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.

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

BACKGROUND/PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

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.

Protocols and programs for high-throughput growth and aging phenotyping in yeast.

In microorganisms, and more particularly in yeasts, a standard phenotyping approach consists in the analysis of fitness by growth rate determination in different conditions. One growth assay that combines high throughput with high resolution involves the generation of growth curves from 96-well plate microcultivations in thermostated and shaking plate readers. To push the throughput of this method to the next level, we have adapted it in this study to the use of 384-well plates. The values of the extracted growth parameters (lag time, doubling time and yield of biomass) correlated well between experiments carried out in 384-well plates as compared to 96-well plates or batch cultures, validating the higher-throughput approach for phenotypic screens. The method is not restricted to the use of the budding yeast Saccharomyces cerevisiae, as shown by consistent results for other species selected from the Hemiascomycete class. Furthermore, we used the 384-well plate microcultivations to develop and validate a higher-throughput assay for yeast Chronological Life Span (CLS), a parameter that is still commonly determined by a cumbersome method based on counting "Colony Forming Units". To accelerate analysis of the large datasets generated by the described growth and aging assays, we developed the freely available software tools GATHODE and CATHODE. These tools allow for semi-automatic determination of growth parameters and CLS behavior from typical plate reader output files. The described protocols and programs will increase the time- and cost-efficiency of a number of yeast-based systems genetics experiments as well as various types of screens.

Sysmex UF-1000i performance for screening yeasts in urine.

We tested the capacity of the Sysmex UF-1000i system to detect yeasts in urine by screening a total of 22 132 urine samples received for culture in our microbiology laboratory during 1 year. We also analyzed different dilutions of previously filtered urine inoculated with a strain of Candida albicans. With clinical samples, a single cut-off point of 50 yeast-like cells (YLCs)/µL detected candiduria ≥10 000 colony forming units (CFU)/mL and >100 000 CFU/mL with a sensitivity of 87.3%/95.4%, a specificity of 97%, a negative predictive value of 95.9%, and a positive predictive value of 9.3%/5.7%. With the simulated samples, a linear relationship was observed between the dilution factor and the number of cells detected by UF-1000i. This instrument appears to be able to reliably rule out candiduria of a magnitude of at least 10 000 CFU/mL and facilitate urine sample screening, thereby providing fast results. The Sysmex UF1000i system can be adapted for candiduria screening by the use of an appropriate YLCs/µL cut-off point that takes account of the prevalence of candiduria in the population.

Comparative study of nail sampling techniques in onychomycosis.

Onychomycosis is a common problem. Obtaining accurate laboratory test results before treatment is important in clinical practice. The purpose of this study was to compare results of curettage and drilling techniques of nail sampling in the diagnosis of onychomycosis, and to establish the best technique and location of sampling. We evaluated 60 patients suffering from distal and lateral subungual onychomycosis and lateral subungual onychomycosis using curettage and vertical and horizontal drilling sampling techniques from three different sites of the infected nail. KOH examination and fungal culture were used for detection and identification of fungal infection. At each sample site, the horizontal drilling technique has a better culture sensitivity than curettage. Trichophyton rubrum was by far the most common pathogen detected by both techniques from all sampling sites. The drilling technique was found to be statistically better than curettage at each site of sampling, furthermore vertical drilling from the proximal part of the affected nail was found to be the best procedure for nail sampling. With each technique we found that the culture sensitivity improved as the location of the sample was more proximal. More types of pathogens were detected in samples taken by both methods from proximal parts of the affected nails.

Comparative study of direct polymerase chain reaction, microscopic examination and culture-based morphological methods for detection and identification of dermatophytes in nail and skin samples.

The positive rates of dermatophytes isolated and identified by conventional methods are rather low. Moreover, clinical isolates sometimes show atypical morphology, and in such cases microscopic methods are not applicable for identification. The present study was performed to assess the utility of specific polymerase chain reaction (PCR)-based methods for Trichophyton rubrum and Trichophyton mentagrophytes as diagnostic tools for dermatophytoses. Both conventional morphological identification and specific PCR methods based on the nuclear ribosomal internal transcribed spacer (ITS)1 DNA sequence were performed to identify dermatophyte species from clinical specimens of patients who visited Kawasaki Social Insurance Hospital between 16 May and 17 August 2005. Specific PCR methods were also directly applied to clinical specimens, and the results of the two methods were compared. The clinical samples examined consisted of 126 skin scale specimens and 80 nail specimens. The positive rates of culture isolation from clinical specimens were 67% and 33% for skin scale and nail specimens, respectively. In contrast, PCR analysis yielded a positive rate of 100% for clinical isolates from both skin scales and nails, and rates of 95% and 99% were obtained by direct application to clinical specimens. The results of the present study indicated that specific PCR is highly advantageous as a diagnostic tool for detection and identification of dermatophytes on direct application to skin scale or nail specimens.

Autofluorescence: a screening test for mycotic infection in tissues.

Fungal infection is a major health concern as the clinical features are not very distinctive. Lack of rapid diagnostic techniques results in delay in diagnosis, which may even culminate in a fatal outcome. The fact that many pathogenic fungal organisms autofluoresce in hematoxylin and eosin (H and E)-stained sections under ultraviolet illumination led us to evaluate the role of autofluorescence as a rapid screening technique for fungal infections. The aim of the present study was to assess the value of autofluorescence as a screening method for detecting fungi on tissue sections and to compare the results of autofluorescence with conventional histochemical stains for fungi. Hematoxylin and eosin-stained slides of mycotic lesions were examined under fluorescent microscope and the findings were compared with results of Gomori's methenamine silver and periodic acid-Schiff stains. We found fungal autofluorescence in 63 out of 64 cases studied, with a sensitivity of 97.8% and specificity of 100% in comparison with fungal stains. This was statistically significant (P < 0.05). We conclude that autofluorescence can be used as a rapid screening method for identification of fungi in tissue sections as it does not require any other specialized staining procedure.

Rhythmic conidiation in Neurospora crassa.

In the filamentous fungus Neurospora crassa the production of asexual spores (conidia) is regulated by its circadian clock. When the fungus is grown on a thin layer of agar medium in long growth tubes (so-called "race tubes"), restricting its growth to one direction only, bright orange bands are clearly visible. This banding pattern persists with a periodicity of approx 24 h in the absence of any environmental stimuli. The bands are formed by alternating zones of nonsporulating mycelium and mycelium laden with orange conidia. Assaying Neurospora conidiation on race tubes is a simple yet powerful and versatile tool for studying the circadian clock of this model organism.

Simultaneous detection and identification of Candida, Aspergillus, and Cryptococcus species by reverse line blot hybridization.

We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit cross-hybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10(0.5) cells/ml and 10(2) conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.

Quantitative TaqMan PCR for detection of Pneumocystis jiroveci.

We developed a quantitative real-time PCR assay for detection and quantification of Pneumocystis jiroveci in bronchoalveolar lavage (BAL) specimens based on primers and probe targeting the gene encoding beta-tubulin. The assay was able to detect 50 DNA copies per ml of a standard plasmid containing the target sequence. The intra- and interassay coefficients of variation were 0.46%-4.27% and 0.05-2.00% over 5 log(10) values. Fifty-seven controls of human, viruses, bacteria and fungi DNA samples were amplified and found negative. Fifty-three BAL samples sent to the laboratory for diagnosis of pneumocystosis were prospectively investigated by real-time PCR and direct microscopic examinations (DME) using Giemsa stain and direct immunofluorescence. All PCR negative samples were negative by microscopy. Among the 24 (45%) BAL found PCR positive, 8 were positive by microscopy (35%). The copy numbers of the target gene were between 4.4 x 10(3) and 2.8 x 10(6) per ml for the microscopically positive samples and between 8 and 9.2 x 10(3) per ml for the microscopically negative samples. In conclusion, we developed a rapid, sensitive and specific real time PCR for the diagnosis and quantification of Pneumocystis jiroveci in BAL samples.

A rapid and highly sensitive method for measuring enzyme activities in single mycorrhizal tips using 4-methylumbelliferone-labelled fluorogenic substrates in a microplate system.

A microplate fluorimetric assay was developed for measuring potential activities of extracellular enzymes of individual ectomycorrhizal (EM) roots using methylumbelliferone (MU)-labelled fluorescent substrate analogues and microsieves to minimise damage due to manipulation of excised mycorrhizal roots. Control experiments revealed that enzyme activities remained stable over the whole time of the experiment suggesting a strong affinity of the studied enzymes to the fungal cell walls. The same mycorrhizal tips thus could be used repeatedly for enzyme detection and subsequently analysed for the projection area by automated image analysis. The developed system was evaluated on four different EM species measuring pH optimum and substrate saturation of phosphatase, chitinase and beta-glucosidase. The four EM species studied were Lactarius subdulcis, Russula ochroleuca, Cortinarius obtusus and Xerocomus cf. chrysenteron. Depending upon the enzyme, each species exhibited different levels of enzymatic activities as well as enzyme kinetics and showed also differences in pH optima.

Detection of Candida in concentrated oral rinse cultures by real-time PCR.

The incidence of oral candidosis has increased in recent years, largely as a result of the emergence of human immunodeficiency virus infection and the more widespread use of immunosuppressive chemotherapy. This development has been associated with a need for more reliable methods for the detection of Candida. The present study assessed the performance of a real-time PCR and two block-based PCRs for the detection of Candida in 193 concentrated oral rinse culture (CRC) specimens. A total of 102 CRC specimens were positive by culture for Candida; and 96, 90, and 75 of these were also positive by real-time, N18-specific, and internal transcribed spacer (ITS)-specific PCRs, respectively. The five false-negative results by the real-time PCR were all non-Candida albicans positive by culture. Of the 91 culture-negative CRC specimens, 20, 41, and 44 were positive by the real-time PCR and the N18- and ITS-specific PCRs, respectively. All three PCRs detected fungal DNA in 8 culture-negative CRC specimens, with a further 30 being positive by two of the three PCRs. A total of 32 CRC specimens were Candida free by all methods. In summary, a real-time PCR that provides a sensitive, specific, and rapid alternative technique for detection of Candida in the mouth is described.

Rapid quantification of drug resistance gene expression in Candida albicans by reverse transcriptase LightCycler PCR and fluorescent probe hybridization.

We developed a rapid, sensitive, and reproducible assay to quantify Candida albicans ACT1, CDR1, CDR2, ERG11, and MDR1 mRNA using a two-step reverse transcription and LightCycler real-time PCR (RT-LightCycler PCR) method with sequence-specific hybridization probes. We compared RT-LightCycler PCR with Northern hybridization for quantitative analysis of gene expression in isolates with various fluconazole susceptibilities. Specificity of each LightCycler PCR was verified by LightCycler melting curve analysis and agarose gel electrophoresis of amplified products. Correlation of quantification results between RT-LightCycler PCR and Northern hybridization yielded correlation coefficients of > or = 0.91 for all genes except MDR1 (0.74). In this case, reduced correlation was due to the inability of Northern hybridization to accurately quantify the high MDR1 expression in a susceptible dose-dependent isolate which was shown by RT-LightCycler PCR to overexpress MDR1 >200-fold relative to the other isolates tested. In four isolates, low levels of CDR2 mRNA were detected by RT-LightCycler PCR but were undetectable by Northern hybridization. mRNA quantification by RT-LightCycler PCR correlates with Northern hybridization and offers additional advantages, including increased sensitivity and speed of analysis, along with lower RNA concentration requirements and an increased dynamic range of signal detection.

Detection of circulating Aspergillus fumigatus galactomannan: value and limits of the Platelia test for diagnosing invasive aspergillosis.

The effectiveness of galactomannan detection with the Platelia test was evaluated in a prospective study of 3,327 sera from 807 patients. The specificity was 99.6% (748 of 751 cases). For the groups of patients with proven and probable invasive aspergillosis, the sensitivity was 50.0% (17 of 34 cases). The disappointing sensitivity associated with the presence of rare false-positive cases underlines the limits of this test.

Seminested PCR for diagnosis of candidemia: comparison with culture, antigen detection, and biochemical methods for species identification.

The rapid detection and identification of Candida species in clinical laboratories are extremely important for the management of patients with hematogenous candidiasis. The presently available culture and biochemical methods for detection and species identification of Candida are time-consuming and lack the required sensitivity and specificity. In this study, we have established a seminested PCR (snPCR) using universal and species-specific primers for detection of Candida species in serum specimens. The universal outer primers amplified the 3' end of 5.8S ribosomal DNA (rDNA) and the 5' end of 28S rDNA, including the internally transcribed spacer 2 (ITS2), generating 350- to 410-bp fragments from the four commonly encountered Candida species, viz., C. albicans, C. tropicalis, C. glabrata, and C. parapsilosis. The species-specific primers, complementary to unique sequences within the ITS2 of each test species, amplified species-specific DNA in the reamplification step of the snPCR. The sensitivity of Candida detection by snPCR in spiked serum specimens was close to 1 organism/ml. Evaluation of snPCR for specific identification of Candida species with 76 clinical Candida isolates showed 99% concordant results with the Vitek and/or ID32C yeast identification system. Further evaluation of snPCR for detection of Candida species in sera from culture-proven (n = 12), suspected (n = 16), and superficially colonized (n = 10) patients and healthy subjects (n = 12) showed that snPCR results were consistently negative with sera from healthy individuals and colonized patients. In culture-proven candidemia patients, the snPCR results were in full agreement with blood culture results with respect to both positivity and species identity. In addition, snPCR detected candidemia due to two Candida species in five patients, compared to three by blood culture. In the category of suspected candidemia with negative blood cultures for Candida, nine patients (56%) were positive by snPCR; two of them had dual infection with C. albicans and either C. tropicalis or C. glabrata. In conclusion, the snPCR developed in this study is specific and more sensitive than culture for the detection of Candida species in serum specimens. Moreover, the improved detection of cases of candidemia caused by more than one Candida species is an additional advantage.

Training medical myocologists in developing countries.

Although there has over recent years been a marked rise in the incidence of serious fungal infections, many of which are prevalent in developing countries, few facilities exist for diagnosis and research in medical mycology. In most countries, medical mycology is not taught adequately to medical students and consequently there is little awareness of the importance of fungal infections. Model teaching programmes need to be developed. Practical knowledge of mycoses, their diagnosis and treatment and also basic mycology can be disseminated through well-constructed courses and workshops. Formalized training in mycology research also needs to be introduced. To achieve all of this, expertise and additional resources need to be made available. In this regard, ISHAM can and should help.

The spheroplast lysis assay for yeast in microtiter plate format.

A yeast lysis assay in the microtiter plate format improved precision and throughput and led to an improved algorithm for estimating lag time. The assay reproducibly revealed differences of 10% or greater in the maximal lysis rate and 50% or greater in the lag time. Clonal differences were determined to be the major source of variation. Microtiter-based assays should be useful for screening for drug susceptibility and for analyzing mutant phenotypes.

Species identification and strain differentiation of dermatophyte fungi by analysis of ribosomal-DNA intergenic spacer regions.

Restriction fragment length polymorphisms (RFLPs) identified in the ribosomal-DNA (rDNA) repeat were used for molecular strain differentiation of the dermatophyte fungus Trichophyton rubrum. The polymorphisms were detected by hybridization of EcoRI-digested T. rubrum genomic DNAs with a probe amplified from the small-subunit (18S) rDNA and adjacent internal transcribed spacer (ITS) regions. The rDNA RFLPs mapped to the nontranscribed spacer (NTS) region of the rDNA repeat and appeared similar to those caused by short repetitive sequences in the intergenic spacers of other fungi. Fourteen individual RFLP patterns (DNA types A to N) were recognized among 50 random clinical isolates of T. rubrum. A majority of strains (19 of 50 [38%]) were characterized by one RFLP pattern (DNA type A), and four types (DNA types A to D) accounted for 78% (39 of 50) of all strains. The remaining types (DNA types E to N) were represented by one or two isolates only. A rapid and simple method was also developed for molecular species identification of dermatophyte fungi. The contiguous ITS and 5.8S rDNA regions were amplified from 17 common dermatophyte species by using the universal primers ITS 1 and ITS 4. Digestion of the amplified ITS products with the restriction endonuclease MvaI produced unique and easily identifiable fragment patterns for a majority of species. However, some closely related taxon pairs, such as T. rubrum-T. soudanense and T. quinkeanum-T. schoenlenii could not be distinguished. We conclude that RFLP analysis of the NTS and ITS intergenic regions of the rDNA repeat is a valuable technique both for molecular strain differentiation of T. rubrum and for species identification of common dermatophyte fungi.

Fluconazole disk diffusion susceptibility testing of Candida species.

We describe a simple procedure for detecting fluconazole-resistant yeasts by a disk diffusion method. Forty clinical Candida sp. isolates were tested on RPMI-glucose agar with either 25- or 50-microgram fluconazole disks. With 25-microgram disks, zones of inhibition of >/=20 mm at 24 h accurately identified 29 of 29 isolates for which MICs were /=27 mm identified 28 of 29 such isolates. All 11 isolates for which MICs were >8 microgram/ml were identified by using either disk. Disk diffusion may be a useful screening method for clinical microbiology laboratories.