A Practical Workflow for the Identification of Aspergillus, Fusarium, Mucorales by MALDI-TOF MS: Database, Medium, and Incubation Optimization.

There is an increasing body of literature on the utility of MALDI-TOF MS in the identification of filamentous fungi. However, the process still lacks standardization. In this study, we attempted to establish a practical workflow for the identification of three clinically important molds: Aspergillus, Fusarium, and Mucorales using MALDI-TOF MS. We evaluated the performance of Bruker Filamentous Fungi database v3.0 for the identification of these fungi, highlighting when there would be a benefit of using an additional database, the MSI-2 for further identification. We also examined two other variables, namely, medium effect and incubation time on the accuracy of fungal identification. The Bruker database achieved correct species level identification in 85.7% of Aspergillus and 90% of Mucorales, and correct species-complex level in 94.4% of Fusarium. Analysis of spectra using the MSI-2 database would also offer additional value for species identification of Aspergillus species, especially when suspecting species with known identification limits within the Bruker database. This issue would only be of importance in selected cases where species-level identification would impact therapeutic options. Id-Fungi plates (IDFP) had almost equivalent performance to Sabouraud dextrose agar (SDA) for species-level identification of isolates and enabled an easier harvest of the isolates with occasional faster identification. Our study showed accurate identification at 24 h for Fusarium and Mucorales species, but not for Aspergillus species, which generally required 48 h.

Assessment of panfungal PCR performance with formalin-fixed paraffin-embedded tissue specimens†.

We reviewed the performance of a panfungal ITS-2 PCR and Sanger sequencing assay performed on 88 FFPE specimens at The Hospital for Sick Children (Toronto, Canada) in 2019. A potential fungal pathogen was identified by ITS PCR in 62.7 and 2.9% of positive and negative direct slide examination of tissue specimens, respectively. ITS amplicons were detected in 87/88 specimens, with 53/88 (60.2%) considered as 'positive-contaminants' and 34/88 (38.6%) as 'positive-potential pathogen' upon sequencing. Potential pathogens included Blastomyces dermatitidis (17.1%), Cryptococcus neoformans (17.1%), Histoplasma capsulatum (14.3%) and Mucormycetes (11.4%). Laboratories should only perform ITS PCR on FFPE tissues if fungal elements have been confirmed on histopathology slides. LAY SUMMARY: In this study, we examined how well a DNA-based test could detect DNA from fungi in archived human biopsy tissues. The best performance was achieved if fungi were seen in the tissue under a microscope before being tested. Our results indicate that we should only use this test if these conditions are met.

Role of respiratory viruses in pulmonary exacerbations in children with cystic fibrosis.

BACKGROUND: The role of respiratory viruses in cystic fibrosis (CF) exacerbations is incompletely understood. METHODS: Cross-sectional study of CF children with a pulmonary exacerbation. Mid-turbinate swabs were tested by a direct immunofluorescent antibody assay and a multiplex PCR panel (ResPlex II v2.0, Qiagen). Resplex II was also applied to sputum or throat swab samples. Pulmonary function tests and quality of life and severity scores were recorded. Sputum cell counts, bacterial density and cytokines were measured. RESULTS: 26/43 (60.5%) subjects tested positive for at least one respiratory virus by any diagnostic method applied to any sample type. Virus-positive patients were younger (p=0.047), more likely to be male (p=0.029), and had higher CF clinical severity (p=0.041) and lower quality of life (physical) scores (p=0.023) but similar IL-8, neutrophil percentage and elastase levels. CONCLUSIONS: Compared to non-viral exacerbations, viral-related exacerbations were associated with worse severity and quality of life scores but similar pulmonary inflammation.

Molecular identification of phaeohyphomycosis due to Alternaria infectoria in a patient with acute myeloid leukemia--a case report.

We report the case of a 15-year old with acute myeloid leukemia who developed breakthrough invasive fungal rhinitis. The fungus was identified as Alternaria infectoria by polymerase chain reaction (PCR) and successfully treated by surgical excision and combination antifungal therapy, emphasizing the utility of fungal PCR in timely diagnosis of invasive fungal infections.

Comparison of a commercial qualitative real-time RT-PCR kit with direct immunofluorescence assay (DFA) and cell culture for detection of influenza A and B in children.

BACKGROUND: Institutional pandemic planning prompted a study of the molecular detection of influenza virus from respiratory specimens in children, compared to conventional diagnostics. OBJECTIVE: To evaluate the performance of a commercial qualitative real-time RT-PCR kit (rRT-PCR), the artus Influenza LC RT-PCR (Qiagen). STUDY DESIGN (METHODS): Specimens were pre-selected to include a high percentage of positives by direct immunofluorescence assay (DFA) or culture. The sensitivity and specificity of the kit for detection of influenza A and B in children were determined against the gold standard, DFA and culture. Specimens yielding discordant results between artus and the gold standard were tested against a reference rRT-PCR assay (Centers for Disease Control) to create an "expanded gold standard". RESULTS: When compared to DFA or cell culture, the sensitivity of the rRT-PCR artus kit was 96.2% and the specificity was 94%. It detected influenza RNA in 6.0% of clinical samples negative by DFA or culture. Using the expanded gold standard, the revised sensitivity was 98.7% (98.6% for influenza A and 97.6% for influenza B) and the specificity was 100%. CONCLUSION: The artus Influenza LC RT-PCR kit is an effective alternative to virus isolation and DFA for the detection of influenza A and B in pediatric clinical specimens.

A 24-hour screening protocol for identification of vancomycin-resistant Enterococcus faecium.

We describe a 24-h protocol for the identification of patients who are positive for vancomycin-resistant Enterococcus faecium (VRE), using stool and rectal swab samples and VRE screening broth, automated DNA extraction, and real-time PCR for vanA and vanB genes. Compared to conventional screening methods, this protocol had a high sensitivity and specificity and a negative predictive value.

Rapid detection of single nucleotide polymorphisms associated with spinal muscular atrophy by use of a reusable fibre-optic biosensor.

Rapid (<2 min) and quantitative genotyping for single nucleotide polymorphisms (SNPs) associated with spinal muscular atrophy was done using a reusable (approximately 80 cycles of application) fibre-optic biosensor over a clinically relevant range (0-4 gene copies). Sensors were functionalized with oligonucleotide probes immobilized at high density (approximately 7 pmol/cm2) to impart enhanced selectivity for SNP discrimination and used in a total internal reflection fluorescence detection motif to detect 202 bp PCR amplicons from patient samples. Real-time detection may be done over a range of ionic strength conditions (0.1-1.0 M) without stringency rinsing to remove non-selectively bound materials and without loss of selectivity, permitting a means for facile sample preparation. By using the time-derivative of fluorescence intensity as the analytical parameter, linearity of response may be maintained while allowing for significant reductions in analysis time (10-100-fold), permitting for the completion of measurements in under 1 min.