Conventional PCR as a reliable method for diagnosing invasive mucormycosis in resource-limited settings

Introduction. Invasive mucormycosis (IM) is a life-threatening infection caused by fungi belonging to the order Mucorales. Histopathology, culture and radiology are the mainstay of diagnosis but lack sensitivity, leading to a delay in timely diagnosis and intervention. Recently, PCR-based approaches have been shown to be a promising method in diagnosing IM. Hypothesis/Gap Statement. Molecular-based approaches may be a valuable adjunct to standard conventional methods for diagnosing IM, especially among culture negatives and patients on antifungal therapy. Aim. In the present study we aimed to evaluate the clinical utility of panfungal and Mucorales-specific PCR for diagnosing IM from various clinical specimens. Methodology. This was a prospective study in which 239 clinically suspected cases of IM attending our tertiary care hospital from August 2015 to March 2018 were enrolled. All the cases were defined as ‘proven’, ‘probable’ or ‘possible’ based on EORTC/MSGERC guidelines. In addition to conventional diagnostics (KOH-calcofluor stain and culture), panfungal and Mucorales-specific PCR assays were also performed. The amplified products were sequenced for species identification. In vitro antifungal susceptibility was performed on all the culture-positive isolates. Results. Among 239 clinically suspected cases of IM, only 140 cases were diagnosed by the demonstration of aseptate ribbon-like hyphae on direct microscopy. Culture was positive in 35.7 % (54/140) of direct microscopy-positive samples. Among the proven cases (n=11), the sensitivity for both Mucorales-specific nested PCR and panfungal PCR was 100 %, but specificity was 91.9 and 73.7% respectively. In probable cases (n=129), the sensitivity of both the PCRs was 98.5 % and specificity for panfungal PCR was 73.7 and 91.9 % for Mucorales-specific PCR. Conclusion. Pan fungal PCR in combination with Mucorales-specific PCR, followed by sequencing, may play a significant role in IM diagnosis especially among those negative for both direct microscopy and culture.

Low sensitivity of conventional fungal agars in fungemia by Rhodotorula mucilaginosa: description of two cases

Background Although most bloodstream yeast infections are caused by Candida spp., infections by rare or less common species have increased in recent years. Diagnosis of infections caused by these species is difficult due to the lack of specific symptoms and adequate diagnostic tools. Cases presentation We describe two cases of fungemia by Rhodotorula mucilaginosa within a few months of each other, in a secondary Spanish hospital. In both cases, diagnosis was challenging. Blood subcultures in conventional fungal media were persistently negatives and the use of non-conventional fungal media was essential for isolating the yeasts and achieving a correct diagnosis. 1–3 beta-d-glucan detection and a panfungal PCR assay were helpful techniques to confirm the diagnosis Conclusion It is highly important to establish an early diagnosis for fungemia. The process is challenging because often non-specific symptoms are presents. When yeasts grow in blood cultures other genera than Candida spp. could be the cause of infection. Patient risk factors should be assessed to incorporate alternative culture media and the available rapid diagnostic test, in order to provide an early recognition of the pathogen.

Diagnostic Accuracy of a Direct Panfungal Polymerase Chain Reaction Assay Performed on Stained Cytology Slides

Molecular techniques are increasingly being applied to stained cytology slides for the diagnosis of neoplastic and infectious diseases. Such techniques for the identification of fungi from stained cytology slides have not yet been evaluated. This study aimed to assess the diagnostic accuracy of direct (without nucleic acid isolation) panfungal polymerase chain reaction (PCR) followed by sequencing for identification of fungi and oomycetes on stained cytology slides from dogs, cats, horses, and other species. Thirty-six cases were identified with cytologically identifiable fungi/oomycetes and concurrent identification via fungal culture or immunoassay. Twenty-nine controls were identified with no cytologically or histologically visible organisms and a concurrent negative fungal culture. Direct PCR targeting the internal transcribed spacer region followed by sequencing was performed on one cytology slide from each case and control, and the sensitivity and specificity of the assay were calculated. The sensitivity of the panfungal PCR assay performed on stained cytology slides was 67% overall, 73% excluding cases with oomycetes, and 86% when considering only slides with abundant fungi. The specificity was 62%, which was attributed to amplification of fungal DNA from control slides with no visible fungus and negative culture results. Direct panfungal PCR is capable of providing genus- or species-level identification of fungi from stained cytology slides. Given the potential of panfungal PCR to amplify contaminant fungal DNA, this assay should be performed on slides with visible fungi and interpreted in conjunction with morphologic assessment by a clinical pathologist.

Case Report: Successful Management of Conidiobolus Lamprauges Rhinitis in a Dog

This is a case of Conidiobolus lamprauges rhinitis in a Goldendoodle, that was presented for evaluation of sneezing, coughing, lethargy, as well as right-sided epistaxis and clear ocular discharge. Computed tomography revealed a large amount of soft tissue within the right nasal passage that obscured the osseous turbinates from the right maxillary canine tooth to the right side of the choanae. Biopsies revealed eosinophilic granulomas with variable number of basophilic to negatively staining, septate, fungal hyphae with non-parallel walls and irregular branching that were subsequently determined to be Conidiobolus lamprauges via panfungal PCR and sequencing. Complete and sustained resolution of clinical disease was achieved after 75 days of systemic antifungal therapy. This report describes for the first time, important clinical features of a dog with nasal conidiobolomycosis that will facilitate its recognition, prognostication, and treatment in clinical practice.

Scedosporium and Lomentospora Infections: Contemporary Microbiological Tools for the Diagnosis of Invasive Disease

Scedosporium/Lomentospora fungi are increasingly recognized pathogens. As these fungi are resistant to many antifungal agents, early diagnosis is essential for initiating targeted drug therapy. Here, we review the microbiological tools for the detection and diagnosis of invasive scedosporiosis and lomentosporiosis. Of over 10 species, Lomentospora prolificans, Scedosporium apiospermum, S. boydii and S. aurantiacum cause the majority of infections. Definitive diagnosis relies on one or more of visualization, isolation or detection of the fungus from clinical specimens by microscopy techniques, culture and molecular methods such as panfungal PCR or genus-/species-specific multiplex PCR. For isolation from respiratory tract specimens, selective media have shown improved isolation rates. Species identification is achieved by macroscopic and microscopic examination of colonies, but species should be confirmed by ITS with or without β-tubulin gene sequencing or other molecular methods. Matrix-assisted laser desorption ionization-time of flight mass spectrometry databases are improving but may need supplementation by in-house spectra for species identification. Reference broth microdilution methods is preferred for antifungal susceptibility testing. Next-generation sequencing technologies have good potential for characterization of these pathogens. Diagnosis of Scedosporium/Lomentospora infections relies on multiple approaches encompassing both phenotypic- and molecular-based methods.

Clinical evaluation of an in-house panfungal real-time PCR assay for the detection of fungal pathogens

Purpose Due to an increasing incidence of invasive fungal infections, the availability of reliable diagnostic tools for the fast detection of a wide spectrum of fungal pathogens is of vital importance. In this study, we aimed to conduct an extensive clinical evaluation of a recently published in-house panfungal PCR assay on samples from suspected invasive fungal infections. Methods Overall 265 clinical samples from 232 patients with suspected invasive fungal disease (96 deep airway samples, 60 sterile fluids, 50 tissue biopsies, and 59 blood samples) were included. All samples underwent standard culture-based diagnostics and were additionally analyzed with our panfungal PCR assay. Results Overall, 55.1% of agreement between culture and the panfungal PCR was observed; in 17% of all samples partial concordance was noted, while results between culture and our PCR assay were not in agreement in 27.9%. Our panfungal assay performed better in samples from normally sterile sites, while samples from the deep airways yielded the highest rate of discordant (39.6%) results. In two tissue and three blood samples an invasive pathogen was only detected by PCR while cultures remained negative. Conclusion In combination with routine methods, our panfungal PCR assay is a valuable diagnostic tool. Patients at risk for invasive fungal infections might profit from the reduced time to pathogen identification.

Molecular Detection of Aspergillus: Application of a Real-Time PCR Multiplex Assay in Tissue Samples

Diagnosis of invasive fungal infections is complex, and the lack of standardization of molecular methods is still a challenge. Several methods are available for the diagnosis of invasive aspergillosis, but their effectiveness will depend on the studied population, the patients’ comorbidities, and the use of mold active prophylaxis, among others. The ability to determine the identity of the infecting Aspergillus species, and to detect mutations conferring specific resistance patterns directly from DNA extracted from the biological product, is an advantage of nucleic acid testing compared with antigen-based assays. In this study, we present laboratory cases where the diagnosis of aspergillosis was performed using a real-time multiplex PCR for the detection of Aspergillus DNA in tissue samples, showing its usefulness as one more tool in the diagnosis of aspergillosis in tissue samples. Aspergillus real-time multiplex PCR was also used to detect azole-resistance in some cases. In the majority of the PCR positive cases, cultures remained negative after 60 days. The PCR assay directed to Aspergillus gave positive signals for Aspergillus fumigatus sensu stricto. Results were confirmed by panfungal PCR, followed by sequencing, revealing 100% homology with Aspergillus fumigatus sensu stricto. Mutations conferring azole resistance were not detected.

Panfungal PCR assays using fresh frozen paraffin embedded tissue specimens for fungal species identification and the detection of azole-resistance mutations in the A. fumigatus cyp51A gene at a South Korean hospital

Background: With rising concerns about changing fungal epidemiology and azole resistance in Aspergillus species, identifying fungal species and susceptibility patterns of mucorales and aspergillosis are crucial in the management of these diseases. The objectives of this study were to evaluate performance of panfungal PCR assays on formalin-fixed paraffin embedded (FFPE) samples for fungal species identification, and the detection of azole-resistance mutations in the Aspergillus fumigatus ( A.fumigatus ) cyp51A gene at a South Korean hospital. Methods: A total of 75 FFPE specimens with a histopathological diagnosis of aspergillosis or mucormycosis were identified during the 10-year study period (2006-2015). After deparaffinization and DNA extraction, panfungal PCR assays were conducted on FFPE samples for fungal species identification. The identified fungal species were compared with histopathological diagnosis. On samples identified as A.fumigatus , sequencings to identify frequent mutations in the cyp51A gene (tandem repeat 46 [TR46], L98H, and M220 alterations) that confer azole resistance were performed. Results: Specific fungal DNA was identified in 31 (41.3%) FFPE samples, and of these, 16 samples of specific fungal DNA were in accord with histopathological diagnosis of aspergillosis or mucormycosis. 15 samples had discordant histopathology and PCR results. No azole-mediating cyp51A gene mutation was revealed among nine cases of A. fumigatus . Moreover, no cyp51A mutations were identified among three cases with history of prior azole use. Conclusion: The pan-fungal PCR assay with FFPE sample may provide additional information on fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.