National production of certified reference fungal cultures.

Reference fungal cultures (RFCs) are essential for the internal quality control of laboratories. The production of these cultures requires standardized procedures (IRAM 14950:2016 and ISO 17034:2016 standards) carried out by a recognized and accredited laboratory. The aim of this work was to produce RFC in paper disks of autochthonous strains, characterized by two, homogeneous and stable reference methods traceable at species level. RFC were produced using 14 regional species (7 yeasts and 7 filamentous fungi) from the fungal culture collection (DMic). Paper disks were impregnated with a culture suspension, dried and packed. Homogeneity, viability, identity and purity were verified. Short- and long-term stability at different temperatures and storage times were studied. Characterization of each strain allowed to confirm its identity and to ensure its traceability at international level. Produced batches were homogeneous and stable at -20±5°C for 30 months. This method of production was adequate to produce homogeneous and stable RFC with phenotypic and genotypic characteristics correctly defined and internationally traceable. Standardized procedures were developed for the production of certified RFC that could be transferred to other microorganisms. Providing RFC that represent regional strains allows laboratories to produce more reliable results with a favorable impact on medical diagnosis, the environment or the food industry.

Survey of laboratory practices for diagnosis of fungal infection in seven Asian countries: An Asia Fungal Working Group (AFWG) initiative.

An online survey of mycology laboratories in seven Asian countries was conducted to assess the status, competence, and services available. Country representatives from the Asia Fungal Working Group (AFWG) contacted as many laboratories performing mycology diagnosis as possible in their respective countries, requesting that the laboratory heads complete the online survey. In total, 241 laboratories responded, including 71 in China, 104 in India, 11 in Indonesia, 26 in the Philippines, four in Singapore, 18 in Taiwan, and seven in Thailand. Overall, 129/241 (53.5%) surveyed mycology laboratories operate as separate designated mycology laboratories, 75/241 (31.1%) conduct regular formal staff training, 103/241 (42.7%) are accredited, and 88/157 (56.1%) participate in external quality assurance scheme (EQAS) programs. Microscopy and culture methods are available in nearly all laboratories, although few perform DNA sequencing (37/219; 16.9%) or use matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) (27/219; 12.3%) for isolate identification. Antifungal susceptibility testing is performed in 142/241 (58.9%) laboratories, mainly for yeasts. The most commonly performed nonculture diagnostic is cryptococcal antigen testing (66 laboratories), followed by galactomannan testing (55), polymerase chain reaction (PCR) diagnosis (37), and beta-D-glucan testing (24). Therapeutic drug monitoring is conducted in 21 laboratories. There is almost no access to advanced diagnostic tests, like galactomannan, ß-D-glucan, and PCR, in the surveyed laboratories in Indonesia, the Philippines, and Thailand. These results highlight the need for development of quality laboratories, accreditation and training of manpower in existing laboratories, and access to advanced non-culture-based diagnostic tests to facilitate the diagnosis of fungal infections in Asia.

Reproducibility of CSF quantitative culture methods for estimating rate of clearance in cryptococcal meningitis.

Quantitative cerebrospinal fluid (CSF) cultures provide a measure of disease severity in cryptococcal meningitis. The fungal clearance rate by quantitative cultures has become a primary endpoint for phase II clinical trials. This study determined the inter-assay accuracy of three different quantitative culture methodologies. Among 91 participants with meningitis symptoms in Kampala, Uganda, during August-November 2013, 305 CSF samples were prospectively collected from patients at multiple time points during treatment. Samples were simultaneously cultured by three methods: (1) St. George's 100 mcl input volume of CSF with five 1:10 serial dilutions, (2) AIDS Clinical Trials Group (ACTG) method using 1000, 100, 10 mcl input volumes, and two 1:100 dilutions with 100 and 10 mcl input volume per dilution on seven agar plates; and (3) 10 mcl calibrated loop of undiluted and 1:100 diluted CSF (loop). Quantitative culture values did not statistically differ between St. George-ACTG methods (P= .09) but did for St. George-10 mcl loop (P< .001). Repeated measures pairwise correlation between any of the methods was high (r≥0.88). For detecting sterility, the ACTG-method had the highest negative predictive value of 97% (91% St. George, 60% loop), but the ACTG-method had occasional (∼10%) difficulties in quantification due to colony clumping. For CSF clearance rate, St. George-ACTG methods did not differ overall (mean -0.05 ± 0.07 log10CFU/ml/day;P= .14) on a group level; however, individual-level clearance varied. The St. George and ACTG quantitative CSF culture methods produced comparable but not identical results. Quantitative cultures can inform treatment management strategies.

[Analysis of the results of the SEIMC External Quality Control Program. Year 2014]. / Análisis de resultados del Programa de Control de Calidad Externo SEIMC. Año 2014.

The External Quality Control Program of the Spanish Society of Infectious Diseases and Clinical Microbiology (SEIMC) include controls for bacteriology, serology, mycology, parasitology, mycobacteria, virology and molecular microbiology. This article presents the most relevant conclusions and lessons from the 2014 controls. As a whole, the results obtained in 2014 confirm the excellent skill and good technical standards found in previous editions. However, erroneous results can be obtained in any laboratory and in clinically relevant determinations. Once again, the results of the SEIMC program highlighted the need to implement both internal and external controls in order to assure the maximal quality of the microbiological tests.

[Requirements for mycological diagnostics in accordance with the guideline of the German Medical Association for quality assurance of medical laboratory tests]. / Anforderungen für die mykologische Diagnostik entsprechend der Richtlinie der Bundesärztekammer (Rili-BÄK) zur Qualitätssicherung laboratoriumsmedizinischer Untersuchungen.

The ability of recognizing various clinical manifestations of mucocutaneous mycosis, making a diagnosis, and establishing a treatment is part of a dermatologist's daily routine. However, due to the fact that clinical manifestations, laboratory diagnostics, and treatment are performed in one hand, laboratory findings are properly classified and interpreted. Since new binding guidelines of the German Medical Association on quality assurance measures in medical laboratory testing came into force, there is much concern among dermatologists of how to comply with these new regulations. It is the intention of the authors to help our readers to implement these new rules in order to make sure that mycological diagnostics continue to be part of a dermatologist's professional work.

The aspHS gene as a new target for detecting Aspergillus fumigatus during infections by quantitative real-time PCR.

Invasive aspergillosis (IA) is a serious nosocomial infection caused by Aspergillus spp. which has a high mortality rate due to the fact, among other factors, that it is difficult to diagnose. Within the Aspergillus genus, A. fumigatus is the main species causing IA. We propose a virulence factor, the aspHS gene, as a novel target for the specific detection of A. fumigatus by quantitative real-time PCR (qPCR). This target gene encodes a haemolysin, which is overexpressed in vivo during infection. We have designed specific primers and hydrolysis (Taqman) probes for the detection of this target and a chimeric internal amplification control (IC), designed to detect false negative results due to PCR inhibition. This qPCR assay was tested with DNA extracted from a wide collection of microorganisms, tissues from infected mice, and human bronchoalveolar lavage (BAL) samples. Results showed that it, together with the DNA extraction method, could detect A. fumigatus with high specificity. Furthermore, it can distinguish between germinated (first step to the development of infection) and non-germinated conidia (not detected). Our data indicate that these techniques could be sufficiently sensitive and rapid to help clinicians establish an earlier diagnosis, but the presence of PCR inhibitors in clinical samples such as BAL fluids needs to be addressed.

Assessment of accuracy of identification of pathogenic yeasts in microbiology laboratories in the United kingdom.

Rapid, accurate identification of yeast isolates from clinical samples has always been important given their innately variable antifungal susceptibility profiles. Recently, this has become paramount with the proposed introduction of species-specific interpretive breakpoints for MICs obtained in yeast antifungal susceptibility tests (M. A. Pfaller, D. Andes, D. J. Diekema, A. Espinel-Ingroff, D. Sheehan, and CLSI Subcommittee for Antifungal Susceptibility Testing, Drug Resist. Updat. 13:180-195, 2010). Here, we present the results of a 12-month evaluation of the accuracy of identifications that accompany yeast isolates submitted to the Mycology Reference Laboratory (United Kingdom) for either confirmation of identity or susceptibility testing. In total, 1,781 yeast isolates were analyzed, and the robustness of prior identifications obtained in microbiology laboratories throughout the United Kingdom was assessed using a combination of culture on chromogenic agar, morphology on cornmeal agar, and molecular identification by pyrosequencing. Over 40% of isolates (755) were submitted without any suggested identification. Of those isolates with a prior identification, 100 (9.7%) were incorrectly identified. Error rates ranged from 5.2% (for organisms submitted for antifungal susceptibility testing) to 18.2% (for organisms requiring confirmation of identity) and varied in a strictly species-specific manner. At least 50% of identification errors would be likely to affect interpretation of MIC data, with a possible impact on patient management. In addition, 2.3% of submitted cultures were found to contain mixtures of at least two yeast species. The vast majority of mixtures had gone undetected in the referring laboratory and would have impacted the interpretation of antifungal susceptibility profiles and patient management. Some of the more common misidentifications are discussed according to the identification method employed, with suggestions for avoiding such misinterpretations.

Evaluation of Aspergillus PCR protocols for testing serum specimens.

A panel of human serum samples spiked with various amounts of Aspergillus fumigatus genomic DNA was distributed to 23 centers within the European Aspergillus PCR Initiative to determine analytical performance of PCR. Information regarding specific methodological components and PCR performance was requested. The information provided was made anonymous, and meta-regression analysis was performed to determine any procedural factors that significantly altered PCR performance. Ninety-seven percent of protocols were able to detect a threshold of 10 genomes/ml on at least one occasion, with 83% of protocols reproducibly detecting this concentration. Sensitivity and specificity were 86.1% and 93.6%, respectively. Positive associations between sensitivity and the use of larger sample volumes, an internal control PCR, and PCR targeting the internal transcribed spacer (ITS) region were shown. Negative associations between sensitivity and the use of larger elution volumes (≥100 µl) and PCR targeting the mitochondrial genes were demonstrated. Most Aspergillus PCR protocols used to test serum generate satisfactory analytical performance. Testing serum requires less standardization, and the specific recommendations shown in this article will only improve performance.

Quality control guidelines for amphotericin B, Itraconazole, posaconazole, and voriconazole disk diffusion susceptibility tests with nonsupplemented Mueller-Hinton Agar (CLSI M51-A document) for nondermatophyte Filamentous Fungi.

Although Clinical and Laboratory Standards Institute (CLSI) disk diffusion assay standard conditions are available for susceptibility testing of filamentous fungi (molds) to antifungal agents, quality control (QC) disk diffusion zone diameter ranges have not been established. This multicenter study documented the reproducibility of tests for one isolate each of five molds (Paecilomyces variotii ATCC MYA-3630, Aspergillus fumigatus ATCC MYA-3626, A. flavus ATCC MYA-3631, A. terreus ATCC MYA-3633, and Fusarium verticillioides [moniliforme] ATCC MYA-3629) and Candida krusei ATCC 6258 by the CLSI disk diffusion method (M51-A document). The zone diameter ranges for selected QC isolates were as follows: P. variotii ATCC MYA-3630, amphotericin B (15 to 24 mm), itraconazole (20 to 31 mm), and posaconazole (33 to 43 mm); A. fumigatus ATCC MYA-3626, amphotericin B (18 to 25 mm), itraconazole (11 to 21 mm), posaconazole (28 to 35 mm), and voriconazole (25 to 33 mm); and C. krusei, amphotericin B (18 to 27 mm), itraconazole (18 to 26 mm), posaconazole (28 to 38 mm), and voriconazole (29 to 39 mm). Due to low testing reproducibility, zone diameter ranges were not proposed for the other three molds.

Naming Aspergillus species: progress towards one name for each species.

The nomenclature of fungi is governed by the International Code of Botanical Nomenclature. That Code is revised at each International Botanical Congress. This Code has permitted most fungi expressing both sexual and asexual states (i.e., pleomorphic fungi) to be accorded separate name(s) for the asexual states. Prior to 1981, the rules on naming pleomorphic fungi had become complicated and were not being applied consistently by mycologists. The changes made in 1981 simplified procedures but resulted in numerous name changes in Aspergillus. Molecular data in particular can now resolve the phylogenetic position of a fungus regardless of whether it expresses sexual or asexual structures. A growing consensus now wishes to either remove entirely or drastically amend the provision to permit separate names to be used for different states of the same species. Some initial changes towards that eventual goal were made at the 2005 International Botanical Congress, and a Special Committee then appointed is debating the most appropriate action to take. In the interim, in order to minimize confusion, mycologists working with Aspergillus and other affected genera are urged to refrain from both introducing new scientific names for further states of already known species, and also from using any such names proposed.

[Evaluation of the methodological quality of the Rémic (microbiology guidelines - bacteriology and mycology) of the Société française de microbiologie]. / Évaluation de la qualité méthodologique du Rémic (référentiel en microbiologie médicale - bactériologie et mycologie) de la Société française de microbiologie, 3(e) édition (2007).

We have evaluated the methodological quality of the Rémic (microbiology guidelines - bacteriology and mycology) of the Société française de microbiologie (edition2007), using to AGREE criteria, which are consensual at an international level, in particular at the the World Health Organisation (WHO) and at the European Union. The methodological quality of the Rémic appears to be sub-optimal. These shortcomings in quality are mainly observed in AGREE domain n° 5 (applicability), in AGREE item n° 5 (patients' opinions were not considered), and in AGREE item n° 23 (conflicts of interest were not declared). The users of the Rémic must be aware of these few methodological shortcomings in order for them to be careful before they put its recommendation in practice. In conclusion, we advise the editors of the Rémic to insert at least a methodological chapter in their next edition.

Critical stages of extracting DNA from Aspergillus fumigatus in whole-blood specimens.

A standardized protocol for extracting DNA from Aspergillus fumigatus has been proposed by the European Aspergillus PCR Initiative (EAPCRI). Using meta-regression analysis, the EAPCRI showed certain stages of the process to be critical to providing a satisfactory analytical sensitivity. The study investigated each step of the EAPCRI protocol by elimination and monitored the influence on Aspergillus PCR performance.

Antifungal susceptibility testing practices in mycology laboratories in France, 2018.

A survey of mycology laboratories for antifungal susceptibility testing (AFST) was undertaken in France in 2018, to better understand the difference in practices between the participating centers and to identify the difficulties they may encounter as well as eventual gaps with published standards and guidelines. The survey captured information from 45 mycology laboratories in France on how they perform AFST (number of strains tested, preferred method, technical and quality aspects, interpretation of the MIC values, reading and interpretation difficulties). Results indicated that 86% of respondents used Etest as AFST method, with a combination of one to seven antifungal agents tested. Most of the participating laboratories used similar technical parameters to perform their AFST method and a large majority used, as recommended, internal and external quality assessments. Almost all the participating mycology laboratories (98%) reported difficulties to interpret the MIC values, especially when no clinical breakpoints are available. The survey highlighted that the current AFST practices in France need homogenization, particularly for MIC reading and interpretation.

Audit of laboratory mycology services for the management of patients with fungal infections in the northwest of England.

BACKGROUND: Fungal infection is increasingly recognised as an important cause of morbidity and mortality, especially in immunocompromised patients. Little information exists on laboratory services available and the methods used by general microbiology laboratories to diagnose these important infections. AIM: To investigate the services microbiology laboratories in northwest England provide towards the diagnosis and management of superficial and deep fungal infections. METHODS: A questionnaire was sent to laboratories to get a holistic view of the support given to clinicians looking after patients with fungal infections. The aim was not to investigate details of each laboratory's standard operating procedures. The completed questionnaires, which formed the basis of this report, were returned by all 21 laboratories which were recruited. This study was conducted between March 2004 and September 2004. RESULTS: Services were provided to District General Hospitals and to six tertiary centres, including eight teaching hospitals by 16 laboratories. Their bed capacity was 250-1300 beds. Total specimens (including bacterial and viral) processed annually were 42 000-500,000 whereas fungal ones were 560-5400. CONCLUSION: In most microbiology laboratories of northwest England, clinicians were aware of the potential of fungal pathogens to cause infections especially in immunocompromised patients. Additional measures such as prolonged incubation of samples were introduced to improve fungal yield from patients at high risk. It is necessary to train and educate laboratory and medical staff about the role of serology and molecular methods in diagnosis and management of patients with fungal infection.

Standardisation of methods for assessing mould germination: a workshop report.

The first workshop on predictive mycology was held in Marseille, France, 2--4 February 2005 under the auspices of the French Microbiological society. The purpose of the workshop was to list the different techniques and definitions used by scientists for assessing mould germination and to evaluate the influence of the different techniques on the experimental results. Recommendations were made when a large consensus was obtained. In order to facilitate the study of germination, alternative methods to microscopic examination were examined.

[Conservation of Malassezia strains in blotting paper]. / Empleo de discos de papel secante para la conservación de cepas de Malassezia spp.

Reference strains belonging to the genus Malassezia were analyzed to evaluate, by comparison, different preservation systems such us subculture, freezing at -80 degrees C in glycerol, and blotting paper-disc conservation. The viability, phenotypic and genotypic characteristics of the strains used in this study was evaluated. The blotting paper method was found to be advantageous to preserve Malassezia spp strains due to both, its simple implementation in the laboratory and its efficiency.

Evaluation of a fungal collection as certified reference material producer and as a biological resource center.

Considering the absence of standards for culture collections and more specifically for biological resource centers in the world, in addition to the absence of certified biological material in Brazil, this study aimed to evaluate a Fungal Collection from Fiocruz, as a producer of certified reference material and as Biological Resource Center (BRC). For this evaluation, a checklist based on the requirements of ABNT ISO GUIA34:2012 correlated with the ABNT NBR ISO/IEC17025:2005, was designed and applied. Complementing the implementation of the checklist, an internal audit was performed. An evaluation of this Collection as a BRC was also conducted following the requirements of the NIT-DICLA-061, the Brazilian internal standard from Inmetro, based on ABNT NBR ISO/IEC 17025:2005, ABNT ISO GUIA 34:2012 and OECD Best Practice Guidelines for BRCs. This was the first time that the NIT DICLA-061 was applied in a culture collection during an internal audit. The assessments enabled the proposal for the adequacy of this Collection to assure the implementation of the management system for their future accreditation by Inmetro as a certified reference material producer as well as its future accreditation as a Biological Resource Center according to the NIT-DICLA-061.

Office practice-based confirmation of onychomycosis: a US nationwide prospective survey.

BACKGROUND: Onychomycosis is sufficiently prevalent to be seen and treated by primary care physicians. The diagnosis of onychomycosis is most often confirmed from nail specimens by microscopy and fungal culture done at a central laboratory; these are relatively expensive tests with a turnaround time of a month or more. This study was conducted (1) to evaluate the use of in-office dermatophyte test medium (DTM) culture, and (2) to determine the epidemiology of onychomycosis in a large, nationwide sample of patients who were not participants in a clinical trial. METHODS: A nationwide sample of primary care physicians and podiatrists enrolled 670 patients with clinical signs of toenail onychomycosis. Dermatophyte test medium cultures were performed in the office and the results were compared with fungal cultures performed by a central laboratory. RESULTS: Central laboratory fungal cultures were positive in 44% (n = 297) of patients and DTM cultures in 51% (n = 345). Dermatophytes accounted for 93% of the confirmed infections and nondermatophyte molds the rest. In the 617 patients with paired dermatophyte test medium and laboratory fungal culture results, the 2 tests were in agreement (both positive or both negative) in 68% of cases (kappa, 0.37; asymptotic SE, 0.04; 95% confidence interval, 0.299-0.441). CONCLUSIONS: A DTM culture is a relatively rapid, easy, and inexpensive method to confirm dermatophyte infections in patients with signs of onychomycosis in the primary care setting. Because the available drugs for treating onychomycosis are effective against all dermatophyte species, the confirmation of dermatophyte infection, without further identification of genus and species, is sufficient evidence to begin treatment.

Evaluation of the Microring YT system for identifying clinical yeast isolates.

The Microring YT is a commercial system for identifying clinical yeast isolates. The system uses a series of discs impregnated with inhibitory agents mounted on a ring. The pattern of growth and inhibition produced provides a six digit code which can be compared with a table provided by the manufacturer. The performance of this system was compared with the API 32C in the identification of 606 yeast isolates (355 clinical and 251 environmental strains). The Microring YT system was in 72.6% agreement with the API 32C system. The sensitivity of identification of different species varied from 38% to 100%. The API 32C system has a more extensive database than the Microring YT and is thus more reliable for use, but it is considerably more expensive. It is concluded that although the Microring YT is cheap, easy, and convenient to use, it is inadequate for many common Candida species.