Clinical Mycology Laboratory Research Articles

A simple culture medium for phenotypic characterization and long-term storage of medically relevant fusarioid fungi

Fusarioid fungi, particularly Neocosmospora solani and Fusarium oxysporum, are emerging as significant human pathogens, causing infections ranging from localized mycoses to life-threatening systemic diseases. Accurate identification and preservation of these fungi in clinical laboratories remain challenging because of their diverse morphologies and specific growth requirements. This study evaluated a novel milk-honey and malt agar (MHM) against conventional media for cultivating and preserving 60 clinical fusarioid isolates, including Neocosmospora spp. (n = 47), Bisifusarium spp. (n = 5), and Fusarium spp. (n = 8). Compared with Sabouraud dextrose 2 % agar (SDA) and malt extract agar (ME2), MHM significantly increased conidia production (p < 0.0001, mean = 3.4 × 103, standard deviation (SD) = ±1.3 × 103), with results similar to those of carnation leaf agar (CLA). MHM facilitated superior preservation of fusarioid viability for up to one year at room temperature on slant cultures and over two years on swabs in Amies gel with charcoal, outperforming current methods such as Castellani (water) or cryopreservation. Morphological characterization of fusarioid fungi grown on MHM revealed distinct growth patterns and conidial structures for Neocosmospora, Bisifusarium, and Fusarium species, aiding in identifying these genera. The superior performance of MHM in stimulating conidiation, maintaining viability, and preserving morphology underscore its potential as a reference medium for medically relevant fusarioid fungi, with broad implications for clinical mycology laboratories and resource-limited settings.

Development of a novel mycobiome diagnostic for fungal infection

BackgroundAmplicon-based mycobiome analysis has the potential to identify all fungal species within a sample and hence could provide a valuable diagnostic assay for use in clinical mycology settings. In the last decade, the mycobiome has been increasingly characterised by targeting the internal transcribed spacer (ITS) regions. Although ITS targets give broad coverage and high sensitivity, they fail to provide accurate quantitation as the copy number of ITS regions in fungal genomes is highly variable even within species. To address these issues, this study aimed to develop a novel NGS fungal diagnostic assay using an alternative amplicon target.MethodsNovel universal primers were designed to amplify a highly diverse single copy and uniformly sized DNA target (Tef1) to enable mycobiome analysis on the Illumina iSeq100 which is a low cost, small footprint and simple to use next-generation sequencing platform. To enable automated analysis and rapid results, a streamlined bioinformatics workflow and sequence database were also developed. Sequencing of mock fungal communities was performed to compare the Tef1 assay and established ITS1-based method. The assay was further evaluated using clinical respiratory samples and the feasibility of using internal spike-in quantitative controls was assessed.ResultsThe Tef1 assay successfully identified and quantified Aspergillus, Penicillium, Candida, Cryptococcus, Rhizopus, Fusarium and Lomentospora species from mock communities. The Tef1 assay was also capable of differentiating closely related species such as A. fumigatus and A. fischeri. In addition, it outperformed ITS1 at identifying A. fumigatus and other filamentous pathogens in mixed fungal communities (in the presence or absence of background human DNA). The assay could detect as few as 2 haploid genome equivalents of A. fumigatus from clinical respiratory samples. Lastly, spike-in controls were demonstrated to enable semi-quantitation of A. fumigatus load in clinical respiratory samples using sequencing data.ConclusionsThis study has developed and tested a novel metabarcoding target and found the assay outperforms ITS1 at identifying clinically relevant filamentous fungi. The assay is a promising diagnostic candidate that could provide affordable NGS analysis to clinical mycology laboratories.

Performance of a Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry Testing Algorithm for the Rapid Identification of Clinical Filamentous Molds.

One of the most significant challenges in the treatment of fungal infections is the relatively long turnaround time (TAT) required for fungal species identification. The length of TAT to identification can impact patient clinical outcomes by delaying appropriate targeted therapy. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) has demonstrated exceptional utility in the rapid identification of bacteria and yeasts in the clinical microbiology laboratory. The capability of MALDI-TOF MS for rapid identification of clinical isolates presents an opportunity for significant advancement in the identification of filamentous molds. In this study, we employed a diagnostic algorithm using MALDI-TOF MS for the rapid identification of filamentous molds in order to assess the impact of this technology on TATs. The majority of isolates included in this study were able to be identified by MALDI-TOF MS (78%). Further, these isolates were identified in less than three days from first detection of colony growth. This study demonstrates the utility of MALDI-TOF MS in the rapid identification of filamentous molds in the clinical mycology laboratory.

Effectiveness of FastFung agar in the isolation of Malassezia furfur from skin samples.

BackgroundLipophilic basidiomycetous yeasts of the Malassezia genus can cause various skin diseases, such as seborrheic dermatitis, pityriasis versicolor, folliculitis and atopic dermatitis, and even life‐threatening fungemia in newborns and immunocompromised individuals. Routine mycological media used in clinical practice do not contain sufficient lipid ingredients required for the growth of Malassezia species. A recently developed medium, FastFung agar, is promising for culturing fastidious fungal species.MethodsIn this study, we compared FastFung agar and mDixon agar for culturing Malassezia species from nasolabial fold and retroauricular specimens of 83 healthy individuals and 187 and 57 patients with acne vulgaris and seborrheic dermatitis, respectively.Results Malassezia species were identified using conventional tests and matrix‐assisted laser desorption/ionisation mass spectrometry. In total, 96 of 654 samples (14.6%) contained Malassezia species. The total isolation rate was significantly higher in patients with seborrheic dermatitis (40.4%) than in healthy volunteers (21.7%; p < .05), and the rate of M. furfur isolation was significantly higher for patients with acne vulgaris (13.9%) and seborrheic dermatitis (24.6%) than for healthy individuals (1.5%; p < .05). FastFung agar was superior to mDixon agar in M. furfur isolation (p = .004) but showed similar performance in the case of non‐M. furfur species (p > .05). Among cultured Malassezia species, perfect agreement between mDixon agar and FastFung agar was found only for M. globosa (κ = 0.90).ConclusionOur results indicate that FastFung agar favours the growth of Malassezia species and should be useful in clinical mycology laboratories.

Mass spectrometry in research laboratories and clinical diagnostic: a new era in medical mycology.

Diagnosis by clinical mycology laboratory plays a critical role in patient care by providing definitive knowledge of the cause of infection and antimicrobial susceptibility data to physicians. Rapid diagnostic methods are likely to improve patient. Aggressive resuscitation bundles, adequate source control, and appropriate antibiotic therapy are cornerstones for success in the treatment of patients. Routine methods for identifying clinical specimen fungal pathogen are based on the cultivation on different media with the subsequent examination of its phenotypic characteristics comprising a combination of microscopic and colony morphologies. As some fungi cannot be readily identified using these methods, molecular diagnostic methods may be required. These methods are fast, but it can cost a lot. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is suitable for high-throughput and rapid diagnostics at low costs. It can be considered an alternative for conventional biochemical and molecular identification systems in a microbiological laboratory. The reliability and accuracy of this method have been scrutinized in many surveys and have been compared with several methods including sequencing and molecular methods. According to these findings, the reliability and accuracy of this method are very high and can be trusted. With all the benefits of this technique, the libraries of MALDI-TOF MS need to be strengthened to enhance its performance. This review provides an overview of the most recent research literature that has investigated the applications and usage of MT-MS to the identification of microorganisms, mycotoxins, antifungal susceptibility examination, and mycobiome research.

Identification of filamentous fungi including dermatophytes using MALDI-TOF mass spectrometry.

Identification of filamentous fungi based on morphological features is the most available approach used in clinical mycology laboratories. However, MALDI-TOF mass spectrometry is currently invaluable for identification of microorganisms because of its rapidity, simplicity, and accuracy. This study aimed to find the optimal way of identifying filamentous fungi using MALDI-TOF MS.The sample comprised 193 isolates of filamentous fungi. The identification started with morphological assessment. Then isolates were identified using MALDI-TOF MS, both directly from culture and following culture in liquid media with extraction. Subsequently, identification of 20 selected isolates was compared by sequencing of the benA gene, ITS1-5,8-ITS2, and D1-D2 LSU regions.Based on morphological criteria, 17 genera of fungi were identified. With MALDI-TOF MS performed directly from culture, nine isolates were identified to the genus level and 184 to the species level, with a total of 75 species being noted. With the MALDI-TOF MS extraction method, 190 isolates were identified to the species level, with 43 species being noted. The rates of agreement between identification using morphology and the MALDI-TOF MS direct method were 58.55% at the genus level and 22.24% at the species level. The rates of agreement between identification using morphology and the MALDI-TOF MS extraction method were 84.97% at the genus level and 46.11% at the species level. Using sequencing, 87.5% agreement was found for identification with the MALDI-TOF MS extraction method, as compared with only 43.75% for the direct method.The results suggest that the optimal approach to identification of filamentous fungi is a combination of morphological features and MALDI-TOF MS using the extraction method.

Epidemiology of urinary candidiasis and antifungal susceptibility pattern of various Candida species at a rural tertiary health care centre of Puducherry, South India- An observational study

Urinary tract infection (UTI) is a common health problem across the globe. Although majority of them are bacterial in origin, there has been an increasing trend in the incidence of UTI due to the yeast like fungi, . UTI due to are in large proportion noscomially acquired and of growing concern is the development of resistance to the commonly used azole group of drugs for their treatment. Since the resistance is more commonly reported among non- albicans , routine species identification and antifungal susceptibility testing is crucial for successful clinical outcomes. This study was conducted to analyse the distribution and risk factors associated with Urinary candidiasis and also to determine the resistance patterns of different to various antifungal agents using phenotypic methods. : A hospital based observational study was conducted from September 2016 to December 2017 on patients presenting with symptomatic UTI. isolates were speciated using phenotypic methods like germ tube test and growth character on chromagar . Antifungal susceptibility to fluconazole, voriconazole, ketoconazole and amphotericin B were determined using disc diffusion method. All data were anlyzed using EpiData Analysis software version 2.2.2.186. A total of 101 were isolated. The incidence of Urinary candidiasis was more among females (66.4%) than in males (33.6%). People above 50 years (38.6%) were commonly affected followed by people in the age group of 21-30 years (22.7%). The incidence among hospitalized patients was 86.7% and urinary catheterization (43.5%) was the most commonly associated risk factor. The most common isolates were tropicalis (31.6%) followed by albicans (21.7%). The overall resistance patterns among various were 50.5%, 32.7%, 19.9% and 2% for fluconazole, ketoconazole, voriconazole and amphotericin B respectively. Hospitalization and urinary catheterization are the important risk factors for developing urinary candidiasis. The antifungal susceptibility varies among different and hence, identification of to species level along with antifungal susceptibility testing should be practiced as a routine in all clinical mycology laboratories.

MALDI-TOF MS in a Medical Mycology Laboratory: On Stage and Backstage.

The implementation of MALDI-TOF MS in medical microbiology laboratories has revolutionized practices and significantly reduced turnaround times of identification processes. However, although bacteriology quickly benefited from the contributions of this technique, adjustments were necessary to accommodate the specific characteristics of fungi. MALDI-TOF MS is now an indispensable tool in clinical mycology laboratories, both for the identification of yeasts and filamentous fungi, and other innovative uses are gradually emerging. Based on the practical experience of our medical mycology laboratory, this review will present the current uses of MALDI-TOF MS and the adaptations we implemented, to allow their practical execution in a daily routine. We will also introduce some less mainstream applications, like those for fungemia, or even still under development, as is the case for the determination of sensitivity to antifungal agents or typing methods.

Diagnosis of Breakthrough Fungal Infections in the Clinical Mycology Laboratory: An ECMM Consensus Statement.

Breakthrough invasive fungal infections (bIFI) cause significant morbidity and mortality. Their diagnosis can be challenging due to reduced sensitivity to conventional culture techniques, serologic tests, and PCR-based assays in patients undergoing antifungal therapy, and their diagnosis can be delayed contributing to poor patient outcomes. In this review, we provide consensus recommendations on behalf of the European Confederation for Medical Mycology (ECMM) for the diagnosis of bIFI caused by invasive yeasts, molds, and endemic mycoses, to guide diagnostic efforts in patients receiving antifungals and support the design of future clinical trials in the field of clinical mycology. The cornerstone of lab-based diagnosis of breakthrough infections for yeast and endemic mycoses remain conventional culture, to accurately identify the causative pathogen and allow for antifungal susceptibility testing. The impact of non-culture-based methods are not well-studied for the definite diagnosis of breakthrough invasive yeast infections. Non-culture-based methods have an important role for the diagnosis of breakthrough invasive mold infections, in particular invasive aspergillosis, and a combination of testing involving conventional culture, antigen-based assays, and PCR-based assays should be considered. Multiple diagnostic modalities, including histopathology, culture, antibody, and/or antigen tests and occasionally PCR-based assays may be required to diagnose breakthrough endemic mycoses. A need exists for diagnostic tests that are effective, simple, cheap, and rapid to enable the diagnosis of bIFI in patients taking antifungals.

Improving Confirmatory Testing for the Antimicrobial Resistance Surveillance Network in Ethiopia

Background: In July 2017, the Ethiopian Public Health Institute (EPHI) launched an antimicrobial resistance (AMR) surveillance network at 4 sentinel laboratories. The National Clinical Bacteriology and Mycology Laboratory (NRL) at EPHI performs monthly confirmatory testing on a subset of isolates submitted by these sites. We assessed the existing confirmatory testing program to identify gaps and develop solutions, including a monitoring and evaluation (M&amp;E) system. Methods: We assembled a technical working group (TWG) of key stakeholders. Laboratory site visits included workflow observation, process mapping, document review, and technologist interviews. Proposed solutions to observed gaps were drafted in formats consistent with their intended application. Feedback from the TWG was incorporated into final drafts. Available AMR network staff members were trained remotely, and they will train remaining staff. Results: Table 1 describes major gaps and solutions identified. Conclusions: Confirmatory testing provides a mechanism to evaluate laboratory testing proficiency, target improvements, and estimate surveillance data quality, yet standardized methods were lacking. Our efforts highlight key components of confirmatory testing programs and provide a model for use in laboratories with similar needs.Funding: NoneDisclosures: None

Incorporating Telementorship Into Laboratory Capacity Building Initiatives for Improved AMR Surveillance in Ethiopia

Background: In July 2017, recognizing the threat that antimicrobial resistance poses to the population, the Ethiopian Public Health Institute (EPHI) launched the Ethiopia AMR Surveillance Network at 4 sentinel laboratories. Simultaneously, laboratory capacity building was initiated to ensure the reporting of quality laboratory data to the surveillance system. One initiative, Project ECHO (Extension for Community Healthcare Outcomes) was used to virtually connect subject matter experts with participating laboratories in remote settings to provide ongoing education and telementoring and to foster peer-to-peer learning and problem solving in microbiology. The 10-month project was supported by the Centers for Disease Control and Prevention (CDC) and the American Society for Microbiology (ASM).Methods: Biweekly 1-hour sessions were held by ASM for 2 sentinel sites, Tikur Anbessa Specialized Hospital and the EPHI Clinical Microbiology and Mycology Laboratory, using a videoconferencing platform. Each virtual session consisted of a didactic session, a case presentation by a participating laboratory, open discussion and feedback. Case presentations focused on technical challenges and problems encountered in the preanalytical, analytical, and postanalytical phases of microbiology testing. Experts from CDC and ASM provided feedback along with a summation of key learning objectives. Sessions were recorded and post session reports were shared with participants. To assess participants’ baseline knowledge, a comprehensive pretest was administered prior to the first session. The same instrument was administered as a posttest 2 weeks after the final session. Unstructured interviews were also conducted to assess participants’ perceptions of the value of ECHO to their work. Results: Mean pretest scores were 69.25% and the posttest scores were 71.04%, a difference of 1.79% (P = NS). Participant interviews revealed perceived benefits of ECHO participation to include enhanced critical thinking and problem resolution in microbiology, increased communication and improved working relationships between participating sites, and improved understanding and application of CLSI standards. As a result of Ethiopia’s participation in Project ECHO, 23 case presentations have been added to ECHO Box, a resource bank and web portal, which allows members of the ECHO community to share and access didactics, documents, and learning materials. Conclusions: Despite minimal difference between pretest and posttest scores, the Project ECHO experience of virtual case-based learning and collaborative problem solving has encouraged critical thinking, peer-to-peer learning, networking among participants, and has provided microbiologists with the resources for improved bacterial isolation, identification, and antibiotic susceptibility testing. The lessons learned could be applied as this project is expanded to additional laboratories in the AMR Surveillance Network.Funding: NoneDisclosures: None

Editorial introductions

Editorial introductions

Updates in Laboratory Diagnostics for Invasive Fungal Infections.

Appropriate diagnosis of invasive fungal infections (IFIs) is critical due to the high rates of morbidity and mortality, as well as the substantial economic burden, associated with the management of these diseases. The recognition of IFI and differentiation from other infections with similar clinical presentations can be challenging, which can lead to diagnostic error that not only has an impact on individual patient health outcomes but also on antimicrobial drug usage and the growing threat of antimicrobial resistance in bacteria. Therefore, there is a significant need for improved stewardship related to diagnostic testing for and treatment of IFIs. The purpose of this review is to highlight recent advances related to current fungal diagnostics, as well as explore some of the most innovative technology that has emerged with the potential to shift the paradigm of clinical mycology. In general, this review will discuss research related to enhanced fungal culture utilization and identification techniques, expanded applications of fungal antigen testing, and recently developed molecular assays and other novel nonculture fungal diagnostic approaches. Specifically, the application of mass spectrometry, novel glycobiomarker detection, and detection of fungal-specific volatile organic compounds will be reviewed, along with other key updates, to provide the reader with an updated review that extends beyond the basics of IFI laboratory diagnostics. Where appropriate, the reader will be directed to more comprehensive reviews of certain aspects of clinical mycology laboratory testing to provide a broader context for the critical consideration of these updates.

Use of MALDI-TOF MS for fungal species distribution of interdigital intertrigo in seafarers, Dakar, Senegal

To determine fungal species distribution of interdigital intertrigo among seafarers in Dakar, Senegal, a cross-sectional study was carried out from May to August 2017 among seafarers clinically diagnosed with interdigital intertrigo. A questionnaire was filled to each patient before sampling the affected folds and transporting to Aristide Le Dantec University Hospital where mycological analyses were realized. Species identification by MALDI-TOF MS was performed in Marseille, France. In total, 169 men (21–66 years) were included. Few of them (3%) had a high level of education and the duration of the mycosis exceed 10 years for 88% of patients. Direct microscopic examination (ME) was positive in 34.3%. Among samples with positive ME, 58.6% had positive culture. An overall incidence of 30.2% was found. Patients with confirmed cases aged between 28 and 66 years. Among them, those between 36–50 years were predominant (52.9%). Those with a high level of education were less representative (2%). For 52.1% of patients, the duration of the mycosis was superior to 10 years. Furthermore, 57% of cases were significantly associated with other types of tinea pedis and/or onychomycosis (P=0.03). Culture was positive in 23.7% isolating 43 strains successfully identified at the species level by MALDI-TOF MS for 31 isolates: 20 Candida and 11 dermatophytes. The rest was identified only at the genus level belonged to Fusarium. In definitive, MALDI-TOF MS could be a useful tool for routine and fast identification of dermatophytes, yeasts and NDFF in clinical mycology laboratories.

A national survey on fungal infection diagnostic capacity in the clinical mycology laboratories of tertiary care hospitals in China.

As the incidence of fungal infections in China increases, the demand for rapid and accurate diagnosis of mycoses is growing. Yet, information on current diagnostic capacity is scarce. An online survey was conducted in February 2018 to collect information on mycology testing from tertiary care hospitals across China. Responses from 348 hospitals were analyzed, and a scoring system was designed and employed to assess the overall diagnostic capacity. Most of the surveyed hospitals did not have separate laboratory space, manpower, or equipment dedicated for fungal testing. Conventional staining methods were widely available (>70%), whereas GMS and fluorescent staining were less common. Fungal identification services were offered mostly with chromogenic medium, morphological characterization or automated identification systems, other than more advanced methods such as MALDI-TOF MS and DNA sequencing. Fungal serology testing was available in 81.1%, with G test being the most often used. Though 91.8% of the respondents had the ability to perform antifungal susceptibility testing for yeasts, less than 13% conducted such testing for molds. The percentage of laboratories participating in External Quality Assessment programs and research was 57.5% and 32.5%, respectively. The average score for the 348 surveyed hospitals was 37.2 (out of a maximum of 89 points), with only 15 hospitals scoring >60, suggesting a general lack of high-quality mycology laboratories. The overall clinical testing capacity for fungal infection in China is insufficient. More investment and training efforts are warranted to establish centers of excellence and promote access to high-quality diagnostic services.

Fishing for a Diagnosis, the Impact of Delayed Diagnosis on the Course of Mycobacterium marinum Infection: 21 Years of Experience at a Tertiary Care Hospital.

Background Mycobacterium marinum is a common but underreported mycobacterial infection. We conducted a large retrospective study to determine risk factors and describe the therapeutic interventions and outcomes in patients with uncomplicated and complicated M. marinum infection.MethodsCulture-confirmed M. marinum infection cases were identified from the Mayo Clinic Clinical Mycology Laboratory from January 1998 to December 2018. Complicated M. marinum infection was defined as the presence of tenosynovitis, septic arthritis, or osteomyelitis. Differences in complicated vs uncomplicated M. marinum infections were analyzed using statistical comparisons.ResultsTwelve cases had a complicated M. marinum infection. Patients with a complicated infection were older (64.3 ± 11.1 vs 55.8 ± 14.5; P = .03), had longer duration of symptoms (5 vs 3 months; P = .011), and had more surgical debridements (1 vs 0; P < .001). Length of treatment and number of drugs used were not statistically significant. Complicated M. marinum cases received more medications (2 vs 1; P = .263) and were treated longer (5.7 vs 3.5 months; P = .067). Antibiotic susceptibilities were performed in 59% of the patients. All isolates were susceptible to clarithromycin. From the tetracyclines, doxycycline had a better susceptibility pattern.Conclusions M. marinum infection is an important cause of skin and soft tissue infection. Poor water exposure documentation, unusual clinical presentation, and empiric antibiotic treatment before definitive M. marinum diagnosis often contribute to a delayed diagnosis. Complicated M. marinum cases had longer duration of symptoms and more surgical debridements. No difference in the number of drugs used or clinical outcome was observed.

From the Clinical Mycology Laboratory: New Species and Changes in Fungal Taxonomy and Nomenclature.

Fungal taxonomy is the branch of mycology by which we classify and group fungi based on similarities or differences. Historically, this was done by morphologic characteristics and other phenotypic traits. However, with the advent of the molecular age in mycology, phylogenetic analysis based on DNA sequences has replaced these classic means for grouping related species. This, along with the abandonment of the dual nomenclature system, has led to a marked increase in the number of new species and reclassification of known species. Although these evaluations and changes are necessary to move the field forward, there is concern among medical mycologists that the rapidity by which fungal nomenclature is changing could cause confusion in the clinical literature. Thus, there is a proposal to allow medical mycologists to adopt changes in taxonomy and nomenclature at a slower pace. In this review, changes in the taxonomy and nomenclature of medically relevant fungi will be discussed along with the impact this may have on clinicians and patient care. Specific examples of changes and current controversies will also be given.

Cutaneotrichosporon (Trichosporon) debeurmannianum: A Rare Yeast Isolated from Blood and Urine Samples.

Cutaneotrichosporon (Trichosporon) debeurmannianum is a rarely isolated yeast from clinical samples. Nine isolates of this yeast were identified from clinical samples within a period of 3years from June 2012 to May 2015. These isolates were from blood and urine samples sent to a clinical mycology laboratory of a tertiary care hospital in Assam, North East India. Clinically, the patients were diagnosed as septicemia and urinary tract infection. The age of the patients ranged from 2 to 50years. Identification was made by sequencing the ITS region of ribosomal RNA gene. Antifungal susceptibility test by disk diffusion method (CLSI, M44-A) showed all the isolates to be sensitive to fluconazole and voriconazole. Vitek 2 compact commercial yeast identification system misidentified this yeast as Cryptococcus laurentii and low discrimination Cryptococcus laurentii/Trichosporon mucoides. This species was originally named as Trichosporon debeurmannianum. In 2015, this yeast has been included into new genera Cutaneotrichosporon based on an integrated phylogenetic classification of the Tremellomycetes. To the best of our knowledge, this is the first report of identification of this species from blood and urine samples of clinically suspected cases. We are reporting these isolates because of their rarity in clinical samples. The pathogenic potential and epidemiological relevance of this yeast remains to be seen.

Hypertonic Xylose Agar Medium: A Novel Medium for Differentiation of Candida dubliniensis from Candida albicans

Background:Candida dubliniensis is a pathogenic Candida species which shares many phenotypic features with Candida albicans. These similarities have caused significant problems in the identification of C. dubliniensis in an average clinical mycology laboratory. Several phenotypic-based tests have been developed to distinguish C. albicans from C. dubliniensis but none has been demonstrated being sufficient alone for accurate differentiation of the two species. Aim: To facilitate the differentiation of these species, we evaluated the utility of a novel medium ‘Hypertonic Xylose Agar Medium’ (HXAM). Materials and Methods: A total of 200 Candida spp. were tested in this study which included 186 stock strains of C. albicans and 14 strains of C. dubliniensis. Identification of all these strains was confirmed by polymerase chain reaction-restriction fragment length polymorphism using BlnI (AvrII) enzyme. All isolates were inoculated on HXAM, incubated at 28°C and examined for visible growth every day up to 7 days. Results: On this medium at 28°C, all 186 C. albicans isolates showed visible growth at 48 h of incubation whereas none of the 14 C. dubliniensis isolates did so even on extending the incubation period up to 7 days. Conclusion: Hence, we propose HXAM as a sole phenotypic method for identifying C. dubliniensis from germ-tube-positive isolates or from stock collections of known C. albicans.

Antifungal Susceptibility Testing of Candida and Cryptococcus Species and Mechanisms of Resistance: Implications for Clinical Laboratories

Resistance to antifungal drugs amongst Candida species is a growing concern, and azole resistance may be emerging in Cryptococcus species. This review provides a contemporary perspective, relevant to the clinical mycology laboratory, of antifungal susceptibility testing of these fungi, focussing on the challenges of phenotypic and genotypic methodologies to detect drug resistance. Standardised CLSI and EUCAST broth microdilution (BMD) susceptibility testing methods are the benchmark to determine clinical breakpoints (CBPs) and/or epidemiological cut-off values (ECVs) MICs for Candida and Cryptococcus spp. Commercial methods may be used but caution is required when employing BMD CBPs/ECVs to interpret results. Species-specific CBPs/ECVs for Candida spp. generally correlate well with predicting likelihood of therapeutic failure or of presence of a drug resistance mechanism with the exception of the echinocandins where the presence of specific FKS gene mutations and not the MIC correlates most accurately with clinical outcome. The relationship of presence of one or more mechanisms of azole resistance and drug MICs is uncertain. Next generation sequencing technology is offering insights into the relationships between susceptibility results obtained by phenotypic and genotypic methods. For Cryptococcus spp., CBPs are not established but species- and genetic type-specific EVCs are useful for guiding therapy where clinically indicated. Isolates of genotype VGII appear to exhibit the highest MICs. Antifungal susceptibility testing of yeasts is important to detect drug resistance. For Candida spp., MICs have clinical utility for the azoles but detecting echinocandin resistance by genotypic methods is preferred. For Cryptococcus spp., ECVs are useful in guiding therapy.