Panfungal PCR Research Articles

Clinical Evaluation of a Novel CRISPR-Cas12a-Based RID-MyC Assay for the Diagnosis of Fungal Endophthalmitis

ObjectiveThis study evaluates the RID-MyC (Rapid Identification of Mycoses using CRISPR) assay, a CRISPR/Cas12a-based diagnostic tool, for its efficacy in diagnosing fungal endophthalmitis (FE), comparing it with panfungal PCR and culture methods. DesignA comparative cross-sectional study assessing the performance of the RID-MyC assay against established diagnostic modalities for FE. SubjectsThe study included 133 intraocular samples from 117 patients with suspected microbial endophthalmitis. MethodsThe study compared the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of the RID-MyC assay against panfungal PCR and culture. The Limit of Detection (LoD) for Aspergillus flavus and Candida albicans was determined for both RID-MyC and panfungal PCR across three different media: nuclease-free water (NFW), aqueous humor (AH), and vitreous humor (VH). Discrepancy analysis was conducted for discordant results, incorporating clinical outcomes and responses to antifungal treatment. Main Outcome MeasuresThe study primarily assessed the sensitivity, specificity, PPV, and NPV for clinical samples. Time to diagnosis was also evaluated. ResultsThe RID-MyC assay demonstrated a sensitivity of 88.24% (CI: 63.56% to 98.54%) and specificity of 93.1% (CI: 86.86% to 96.98%), with PPV and NPV of 65.22% (CI: 48.45% to 78.91%) and 98.18% (CI: 93.62% to 99.50%), respectively. Discrepancy analysis enhanced sensitivity to 90.48% (CI: 69.62% to 98.83%) and specificity to 96.43% (CI: 91.11% to 99.02%). The RID-MyC assay was 10 to 1000-fold more sensitive than panfungal PCR in detecting Aspergillus flavus and Candida albicans in intraocular specimens. The time to diagnosis with the RID-MyC assay was consistently under two hours. ConclusionsThe RID-MyC assay may advance the rapid and precise diagnosis of FE, with possible relevance to other invasive fungal conditions.

Successful management of pneumonia and pyogranulomatous cranial mediastinitis due to Coccidioides species infection with fluconazole in a 3‐year‐old horse

SummaryA 3‐year‐old Quarter Horse gelding originating from Texas presented for fever and inappetence. An encapsulated, mixed echogenic cranial mediastinal mass visible ultrasonographically in the left and right fourth intercostal space that displaced the heart caudally was identified. Histological evaluation of needle biopsies obtained under general anaesthesia revealed a severe pyogranulomatous inflammatory process associated with intralesional round, 15–25 μm in diameter, thick double‐contoured cell wall fungal structures. Sequencing of products obtained from DNA extracted from the paraffin‐embedded biopsy sample and a panfungal PCR assay targeting the large ribosomal unit region had a 99.1% identity to either Coccidioides immitis or C. posadessii. Bronchoscopy revealed purulent exudate in the right primary bronchus. Cytological evaluation of bronchial alveolar lavage fluid (BALF) contained 96% moderately degenerative neutrophils and 4% macrophages. Fungal structures were not appreciated in the BALF. However, the mediastinitis could have resulted from inhalation and dissemination of arthroconidia through lymphatics or directly from the lung parenchyma. Quantitative immunodiffusion of serum for the presence of coccidioidal IgG antibodies yielded a titre of 1:256 suggestive of disseminated coccidioidomycosis. After oral fluconazole for 24 months, the horse's general health and athletic performance had returned to normal and the mediastinal mass was not appreciated sonographically.

Diagnosis of invasive pulmonary fungal infections by a real-time panfungal PCR assay in non-neutropenic patients.

This study explored the utility of quantitative real-time panfungal PCR assay in diagnosing invasive pulmonary fungal diseases (IPFD) in non-neutropenic patients. Panfungal PCR assay was performed on respiratory tract specimens from patients whose clinical signs could not exclude fungal infection. At the same time, the samples were subjected to bacterial and fungal culture, microscopic examination and galactomannan antigen (GM) test in order to find the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the 4 diagnostic methods in proven and probable cases. 518 specimens were collected while 63 respiratory tract specimens tested by PCR had positive results. According to diagnostic criteria, 40 patients were diagnosed with IPFD, with 12 proven, 20 probable and 8 possible cases. Among these, 33 patients of PCR results were positive, most of which were from BALF samples (44.12%). 23 cases were caused by Aspergillus species, with Aspergillus fumigatus was the major cause. Other Aspergillus species, including Aspergillus flavus, Aspergillus terreus and Aspergillus nidulans were found in 1 sample respectively. Candida species were found in 5 samples, Pneumocystis jeroveci pneumonia (PJP) in 4 samples and Mucormycosis in 1 sample. An analysis of proven/probable diagnosis showed a sensitivity of 78.13%, specificity of 92.18%, PPV of 39.68% and NPV of 98.46% for PCR and 50%, 85.27%, 35.7%, 95.65% for GM test respectively. The Ct value difference between proven/probable and possible cases had no statistical significance (P = .824). Fungal culture showed a sensitivity of 17.5% while microscopic examination sensitivity of 32.5%. Through stratified analysis, no apparent correlation was found between the Ct value of the PCR assay and GM value (r: 0.223, P = .294). But a conjunction of the 2 tests raised the PPV of Aspergillus to 90%. As shown in this study, the panfungal RT-PCR assay has high sensitivity and consistency with serological test and culture. Its high PPV in the detection of Aspergillus and PJP were also evident.

Screening of Ophidiomyces ophidiicola in the free-ranging snake community annually harvested for the popular ritual of San Domenico e dei Serpari (Cocullo, AQ, Italy)

In the Abruzzi village of Cocullo (Italy), each year, on May 1st, local snake hunters (known as Serpari) display colubrids, captured in the wild, to commemorate the ancient ritual of San Domenico. The ascomycete Ophidiomyces ophidiicola (Oo) is the causative agent of ophidiomycosis, an emerging disease with sublethal effects. Skin lesions, such as dysecdysis, edematous, crusty or necrotic scales, swellings, nodules, and ulcers, are the most common clinical manifestation of the disease. The pathogen and its associated disease are well characterized in wild snakes in North America, whereas broad screenings of free ranging wild ophidians in Europe are rare. In 2019, as part of a multi-year snake health monitoring project, all the Cocullo ophidians were carefully examined for integumentary affections and those showing signs consistent with ophidiomycosis were dry swabbed on the skin and on any visible cutaneous lesions with a single applicator. The extracted DNA underwent a broad-range panfungal PCR targeting the D1-D2 region, as well as two conventional PCRs specific to the ITS2 and IGS regions of Oo DNA. Twenty-three out of 129 snakes (13/82 Elaphe quatuorlineata; 7/31 Hierophis viridiflavus; 3/15 Zamenis longissumus; 0/1 Natrix helvetica) resulted clinically affected, but no specific Oo genomic DNA was detected by PCR. The Cocullo ritual celebration provided a unique opportunity for the first systematic testing of a large sample size of a local snake community for the monitoring of this pathogen in Italy.

Panfungal PCR on formalin-fixed, paraffin-embedded tissue: to proceed or not proceed?

Performance of panfungal PCR-DNA sequencing assays for diagnosis of invasive fungal disease on formalin-fixed, paraffin-embedded tissue (FFPE) is influenced by many variables. Interpretation of a positive result can be challenging due to the need to differentiate colonisers and contaminants from clinically significant pathogens. We conducted a retrospective audit on FFPE tissue specimens that underwent panfungal PCR from January 2021 to August 2022. Panfungal PCR results from samples where fungal elements were visualised on histopathology were compared with results from samples where no fungal elements were visualised. The cost per clinically significant positive sample in each group was calculated. Of the 248 FFPE tissues sampled, 18.1% (45/248) had fungal forms seen on histopathology. Panfungal PCR was positive in 22/45 samples (48.9%), with 16 (35.6%) results deemed clinically significant. For the remaining 203 specimens, panfungal PCR was positive in 19 (9.4%) samples with only six (3.0%) clinically significant. The average cost per clinically significant result was AUD 258.13 in the histopathology positive group and AUD 3,105.22 in the histopathology negative group. Our data suggest panfungal PCR has limited clinical utility in FFPE tissue when no fungal elements are seen. Restricting the assay to only those samples that are positive on histopathological examination aids interpretation of PCR positive results and conserves laboratory resources.

Panfungal PCR on formalin fixed paraffin embedded tissue – to proceed or not proceed?

Performance of panfungal PCR-DNA sequencing assays for diagnosis of invasive fungal disease on formalin fixed paraffin embedded tissue (FFPE) is influenced by many variables Interpretation of a positive result can be challenging due to need to differentiate colonisers, contaminants from clinically significant pathogens. We conducted a retrospective audit on FFPE tissue specimens that underwent panfungal PCR from January 2021 to August 2022. Panfungal PCR results from samples where fungal elements were visualised on histopathology were compared with results from samples where no fungal elements were visualised.

Eumycetoma Caused by Madurella pseudomycetomatis in a Captive Tiger (Panthera tigris).

A captive-kept adult male tiger presented with a large cutaneous and subcutaneous mass on the thigh with a fistula. During sedation, multiple nodules were detected and samples for a histopathological exam were collected. Histologically, granulomatous panniculitis and dermatitis were seen around dense aggregates of pigmented fungal hyphae, and a diagnosis of phaeohyphomycosis was made; considering the clinical features, it was classified as a eumycotic mycetoma. This is a rarely reported subcutaneous fungal infection in humans and animals, caused by dematiaceous fungi. Clinically, it is characterized by tumefaction, fistulous sinus tracts, and the formation of macroscopically visible grains. In the literature, only a few infections in wild felids have been reported. In this case, Fontana-Masson staining better showed pigmentation and panfungal PCR and sequencing identified Madurella pseudomyectomatis (OP623507) as the causative agent. Systemic therapy with oral administration of itraconazole was planned, but the patient died during the first period of treatment. The animal was not submitted for post-mortem examination. Visceral dissemination of the agent cannot be excluded. To the authors' knowledge, this is the first report of eumycotic mycetoma by Madurella pseudomycetomatis in a captive tiger.

The use of readily available laboratory tests for the identification of the emerging yeast Candida auris in Mexico.

Identification of the emerging multidrug-resistant yeast Candida auris is challenging. Here, we describe the role of the Mexico national reference laboratory Instituto de Diagnóstico y Referencia Epidemiológicos Dr. Manuel Martínez Báez (InDRE) and the Mexican national laboratory network in the identification of C. auris. Reference identification of six suspected isolates was done based on phenotypic and molecular laboratory methods, including growth in special media, evaluation of isolate micromorphology, and species-specific PCR and pan-fungal PCR and sequencing. The four C. auris isolates identified were able to grow on modified Sabouraud agar with 10% NaCl incubated at 42 °C. With one exception, isolates of C. auris were spherical to ovoid yeast-like cells and blastoconidia, with no hyphae or pseudohyphae on cornmeal agar. C. auris isolates were resistant to fluconazole. Species-specific and pan-fungal PCR confirmed isolates as C. auris. Sequence analysis revealed the presence of two different C. auris clades in Mexico, clade I (South Asia) and clade IV (South America).

Rapid sourdough yeast identification using panfungal PCR combined with high resolution melting analysis

The microbial composition of the sourdough starter affects the sourdough bread properties. Therefore, it is crucial to find a tool for rapid, time-saving, and economical identification of the sourdough microbiota. We focused on the rapid identification of sourdough yeasts. We designed a panfungal real time-PCR targeting the ITS2 region (ITS-amplicon) and a fragment of D1/D2 region of 26S rRNA gene (U-amplicon) and used high resolution melting analysis (HRM) for subsequent species identification. The sensitivity and specificity of our method were tested on the reference yeast cultures. We obtained divergent melting peaks (Tm). The further analysis of melt curves suggests the possibility to discriminate yeasts on the genus- and some on species-specific level in the mixed sample. The applicability of this method in routine practice was evaluated on nine sourdough samples. Revealed melt curves of U-amplicons were predominantly characteristic of the sourdough. The evaluation of the Tm and the shape of the melt curve was used to assess the sourdough yeasts. Additionally, using the HRM-PCR method the contamination with the ergot fungus DNA was revealed. Our data showed HRM-PCR is a simple, rapid, and inexpensive tool useful in identifying sourdough yeasts.

Development of a Real-Time PCR Assay to Identify and Distinguish between Cryptococcus neoformans and Cryptococcus gattii Species Complexes.

Cryptococcus neoformans and Cryptococcus gattii are the principle causative agents of cryptococcosis. Differences in epidemiological and clinical features, and also treatment, mean it is important for diagnostic laboratories to distinguish between the two species. Molecular methods are potentially more rapid than culture and cryptococcal antigen (CRAG) detection; however, commercial PCR-based assays that target Cryptococcus do not distinguish between species. Here, we developed a real-time PCR assay targeting the multicopy mitochondrial cytochrome b (cyt b) gene to detect C. neoformans and C. gattii in clinical specimens. Assay performance was compared with culture, histopathology, CRAG and panfungal PCR/DNA sequencing. The cyt b-directed assay accurately detected and identified all eight C. neoformans/gattii genotypes. High-resolution melt curve analysis unambiguously discriminated between the two species. Overall, assay sensitivity (96.4%) compared favorably with panfungal PCR (76.9%) and culture (14.5%); assay specificity was 100%. Of 25 fresh frozen paraffin embedded (FFPE) specimens, assay sensitivity was 96% (76% for panfungal PCR; 68% for histopathology). The Cryptococcus-specific PCR is a rapid (~4 h) sensitive method to diagnose (or exclude) cryptococcosis and differentiate between the two major species. It is suitable for use on diverse clinical specimens and may be the preferred molecular method for FFPE specimens where clinical suspicion of cryptococcosis is high.

Fungal Infections of Implantation: More Than Five Years of Cases of Subcutaneous Fungal Infections Seen at the UK Mycology Reference Laboratory.

Subcutaneous fungal infections, which typically result from traumatic introduction (implantation) of fungal elements into the skin or underlying tissues, can present as a range of different clinical entities including phaeohyphomycosis, chromoblastomycosis, subcutaneous nodules or masses, and genuine eumycetoma. Here, we mined our laboratory information management system for such infections in humans and domestic animals for the period 2016–2022, including (i) fungal isolates referred for identification and/or susceptibility testing; (ii) infections diagnosed at our laboratory using panfungal PCR approaches on infected tissue; and (iii) organisms cultured in our laboratory from biopsies. In total, 106 cases were retrieved, involving 39 fungal species comprising 26 distinct genera. Subcutaneous infections with Alternaria species were the most frequent (36 cases), which possibly reflects the ubiquitous nature of this common plant pathogen. A substantial proportion of Alternaria spp. isolates exhibited reduced in vitro susceptibility to voriconazole. Notably, a significant number of subcutaneous infections were diagnosed in renal and other solid organ transplant recipients post transplantation, suggesting that humans may harbour “inert” subcutaneous fungal elements from historical minor injuries that present as clinical infections upon later immunosuppression. The current study underscores the diversity of fungi that can cause subcutaneous infections. While most organisms catalogued here were responsible for occasional infections, several genera (Alternaria, Exophiala, Phaeoacremonuim, Scedosporium) were more frequently recovered in our searches, suggesting that they possess virulence factors that facilitate subcutaneous infections and/or inhabit natural niches that make them more likely to be traumatically inoculated.

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

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 gene sequencing for the diagnosis, tracking and prevention of fungal infections

The precise diagnosis of fungi is utmost important owing to the morbidity and mortality caused especially in various susceptible hosts. Among the diagnostic methods though the phenotypic methods are being routinely used among laboratories but they have inherent hindrances of being tedious, time-consuming and entail experience. These roadblocks acting as a major obstacle in precise identification of fungi, underlined the requisite for implementation of genotypic methods for routine diagnosis. Since sequencing forms the cornerstone of molecular identification of fungi, many sophisticated modalities and platforms have been developed. The role of fungal sequencing isn't limited merely to the identification of known fungal species in routine laboratory, but is of utmost significance in deciphering the emerging pathogenic and saprophytic fungal species that have the potential to infect humans. It was with the use of these sequencing techniques that the complex fungal nomenclature based on presence or absence of sexual form of the fungus, could be simplified and unified effort led to adoption of 'one-fungus--one-name' rule. Panfungal PCR targeting 28S rRNA when used in conjunction with sequencing for detection of etiological agents in patients with invasive fungal disease (IFD) from deep tissue samples has shown encouraging results. Though many sequencing modalities are available, an ideal diagnostic platform is yet awaited to meet the diversity of fungal infections in initial stages itself. The early diagnosis enables the clinician to administer appropriate therapy as and when required. The same helps in delimiting the undesired affects of antifungals as well as indirectly help in antimicrobial stewardship as well.

232 To Report A Rare Case of Fungal Necrotising Otitis Externa (NOE) Centred on The Left Temporomandibular Joint (TMJ)

Abstract Introduction NOE is a rare life-threatening complication of otitis externa, affecting the skull base, mastoid and temporal bones. Pseudomonas aeruginosa account for 95% of cases, making fungal NOE unusual. Complications secondary to NOE include cranial neuropathies, meningitis and dural sinus thrombophlebitis. Case-study A 67-year-old man with stage-5 chronic kidney disease presented with left otalgia and otorrhea. He was treated with antipseudomonal topical antibiotics and microsuction for months as an outpatient. Aspergillus flavus was grown on an initial swab, but subsequent cultures were negative. Computerised Tomography scans revealed inflammatory changes in the left masticator space with mastoid bone involvement suggestive of left NOE. He received three months of intravenous anti-pseudomonal antibiotics, microsuction and topical aminoglycosides. Despite interventions symptoms persisted and magnetic resonance imaging scanning revealed disease progression into the left TMJ, prompting maxillofacial surgical opinion. Following washout of the TMJ, a tissue biopsy was positive for DNA on pan-fungal PCR, and the sequence identified as Aspergillus flavus group. The patient was successfully treated with oral posoconazole and topical amphotericin and discharged home. Conclusions Fungal NOE remains poorly treated as there is limited guidance on antifungal choice and duration of treatment. It should always be considered, particularly in immunocompromised patients with intractable cases of NOE

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

BackgroundAlthough 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 presentationWe 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 diagnosisConclusionIt 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.

Direct Detection and Identification of the Most Common Bacteria and Fungi Causing Otitis Externa by a Stepwise Multiplex PCR.

BackgroundConsidering the importance of differential diagnosis of infectious otitis externa (OE), a stepwise PCR-based assay using universal and genus- or species-specific primers for the detection/identification of the most prevalent bacterial and fungal OE was developed and evaluated on the ear aspiration specimens of clinically suspected patients.Methods and MaterialsA total of 120 ear aspiration specimens with otomycosis suspicion were subjected to manual DNA extraction using phenol–chloroform extraction after tissue digestion with a lysis buffer. The multiplex PCR was initially performed using pan-fungal and bacterial homemade primers. Pseudomonas and Staphylococcus specific primers were simultaneously used in one reaction mixture to identify the bacterial genera. Furthermore, for the identification of fungal agents, Candida species-specific multiplex primers targeting the most clinically important Candida species causing OE (i.e., C. albicans, C. parapsilosis, and C. auris), as well as Aspergillus related multiplex PCR identifying the most prevalent Aspergillus species were used in two separate reaction mixtures. All the results of multiplex PCR were interpreted based on the amplicon size.ResultsThe overall multiplex PCR-based detection rate of bacterial (n = 88; 73.3%) and fungal (n = 97; 81%) OE was documented to be 100% along with and complete consistency with the results of direct examination and Giemsa staining. Double amplicon bands of bacterial and fungal pathogens were evidenced in 76 specimens (63.3%). Moreover, the positivity rate of pan-fungal PCR was higher than that of the culture result. Out of 88 pan-bacterial positive PCR specimens, 66 and 47 ones were positive for Staphylococcus and Pseudomonas, respectively. In addition, 30 samples exhibited mixed infection of both, and five specimens remained negative. Out of 97 pan-fungal positive PCR specimens, 67 and 51 ones contained Candida and Aspergillus species, respectively. It should be noted that dual amplicon bands of Candida and Aspergillus-related multiplex PCR were yielded in 30 specimens.ConclusionThe stepwise multiplex PCR assay proved to be more sensitive, more rapid, as well as less cumbersome in detection and identification of fungal and bacterial OE, compared to culture.

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.

Utility of Panfungal PCR in the diagnosis of invasive fungal infections in febrile neutropenia.

Background:The prevalence of invasive fungal infections (IFIs) is increasing due to the increasing population of immunocompromised patients. Fungal culture is the gold standard for diagnosis but not sensitive and the turnaround time is long. Samples for histopathology are difficult to obtain because of profound cytopenias. We conducted this study with the aim to evaluate panfungal PCR for the diagnosis of IFIs in patients of febrile neutropenia.Methods:This was a single-centre, cross-sectional observational study. Patients of febrile neutropenia suspected of having IFI were included in the study. Panfungal PCR was performed on the blood of included patients along with other investigations for diagnosis of IFI. The sensitivity, specificity, positive predictive value, and negative predictive value of panfungal PCR were calculated using EORTC/MSG 2008 criteria as the gold standard.Results:Fifty patients of febrile neutropenia were included in the study, of which 52% were diagnosed positive by panfungal PCR assay. The sensitivity, specificity, positive predictive value, and negative predictive value of panfungal PCR assay was found to be 82.76%, 90.48%, 92.31% and 79.17% respectively.Conclusion:Panfungal PCR is a promising and highly sensitive diagnostic test for screening at-risk patients suspected to have IFIs. The use of panfungal PCR assay in combination with other diagnostic modalities and clinical judgment can be very helpful in the early diagnosis of IFI.

Knowledge at what cost? An audit of the utility of panfungal PCR performed on bronchoalveolar lavage fluid specimens at a tertiary mycology laboratory

The diagnostic utility and costs of panfungal PCR assays for invasive fungal disease (IFD) from bronchoalveolar lavage fluid (BALF) specimens are incompletely defined. In a retrospective audit, panfungal PCR results from 2014-2018 were matched with information on request forms and the registrar/microbiologist diary of clinical liaison. Identification of a single fungus other than a commensal was considered potentially clinically significant, and assessed for clinical relevance. Of 1002 specimens tested, an estimated 90% were requested in patients without clinical suspicion of IFD. There were 530 (52.9%) PCR-positive results of which 485/530 (91.5%) identified multiple fungal species or commensal fungi; 45 (8.5%) were clinically significant but only in 12 (1.2%) was panfungal PCR the sole diagnostic test leading to IFD diagnosis, all in immunocompromised patients with clinical suspicion of IFD. Costs of panfungal PCR tests averaged AUD 133 per test, or AUD 26,767/annum. However, the average cost-per-diagnosis achieved was AUD 15,978/annum. Limiting testing to patients at risk and with clinical suspicion of IFD, may save over AUD 13,383/annum (assuming 50-90% reduction in testing). The value-added utility of panfungal PCR on BALF is 1.2% (12/1002). We have since introduced pre-analytical stewardship limiting routine panfungal PCR testing of BALF to high-risk patients in our hospital.

Colonic Basidiobolomycosis—An Unusual Presentation of Eosinophilic Intestinal Inflammation

Basidiobolomycosis is a rare fungal disease caused by Basidiobolus ranarum. Involvement of the gastrointestinal tract is unusual and poses both a diagnostic and therapeutic challenge, as clinical signs are non-specific and predisposing risk factors are lacking. It can mimick inflammatory bowel disease, primary immunodeficiency, or a malignancy and should be considered in patients who do not respond to standard therapy. We present the case of a 22 months old boy with confirmed colonic Basidiobolomycosis, who presented with severe eosinophilic inflammation of the gastrointestinal tract. Panfungal PCR performed on DNA extracted directly from a tissue sample confirmed the presence of Basidiobolus. He made a full recovery with a combination of surgery and prolonged targeted antifungal medication.