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.

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.

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.

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.

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.

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.

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.

Oscillatory, stochastic and chaotic growth rate fluctuations in permittistatically controlled yeast cultures.

We describe a continuous culture system related to the turbidostat, but using a feedback system based on biomass estimation from the dielectric permittivity of the cell suspension rather than its optical density. It is shown that this system provides an excellent method of maintaining a constant biomass level within a fermentor. The computer-controlled system was able to effect the essentially continuous registration of growth rate by monitoring the rate of medium addition via the time-dependent activity of the pump. At some biomass setpoints for aerobically grown cultures of baker's yeast substantial time-dependent fluctuations in the growth rate of the culture were thereby observed. At some biomass setpoints, however, or under anaerobic conditions, or when using a non-Crabtree yeast, the growth rate was constant, indicating that the fluctuations were inherent to the biological system and not simply a property of the fermentor and control system. A variety of time series analyses (Fourier transformations, Hurst and Lyapunov exponents, the determination of embedding dimension, and non-linear time series predictions based on the methodology of Sugihara and May) were used to demonstrate, for the first time, that as well as stochastic and periodic components these fluctuations exhibited deterministic chaos. 'Trivial predictors' were unable to give accurate predictions of the growth rate in these cultures. The growth rate fluctuations were studied further by means of offline measurements of changes in percentage viability, bud count, and in the external ethanol and glucose concentrations; these data and other evidence suggested that the growth rate fluctuations were closely linked to the primary respiro-fermentative metabolism of this organism. The identification of chaotic growth rates in cell cultures suggests that there may be novel methods for controlling the growth of such cultures.

[Rapid identification of Candida albicans: evaluation of "Rapidec albicans". Study of 444 yeast strains]. / Identification rapide de Candida albicans: évaluation de "Rapidec albicans". Etude de 444 souches de levures.

Rapid identification of Candida albicans is of great importance as it is the most frequently isolated yeast pathogen. Rapidec albicans, a new 2-h micromethod, performs two fluorescent enzymatic activities: hexosaminidase and proline arylamidase. A total of 444 yeast strains (334 from type culture collections and 110 from recent clinical isolates) were tested. The sensitivity was 98.5% and the specificity 95.8%. When only considering the clinical strains, 47/47 Candida albicans were identified by Rapidec albicans (sensitivity 100%) but only 43/47 by the germ tube test (sensitivity 91.5%). The specificities of the two tests were respectively 98.2% and 100%. This new system is therefore very efficient for the routine diagnosis of Candida albicans in the clinical field. It is easier and quicker than the germ tube test.

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.

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.

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.

Comparison of automated Difco ESP blood culture system with biphasic BBL Septi-Chek system for detection of bloodstream infections in pediatric patients.

We compared the Difco ESP 384 blood culture system with the pediatric Septi-Chek system for the detection of bloodstream infections in pediatric patients. A total of 10,762 blood culture sets included an ESP aerobic bottle and a Septi-Chek bottle. From these cultures, a total of 278 organisms classified as probable pathogens were isolated, including 237 from ESP bottles and 221 from Septi-Chek bottles. This difference was not statistically significant. More organisms classified as possible contaminants were also isolated from ESP bottles (for ESP, 480 bottles; for Septi-Chek, 418 bottles; P < 0.01). The time to detection was shorter for probable pathogens isolated from ESP bottles (median times for organisms isolated from both systems: ESP, 14.0 h; Septi-Chek, 34.5 h; P < 0.001). The proportions of all probable pathogens detected by 24 and 48 h after inoculation were 78 and 96%, respectively, for ESP compared with 31 and 74%, respectively, for Septi-Chek. The time to final identification was also shorter for organisms grown in ESP bottles (median times for organisms isolated from both systems: ESP, 48.8 h; Septi-Chek, 58.5 h; P < or = 0.001). A subset of 4,442 cultures also included an ESP anaerobic bottle in addition to an ESP aerobic bottle and a Septi-Chek bottle. There were no significant differences in the recovery of probable pathogens by any of the possible two bottle combinations, but five anaerobic pathogens were recovered only in the anaerobic bottle. We conclude that the ESP 384 is an excellent system for culturing pediatric blood samples and that it provides for the very rapid detection of bloodstream pathogens.

Value of terminal subcultures for blood cultures monitored by BACTEC 9240.

Blood cultures collected in BACTEC Plus Aerobic/F bottles and BACTEC Plus Anaerobic/F bottles were monitored for 5 days by BACTEC 9240 and subsequent terminal subcultures. Of the 13,471 bottles subcultured, 11.0% (1,477 of 13,471) were culture positive. Of these, 94.0% (1,388 of 1,477) were detected by BACTEC 9240; the additional 6.0% (89 of 1,477) were considered to be false negatives by BACTEC 9240 since they were detected by terminal subculture only. The false-negative bottles consisted of 17 BACTEC Plus Aerobic/F and 72 BACTEC Plus Anaerobic/F bottles, accounting for 2.2 (17 of 786) and 10.4% (72 of 691) of the total positive aerobic and anaerobic bottles, respectively. The positive blood culture bottles most frequently not detected by BACTEC 9240 grew Pseudomonas spp. (24), Staphylococcus spp. (21), and yeasts (24). Of the 86 blood cultures represented by the 89 false-negative bottles, 41 would not have been identified as positive since the other bottle in the blood culture set was either a false negative or a true negative. In general, terminal subcultures of false-negative BACTEC bottles had heavy growth, indicating that BACTEC Plus media were able to support the growth of microorganisms, but the BACTEC 9240 instrument was unable to detect this growth.

G test, a new direct method for diagnosis of Candida infection: comparison with assays for beta-glucan and mannan antigen in a rabbit model of systemic candidiasis.

An indirect method to measure beta-glucan, a major structural component of yeast cell walls, is available, but has the disadvantage of requiring the combined use of two assays. Recent reports describe the fungal index, which measures the difference between the conventional limulus test, in which factors C and G react with endotoxin and beta-glucan, and a new endotoxin-specific test, in which only factor C reacts with endotoxin. The G test was developed as a direct method to measure beta-glucan, and contains only factor G reacting with beta-glucan alone. In this study, the G test was examined in sera of rabbits with experimental systemic candidiasis, and compared with the fungal index and mannan assay. The G test showed positive in all rabbits with systemic candidiasis faster and with higher titers than with the fungal index. Three rabbits with fulminant systemic candidiasis showed higher levels of reactivity with the G test and the fungal index than two rabbits with mild reactions. Mannan was positive by at least one serum in four of five rabbits by the latex agglutination test, and there was a good correlation between these assays. The G test is a good serodiagnostic method for the detection of candidiasis.

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.