Clinical Mycology Research Articles

How to prophylax against invasive fungal infections in adult ALL? An unmet need.

Although the benefit for any type of antifungal prophylaxis in patients with acute myelogenous leukaemia is well accepted, less is known about the risk for invasive fungal infections (IFIs) and the optimal prophylaxis strategies in patients with acute lymphocytic leukaemia (ALL). Based on recent studies, ALL is a disease that appears to be associated with significant risk for IFIs. The pharmacokinetic interactions between azoles and vincristine, an antineoplastic agent that is part of modern combination chemotherapies in ALL, results in clinically significant neurotoxicity that makes the use of azoles problematic. However, a number of questions regarding azole-vincristine interactions remain unanswered. In this viewpoint, we call for a renewed interest in antifungal prophylaxis studies in ALL in view of the availability of several non-azole novel antifungal agents that are under preclinical and/or clinical development. This is clearly a major unmet need in modern clinical mycology.

Breakthrough Invasive Mold Infections in the Hematology Patient: Current Concepts and Future Directions.

Although the widespread use of mold-active agents (especially the new generation of triazoles) has resulted in reductions of documented invasive mold infections (IMIs) in patients with hematological malignancies and allogeneic hematopoietic stem cell transplantation (HSCT), a subset of such patients still develop breakthrough IMIs (bIMIs). There are no data from prospective randomized clinical trials to guide therapeutic decisions in the different scenarios of bIMIs. In this viewpoint, we present the current status of our understanding of the clinical, diagnostic, and treatment challenges of bIMIs in high-risk adult patients with hematological cancer and/or HSCT receiving mold-active antifungals and outline common clinical scenarios. As a rule, managing bIMIs demands an individualized treatment plan that takes into account the host, including comorbidities, certainty of diagnosis and site of bIMIs, local epidemiology, considerations for fungal resistance, and antifungal pharmacological properties. Finally, we highlight areas that require future investigation in this complex area of clinical mycology.

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.

Contribution of the National Mycology Laboratory Network to Surveillance of Cryptococcosis in Argentina

Cryptococcal meningitis is the most common fungal infection of the central nervous system and the third most frequent neurological complication in AIDS patients. To understand the Argentinean epidemiology of cryptococcosis, several efforts have been made by the National Reference Laboratory in Clinical Mycology. In Argentina, reports of distribution and frequency of Cryptococcus neoformans and Cryptococcus gattii isolates are scarce and very little is known about its circulating genotypes and mating types. The National Mycology Laboratory Network and the National Reference Laboratory in Clinical Mycology joined forces to estimate the prevalence of cryptococcosis and to obtain and to analyse epidemiological data of this important fungal infection. Data presented here were recovered from 1998 to 2016 and represents an approximation to the actual situation of cryptococcosis in Argentina. These results could be useful to design future investigations.

Breath-Based Diagnosis of Invasive Mucormycosis (IM)

BackgroundTimely diagnosis of IM remains a major challenge in clinical mycology. Because of the lack of specific diagnostic methods for IM and the frequently fulminant nature of this infection, IM-associated mortality remains high.MethodsWe examined breath volatile metabolite profiles in a neutropenic murine model of IM, using the 3 Mucorales species that most commonly cause human IM - Rhizopus arrhizus var. arrhizus, R. arrhizus var. delemar, and R. microsporus - and for comparison, Aspergillus fumigatus. We infected female balb/c mice (N = 4 per group) treated with cyclophosphamide and cortisone followed by intranasal administration of 106 conidia of each species. 3 days post-infection, we collected breath samples from each mouse via tracheostomy using a flexiVent murine ventilator, examining breath volatile metabolites using thermal desorption gas chromatography/tandem mass spectrometry (GC-MS/MS). We also sampled breath prospectively from five patients eventually diagnosed with proven IM caused by R. microsporus, analyzing breath volatile metabolites using thermal desorption GC-MS/MS.ResultsEach Mucorales species produced a consistent profile of breath sesquiterpene secondary metabolite VOCs in our murine models, which distinguished these species from each other and from murine invasive aspergillosis (Figure A). These fungi shifted their secondary metabolism significantly in vivo, compared with their previously characterized in vitro metabolism. We found overlapping VOC sesquiterpene metabolites between breath samples from the murine model of R. microsporus infection and 5 of 5 patients with R. microsporus IM, with additional sesquiterpene secondary metabolites detected in patient breath, compared with the murine IM model (Figure B). In one patient with serial breath samples, these sesquiterpenes declined in abundance and disappeared with antifungal therapy, in parallel with clinical improvement (Figure C).ConclusionThe three Mucorales species that cause most human IM have distinct breath sesquiterpene profiles that can be used to identify these infections in vivo noninvasively. These profiles distinguish these infections from each other and from aspergillosis, and may be useful in monitoring clinical response to treatment.Disclosures F. M. Marty, Astellas Pharma US: Consultant and Grant Investigator, Consulting fee and Grant recipient; Chimerix: Consultant and Grant Investigator, Consulting fee and Grant recipient; Fate Therapeutics: Scientific Advisor, Consulting fee; Gilead Sciences: Consultant and Grant Investigator, Consulting fee and Grant recipient; LFB: Consultant, Consulting fee; Merck: Consultant, Grant Investigator and Scientific Advisor, Consulting fee and Grant recipient; Roche Molecular Systems: Consultant, Consulting fee; Shire: Consultant and Grant Investigator, Consulting fee and Grant recipient; D. P. Kontoyiannis, Pfizer: Research Contractor, Research support and Speaker honorarium; Astellas: Research Contractor, Research support and Speaker honorarium; Merck: Honorarium, Speaker honorarium; Cidara: Honorarium, Speaker honorarium; Amplyx: Honorarium, Speaker honorarium; F2G: Honorarium, Speaker honorarium

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.

What Can the Clinical Mycology Laboratory Do for Clinicians Today and Tomorrow?

In this review, we discuss the old and new tools available to the clinical mycology laboratory, the “growing pains” in their use, and how they impact patient care and aim to recommend what clinical mycology laboratories need to do to succeed in optimizing patient care. Through the years, studies have shown the importance of the radiologic, molecular, and non-molecular methods for the diagnosis and follow-up of patients with invasive fungal infections (IFIs) and their impact on patient outcome. (1) Accurate fungal species identification is essential. (2) Histopathology can be insightful, and the clinical mycology laboratory needs to collaborate with the pathology department for optimization of care. (3) Rapid diagnosis is important and biomarkers need to carefully replace or reduce spiraling antifungal empiricism. (4) Culture techniques need to be carefully integrated with rapid diagnostic methods. (5) IFIs are deadly but a combination of a receptive and progressive mycology laboratory and a small cadre of antifungal agents can save lives. (6) The clinical mycology laboratory needs to be carefully linked to clinical practices, antifungal stewardship, and infection control challenges.

Molecular Identification and Phenotypic Characterisation of Sporothrix globosa from Clinical Cases of Eastern Assam, North-east India

Sporotrichosis is known to be endemic in the state of Assam, North-east India, which is situated in the Sub-Himalayan region. This disease is an acute or chronic infection caused by Sporothrix schenckii species complex which currently includes several species of clinical relevance such as Sporothrix brasiliensis, Sporothrix globosa, Sporothrix schenckii sensu stricto, Sporothrix albicans, Sporothrix mexicana, Sporothrix pallida and Sporothrix luriei. S. globosa is the prevalent species in India. Eight culture-positive patients were diagnosed from suspected consecutive cases of two lymphocutaneous and six fixed cutaneous forms over a period of 4 years in a clinical mycology laboratory of a tertiary care centre in Eastern Assam. Phenotypic speciation was inconclusive using the criteria of Marimon et al. because of atypical growth pattern shown by the isolates. Our isolates showed good growth at 37°C ranging from 6 to 27 mm; four of the isolates showed growth of 11–27 mm unlike S. globosa strains reported earlier. Molecular identification was done by sequencing both the internal transcribed spacer (ITS) region and the calmodulin (CAL) protein encoding gene (partial). All the isolates were identified as S. globosa. Molecular confirmation of species using ITS region and CAL protein encoding gene (partial) is necessary for isolates of S. globosa showing atypical biopatterns.

The Swiss Society of Microbiology: Small Bugs, Big Questions and Cool Answers.

The Swiss Society for Microbiology (SSM) represents around 700 scientists working in the fields of medical (human and veterinary), microbial biotechnology as well as fundamental, environmental, and food microbiology. Five sections: Clinical Microbiology, Environmental Microbiology, Mycology, Prokaryotic Biology, and Virology reflects the main interests of the membership.

Molecular Diagnostic Testing for Aspergillus.

The direct detection of Aspergillus nucleic acid in clinical specimens has the potential to improve the diagnosis of aspergillosis by offering more rapid and sensitive identification of invasive infections than is possible with traditional techniques, such as culture or histopathology. Molecular tests for Aspergillus have been limited historically by lack of standardization and variable sensitivities and specificities. Recent efforts have been directed at addressing these limitations and optimizing assay performance using a variety of specimen types. This review provides a summary of standardization efforts and outlines the complexities of molecular testing for Aspergillus in clinical mycology.

Hot Topics in Antifungal Susceptibility Testing: a New Drug, a Bad Bug, Sweeping Caspofungin Testing under the Rug, and Solving the Epidemiological Cutoff Value Shrug

There are several new hot topics in antifungals and antifungal susceptibility testing. In this review, four topics of general interest to the clinical microbiology community are discussed. The first topic is the introduction of isavuconazole, a new triazole approved for clinical use in the United States. The second is triazole resistance in Aspergillus fumigatus isolates. A specific set of mutations are being found with greater frequency in isolates globally, including the U.S. The third topic of interest is a word of caution about antifungal susceptibility testing for caspofungin in Candida isolates; some laboratories have reported susceptible isolates with high MIC values that would be interpreted as resistant. The final topic is an introduction to epidemiological cutoff values and their use in the clinical mycology laboratory.

The epidemiology of fungemia in an infectious diseases hospital in Mexico city: A 10-year retrospective review.

The epidemiology of invasive fungal infections has recently changed in immunosuppressed populations as a result of HIV infection, organ transplant, chemotherapy and in elderly patients. The diagnosis of invasive fungal infections by culture is prolonged since fungi grow slowly in vitro. we wanted to estimate the frequency of fungemia diagnoses established through the Clinical Mycology Laboratory over the past 10 years; through a retrospective study; data was obtained from the laboratory patient registry in the Infectious Disease Hospital's laboratory registry of patients with a systemic fungal isolate between 2005 and 2014. One hundred and thirty two (132) systemic fungal infections were identified. They were more prevalent in males, in the age group between 20 and 59 years and in patients with a diagnosis of AIDS. The most frequently isolated agents belonged to the genus Candida and others such as Histoplasma sp., Cryptococcus sp., Aspergillus sp., and Coccidioides sp. Of all blood and bone marrow cultures received 17.9% had fungal development; of these, in 70% of cases it was through blood cultures. In general, fungal agents were not diagnostically suspected. We identified that Sixty percent (60%) of fungemias developed in AIDS patients, followed by patients with sepsis. The most common agents belonged to the genus Candida, predominantly the albicans species. They were more frequently identified by blood culture than by bone marrow culture. Invasive fungal infections have not followed a usual clinical pattern and are not easily recognizable.

The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA.

Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

Incidence and Speciation of Candida Species among Non-gravid young Females in Ilorin, North Central, Nigeria

This study investigated the incidence and speciation of Candida species among non-gravid young females, using commercially available chromogenic Candida speciation media (CHROM agar) for the identification/speciation of medically important yeast and yeastlike organisms in a routine clinical mycology laboratory. High Vaginal Swabs (HVS) were randomly collected from consenting non-gravid-young females for the study. The participants also completed a structured questionnaire assessing demographic data, symptoms, and risk factors of candidiasis. A total of 120 females between the ages of 17 and 31 years were randomly recruited for the study. Standard microbiological techniques such as Gram’s stain, wet mount and culture on Sabouraud Dextrose Agar (SDA) and CHROM agar were used to analyze the swabs. Candida species was isolated from 64 of the 120 females, representing 53.3%. The highest incidence rate of 25.0% was recorded in the 23-25 years age group, followed by 12.5% in the 26 28 years while the lowest incidence (1.7%) was observed in the 17-19 years age group. Candida albicans (35.0%) was the most common Candida species , followed by C. tropicalis (8.3%), C. glabrata (6.7%) and C. krusie (3.3%), whereas, non-Candida infection constituted 46.7% (Negative cultures). Vaginal discharge (85.5%) and itching (52.5%) recorded the high values, with respect to symptoms. The prevalent risk factors associated with vaginal candidiasis in this study were washing of vagina with soap (Vaginal douching) (72.5%) and unprotected casual sex (16.7%). Whitish vaginal discharge (78.3%) was most prevalent followed by creamish vagina discharge (56.7%). In addition to Candida albicans , non-albicans Candida spp were isolated from HVS specimens; therefore, public health education is vital. CHROM agar is a simple, rapid and inexpensive method with good sensitivity and specificity for identification and speciation of Candida species thus, allowing an early and appropriate antifungal therapy. The results of the study will eliminate the ambiguities concerning Candida identification in this country and will contribute to better management and proper treatment of patients. Keywords: Candidiasis, vulvovaginitis, Incidence, Speciation, CHROM agar, Risk Factors

Prevalence of Vulvovaginitis in Sexually Active Women from Riverside Communities in Brazilian Amazonia

Aims: Analyze the prevalence of vulvovaginitis in sexually active adult women, residents of riverside communities in Brazilian Amazonia, receiving health services through the “Luz na Amazonia” program in 2013 and 2014. Study Design: Population based cross-sectional and evaluative study. Place and Duration of Study: “Luz na Amazonia” Program and Laboratory of Clinical and Environmental Mycology from the Federal University of Para (UFPA), between January 2013 and December 2014. Short Communication Vilaca et al.; IJTDH, 15(1): 1-8, 2016; Article no.IJTDH.22816 2 Methodology: The analytical cross-sectional study was made with bacterioscopy and fresh examination of vaginal secretion and the results were statistically analyzed with software BioEstat 5.0. Results: The prevalence of vulvovaginitis was 78,57%, and the prevalence of bacterial vaginosis, genital infection by Candida spp and vaginitis caused by Trichomonas vaginalis was 66,07%, 28,57% and 8,92%, respectively. Statistically significant association was found between the signs, symptoms and vulvovaginitis. Conclusion: Due to the necessities of this population and the high prevalence of vulvovaginitis found, becomes evident the importance of projects which aim the prevention and promotion of health education for women from these communities to be encouraged to seek medical exams more often, in order to minimize the cases of vaginitis and vaginosis in these communities.

Misidentification of a Rare Species, Cryptococcus laurentii, by Commonly Used Commercial Biochemical Methods and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry Systems: Challenges for Clinical Mycology Laboratories.

Forty-two putative Cryptococcus laurentii isolates identified by the Vitek 2 system were collected in China. The gold standard, internal transcribed spacer (ITS) sequencing, confirmed that only two isolates were genuine C. laurentii. Bruker Biotyper matrix-assisted laser desorption ionization-time of flight mass spectrometry was able to identify the C. laurentii isolates with an expanded custom database.

Quantitative Analysis of Single-Nucleotide Polymorphism for Rapid Detection of TR34/L98H- and TR46/Y121F/T289A-Positive Aspergillus fumigatus Isolates Obtained from Patients in Iran from 2010 to 2014.

We employed an endpoint genotyping method to update the prevalence rate of positivity for the TR34/L98H mutation (a 34-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with a substitution at codon L98) and the TR46/Y121F/T289A mutation (a 46-bp tandem repeat mutation in the promoter region of the cyp51A gene in combination with substitutions at codons Y121 and T289) among clinical Aspergillus fumigatus isolates obtained from different regions of Iran over a recent 5-year period (2010 to 2014). The antifungal activities of itraconazole, voriconazole, and posaconazole against 172 clinical A. fumigatus isolates were investigated using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. For the isolates with an azole resistance phenotype, the cyp51A gene and its promoter were amplified and sequenced. In addition, using a LightCycler 480 real-time PCR system, a novel endpoint genotyping analysis method targeting single-nucleotide polymorphisms was evaluated to detect the L98H and Y121F mutations in the cyp51A gene of all isolates. Of the 172 A. fumigatus isolates tested, the MIC values of itraconazole (≥16 mg/liter) and voriconazole (>4 mg/liter) were high for 6 (3.5%). Quantitative analysis of single-nucleotide polymorphisms showed the TR34/L98H mutation in the cyp51A genes of six isolates. No isolates harboring the TR46/Y121F/T289A mutation were detected. DNA sequencing of the cyp51A gene confirmed the results of the novel endpoint genotyping method. By microsatellite typing, all of the azole-resistant isolates had genotypes different from those previously recovered from Iran and from the Dutch TR34/L98H controls. In conclusion, there was not a significant increase in the prevalence of azole-resistant A. fumigatus isolates harboring the TR34/L98H resistance mechanism among isolates recovered over a recent 5-year period (2010 to 2014) in Iran. A quantitative assay detecting a single-nucleotide polymorphism in the cyp51A gene of A. fumigatus is a reliable tool for the rapid screening and monitoring of TR34/L98H- and TR46/Y121F/T289A-positive isolates and can easily be incorporated into clinical mycology algorithms.

Pushing the Limits of MALDI-TOF Mass Spectrometry: Beyond Fungal Species Identification.

Matrix assisted laser desorption ionization time of flight (MALDI-TOF) is a powerful analytical tool that has revolutionized microbial identification. Routinely used for bacterial identification, MALDI-TOF has recently been applied to both yeast and filamentous fungi, confirming its pivotal role in the rapid and reliable diagnosis of infections. Subspecies-level identification holds an important role in epidemiological investigations aimed at tracing virulent or drug resistant clones. This review focuses on present and future applications of this versatile tool in the clinical mycology laboratory.

Evaluation of T3B fingerprinting for identification of clinical and environmental Sporothrix species.

In this study, PCR fingerprinting using the universal primer T3B was applied to distinguish among clinical and environmental species of the Sporothrix complex, Sporothrix brasiliensis, S. globosa, S. mexicana, S. pallida, S. luriei and S. schenckii sensu stricto. The T3B fingerprinting generated clearly distinct banding patterns, allowing the correct identification of all 43 clinical and environmental isolates at the species level, what was confirmed by partial calmodulin gene sequence analyses. This technique is reproducible and provides the identification of all species of the Sporothrix complex with sufficient accuracy to be applied in clinical mycology laboratories as well as in epidemiological studies in order to obtain a better understanding of the epidemiology of sporotrichosis.