated had been treated with amphotericin B (AMB), and four had been treated with itraconazole (ITZ). All isolates were identified as Candida albicans, and all were resistant to both ITZ and voriconazole, though the isolates exhibited susceptibility to AMB and micafungin. Based on our findings, we suspect that the frequency of occurrence of azole-resistant Candida species is increasing in captive dolphins as well as in their aquarium environments.

Candida albicans blastospores and hyphae respond differentially to fluconazole: additional virulence factor of germination process

Background: The morphologic transition from yeasts (blastospores) to hyphae, through the germination process, is a crucial virulence factor of Candida albicans ( C. albicans), as it enhances adherence, tissue invasion and biofilm formation. The study aimed at comparing fluconazole susceptibility patterns of C. albicans yeast and germinated forms. Methods: Six C. albicans strains, including C. albicans ATCC 10231 and 5 clinical isolates were tested in yeast forms and germinated forms for fluconazole susceptibility using CLSI M27-A3 broth microdilution and CLSI M44-A2 disk diffusion reference methods. Results: Fluconazole minimal inhibitory concentrations (MICs) were 0.125-1 µg/ml and 0.25-4 µg/ml for yeasts and germinated forms, respectively. The sensitivities of yeasts were higher than those of their corresponding germinated forms with 1 to 4-fold dilutions differences. The MIC50s of yeasts and germinated forms were 0.125 and 1 µg/ml, respectively, with 3-fold dilution difference, while MIC90s were 1 and 4 µg/ml for yeasts and germinated forms, respectively, with 2-fold dilution difference. Means of inhibition zones were 31-46.7 mm and 29.3-43.7 mm for yeasts and germinated forms, respectively. The germinated forms of all tested strains had inhibition zones smaller than those of their corresponding yeast forms with 0.2-4.8 mm differences. There was significant statistical difference in sizes of inhibition zones between yeasts and germinated forms (p = 0.043). Conclusions: Yeast and hyphae of C. albicans, both in planktonic state, respond differentially to fluconazole, as the hyphae are less sensitive than their yeasts counterparts, which can be considered as a virulence factor for hyphae, which should be considered in treatment plans of candidal infections.

Multi-locus sequence typing reveals genotypic similarity in Nigerian Cryptococcus neoformans AFLP1/VNI of environmental and clinical origin.

IntroductionPigeon droppings are among the major environmental sources of Cryptococcus neoformans AFLP1/VNI, from where the organism infects susceptible humans and animals resulting in cryptococcosis. Until now, C. neoformans AFLP1B/VNII was the only molecular type reported in Nigeria. Effective clinical treatment of this infection has occasionally been stymied by the emergence of antifungal non-susceptible, and resistant strains of C. neoformans AFLP1/VNI.Hypothesis/Gap StatementPigeon droppings harbour C. neoformans and HIV/AIDS patients are among the susceptible population to develop cryptococcal infection. Epidemiological data on cryptococcal prevalence is limited in Nigeria.AimTo investigate the environmental prevalence of C. neoformans in South-eastern Nigeria and compare the isolates with other lineages by using molecular and microbiological tools.MethodologyA total of 500 pigeon droppings and 300 blood samples of HIV/AIDS patients were collected, respectively, from five market squares and three tertiary healthcare centres within the Nsukka area of South-eastern Nigeria. The antifungal susceptibility of the C. neoformans isolates to amphotericin B, fluconazole, 5-fluorocytosine, itraconazole, voriconazole, posaconazole, and isavuconazole was investigated based on the CLSI M27-A3 protocol. Yeasts were identified by MALDI-TOF MS, thereafter Cryptococcus MLST was performed according to the International Society for Human and Animal Mycology (ISHAM) consensus scheme.Results C. neoformans was recovered from 6 (1.2 %) pigeon droppings and 6 (2 %) blood cultures of HIV/AIDS patients. Molecular analyses indicated that all cryptococcal isolates belong to serotype A and the AFLP1/VNI molecular type with sequence type (ST)32. Infection with C. neoformans was independent of sex and age of the patients investigated. All C. neoformans isolates were susceptible to the seven antifungal agents.ConclusionThis is the first report on the prevalence of C. neoformans AFLP1/VNI (ST32) in environmental and clinical samples from Nigeria. The antifungal susceptibility indicates that antifungal resistance by C. neoformans is yet a rare occurrence in Nigeria.