Interlaboratory evaluation of Mucorales PCR assays for testing serum specimens: A study by the fungal PCR Initiative and the Modimucor study group.

Interlaboratory evaluations of Mucorales qPCR assays were developed to assess the reproducibility and performance of methods currently used. The participants comprised 12 laboratories from French university hospitals (nine of them participating in the Modimucor study) and 11 laboratories participating in the Fungal PCR Initiative. For panel 1, three sera were each spiked with DNA from three different species (Rhizomucor pusillus, Lichtheimia corymbifera, Rhizopus oryzae). For panel 2, six sera with three concentrations of R. pusillus and L. corymbifera (1, 10, and 100 genomes/ml) were prepared. Each panel included a blind negative-control serum. A form was distributed with each panel to collect results and required technical information, including DNA extraction method, sample volume used, DNA elution volume, qPCR method, qPCR template input volume, qPCR total reaction volume, qPCR platform, and qPCR reagents used. For panel 1, assessing 18 different protocols, qualitative results (positive or negative) were correct in 97% of cases (70/72). A very low interlaboratory variability in Cq values (SD = 1.89 cycles) were observed. For panel 2 assessing 26 different protocols, the detection rates were high (77-100%) for 5/6 of spiked serum. There was a significant association between the qPCR platform and performance. However, certain technical steps and optimal combinations of factors may also impact performance. The good reproducibility and performance demonstrated in this study support the use of Mucorales qPCR as part of the diagnostic strategy for mucormycosis.

Combined medico-surgical strategy for invasive sino-orbito-cerebral breakthrough fungal infection with Hormographiella aspergillata in an acute leukaemia patient.

Hormographiella aspergillata is a rare causative agent of invasive filamentous breakthrough infection, mostly arising after echinocandin exposure. We report a neutropenic patient who developed a severe sino-orbito-cerebral H. aspergillata infection while receiving empirical caspofungin, successfully controlled by an aggressive strategy associating surgical debridement and combined high-dose regimen of antifungal drugs.

Iterative breakthrough invasive aspergillosis due to TR(34) /L98H azole-resistant Aspergillus fumigatus and Emericella sublata in a single hematopoietic stem cell transplant patient.

We report 3 consecutive episodes of invasive aspergillosis in a single hematopoietic stem cell transplant patient successively attributed to TR(34) /L98H azole-resistant Aspergillus fumigatus and to a first occurrence of invasive Emericella sublata infection. This case illustrates potential selection of resistant molds during antifungal therapy in hematological patient.

Current knowledge and practice of Candida auris screening in France: A nationwide survey from the French Society of Medical Mycology (SFMM).

Due to large outbreaks observed worldwide, Candida auris has emerged as a major threat to healthcare facilities. To prevent these phenomena, a systematic screening should be performed in patients transferred from regions where the pathogen is highly endemic. In this study, we recorded and analyzed French mycologists' current knowledge and practice regarding C. auris screening and diagnosis. Thirty-six centers answered an online questionnaire. Only 11 (30.6 %) participants were aware of any systematic screening for C. auris for patients admitted to their hospital. In the case of post-admission screening, axillae/groins (n = 21), nares (n = 7), rectum (n = 9), and mouth (n = 6) alone or various combinations were the body sites the most frequently sampled. Only six centers (8.3 %) reported using a commercially available plate allowing the differentiation of C. auris colonies from that of other Candida species, while five laboratories (13.8 %) had implemented a C. auris-specific qPCR. Considering the potential impact on infected patients and the risk of disorganization in the care of patients, it is crucial to remember to biologists and clinicians the utmost importance of systematic screening on admission.

Allergic fungal rhinosinusitis and eosinophilic mucin chronic rhinosinusitis: Differential diagnostic criteria. A two-center comparative study following STROBE methodology.

OBJECTIVES: Allergic fungal rhinosinusitis (AFRS) and eosinophilic mucin chronic rhinosinusitis (EMRS) are two forms of chronic sinusitis distinguished by the presence (AFRS) or absence (EMRS) of fungal elements in sinus mucin. Detection of the fungal elements, however, is complex and it is difficult to say whether EMRS is in fact an entity distinct from AFRS. The aim of the present study, based on a retrospective series of AFRS and EMRS, was to identify the specific clinical and radiological elements distinguishing between the two. MATERIALS AND METHODS: A 2-center retrospective observational study following STROBE guidelines included patients managed for AFRS or EMRS between 2009 and 2022. Clinical, mycological, pathologic and radiological data were collected. Type of treatment and disease progression were also analyzed. Intergroup comparison used Student's test for mean values of quantitative variables, with calculation of P-values, and Pearson's Chi2 test or Fisher's exact test for categoric variables, with calculation of relative risk and 95% confidence intervals. RESULTS: The AFRS group comprised 41 patients and the EMRS group 34. Demographic data were comparable between groups. EMRS showed a higher rate of asthma (79.4 vs. 31.4%; P<0.001), more severe nasal symptomatology (rhinorrhea, P=0.01; nasal obstruction, P=0.001), and more frequent bilateral involvement (85.3 vs. 58.5%; P=0.021). AFRS showed more frequent complications (19 vs. 0%; P=0.006). Radiologically, mucin accumulation was greater in AFRS, filling the sinus in 84.2% of cases, versus 26.3% (P<0.001), with more frequent sinus wall erosion (19 vs. 5.8%; P=0.073). The recurrence rate was higher in EMRS: 38.2 vs.21.9% (P=0.087). CONCLUSION: The present retrospective study found a difference in clinical and radiological presentation between AFRS and EMRS, with EMRS more resembling the presentation of severe nasal polyposis.

[Fungal sinusitis]. / Sinusites fongiques.

Although sinusitis affects about 20 % of the population, fungal sinusitis is rare. Aspergillus sp. are most frequently implicated. Fungal sinusitis represents a wide spectrum of disorders, including acute or chronic and invasive or non-invasive forms. Invasive fungal sinusitis may develop in an immunocompromised or diabetic patient, whereas non-invasive fungal sinusitis should be considered in a chronic situation, resistant to antibiotics in immunocompetent patients. Allergic fungal sinusitis is related to hypersensitivity of the host to the fungus. The diagnosis of these infections requires radiological examination and endoscopy with mucosal biopsies examined histologically and mycologically in order to distinguish the different types of sinusitis. In the non-invasive forms, surgical treatment is essential, sometimes combined with antifungal and anti-inflammatory treatment. The invasive forms require antifungal treatment, combined with surgery in some forms, particularly mucormycosis.

Early diagnosis and monitoring of mucormycosis by detection of circulating DNA in serum: retrospective analysis of 44 cases collected through the French Surveillance Network of Invasive Fungal Infections (RESSIF).

The main objective of this study was to assess the diagnostic performance of a set of three Mucorales quantitative PCR assays in a retrospective multicentre study. Mucormycosis cases were recorded thanks to the French prospective surveillance programme (RESSIF network). The day of sampling of the first histological or mycological positive specimen was defined as day 0 (D0). Detection of circulating DNA was performed on frozen serum samples collected from D-30 to D30, using quantitative PCR assays targeting Rhizomucor, Lichtheimia, Mucor/Rhizopus. Forty-four patients diagnosed with probable (n = 19) or proven (n = 25) mucormycosis were included. Thirty-six of the 44 patients (81%) had at least one PCR-positive serum. The first PCR-positive sample was observed 9 days (range 0-28 days) before diagnosis was made using mycological criteria and at least 2 days (range 0-24 days) before imaging. The identifications provided with the quantitative PCR assays were all concordant with culture and/or PCR-based identification of the causal species. Survival rate at D84 was significantly higher for patients with an initially positive PCR that became negative after treatment initiation than for patients whose PCR remained positive (48% and 4%, respectively; p <10-6). The median time for complete negativity of PCR was 7 days (range 3-19 days) after initiation of l-AmB treatment. Despite some limitations due to the retrospective design of the study, we showed that Mucorales quantitative PCR could not only confirm the mucormycosis diagnosis when other mycological arguments were present but could also anticipate this diagnosis. Quantification of DNA loads may also be a useful adjunct to treatment monitoring.

Aspergillus spp. invasive external otitis: favourable outcome with a medical approach.

Aspergillus spp. invasive external otitis (IEO) is a rare infection. We performed a seven-year, single-centre retrospective study from 2007 to 2014 including all patients with proven Aspergillus spp. IEO. Twelve patients were identified. All patients had a poorly controlled diabetes mellitus and one underwent solid organ transplant. The most frequently isolated species was Aspergillus flavus (n = 10) and voriconazole was the first-line therapy in all cases, with a median length of treatment of 338.5 days (158-804 days). None of the patients underwent extensive surgery. The clinical outcome was excellent. However, otological sequelae were reported, including hearing impairment (n = 7) and facial palsy (n = 3).

Molecular identification of Mucorales in human tissues: contribution of PCR electrospray-ionization mass spectrometry.

Molecular methods are crucial for mucormycosis diagnosis because cultures are frequently negative, even if microscopy suggests the presence of hyphae in tissues. We assessed PCR/electrospray-ionization mass spectrometry (PCR/ESI-MS) for Mucorales identification in 19 unfixed tissue samples from 13 patients with proven or probable mucormycosis and compared the results with culture, quantitative real-time PCR, 16S-23S rRNA gene internal transcribed spacer region (ITS PCR) and 18S PCR sequencing. Concordance with culture identification to both genus and species levels was higher for PCR/ESI-MS than for the other techniques. Thus, PCR/ESI-MS is suitable for Mucorales identification, within 6 hours, for tissue samples for which microscopy results suggest the presence of hyphae.

Difficulties with molecular diagnostic tests for mould and yeast infections: where do we stand?

PCR assays have not reached the same level of acceptance for the detection of human fungal pathogens as for other micro-organisms, mainly because the low number of micro-organisms challenges the detection limits of PCR. Therefore, whereas meta-analyses focusing on clinical validation suggest interest in adding PCR results to the diagnostic workup for invasive fungal disease (IFD) along with clinical evaluation, CT scans, classical mycology and antigen detection, no consensual PCR method has emerged. Compared with the end-point format of the 1990s, real-time quantitative PCR is a major breakthrough. This format prevents contamination with previously amplified products, provides the yield of amplification, allows for developing consensus procedures and should therefore be the only format used. An internal control is now mandatory to avoid false-negative results. Primer design strongly impacts on the objectives: pan-fungal primers can provide false-positive results due to environmental fungal DNA contamination; conversely, species-specific primers miss infections caused by untargeted fungi. Unresolved issues include the best specimens to be used; serum is currently preferred to blood because of the ease of the DNA extraction step. Work is in progress to establish standards at least for Aspergillus PCR, and the implementation of quality controls should help centres to improve assays. Eventually, the classical analysis of biomarker performance does not consider the evolving risk factors and changing treatments during IFD, which can lead to variable conclusions. New statistical methods such as event history analysis should be considered.

Antifungal pre-emptive strategy for high-risk neutropenic patients: why the story is still ongoing.

Neutropenic patients with haematological malignancies are at high risk of invasive fungal disease (IFD). Due to limitations in specific procedures to establish an early diagnosis of IFD, two historical unpowered studies suggested, three decades ago, that giving an empirical antifungal treatment to patients with persistent or recurrent fever under broad-spectrum antibacterials, could reduce the risk of IFD. For cost and toxicity reasons, this strategy became debated when modern imaging and indirect biological markers became available. Different pre-emptive strategies, either based on lung imaging, galactomannan antigenaemia, fungal PCR, or a combination of several parameters, were designed with the goal of restricting the administration of antifungals to the more at-risk patients with early signs of IFD. Almost all pre-emptive studies showed or suggested a reduction of administration and cost of antifungals during neutropenic phases. However, the clinical pertinence and safety of the strategy, and mainly its optimal design, are still pending. This paper reviews the evolution of these strategies and how they may be implemented in the haematology ward.

Real-time PCR assay-based strategy for differentiation between active Pneumocystis jirovecii pneumonia and colonization in immunocompromised patients.

Diagnosis of pneumocystosis usually relies on microscopic demonstration of Pneumocystis jirovecii in respiratory samples. Conventional PCR can detect low levels of P. jirovecii DNA but cannot differentiate active pneumonia from colonization. In this study, we used a new real-time quantitative PCR (qPCR) assay to identify and discriminate these entities. One hundred and sixty-three bronchoalveolar lavage fluids and 115 induced sputa were prospectively obtained from 238 consecutive immunocompromised patients presenting signs of pneumonia. Each patient was classified as having a high or a low probability of P. jirovecii pneumonia according to clinical and radiological presentation. Samples were processed by microscopy and by a qPCR assay amplifying the P. jirovecii mitochondrial large-subunit rRNA gene; qPCR results were expressed as trophic form equivalents (TFEq)/mL by reference to a standard curve obtained from numbered suspensions of trophic forms. From 21 samples obtained from 16 patients with a high probability of P. jirovecii pneumonia, 21 were positive by qPCR whereas only 16 were positive by microscopy. Fungal load ranged from 134 to 1.73 × 10(6) TFEq/mL. Among 257 specimens sampled from 222 patients with a low probability of P. jirovecii pneumonia, 222 were negative by both techniques but 35 were positive by qPCR (0.1-1840 TFEq/mL), suggesting P. jirovecii colonization. Two cut-off values of 120 and 1900 TFEq/mL were proposed to discriminate active pneumonia from colonization, with a grey zone between them. In conclusion, this qPCR assay discriminates active pneumonia from colonization. This is particularly relevant for patient management, especially in non-human immunodeficiency virus (HIV)-infected immunocompromised patients, who often present low-burden P. jirovecii infections that are not diagnosed microscopically.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry for fast and accurate identification of clinically relevant Aspergillus species.

New Aspergillus species have recently been described with the use of multilocus sequencing in refractory cases of invasive aspergillosis. The classical phenotypic identification methods routinely used in clinical laboratories failed to identify them adequately. Some of these Aspergillus species have specific patterns of susceptibility to antifungal agents, and misidentification may lead to inappropriate therapy. We developed a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS)-based strategy to adequately identify Aspergillus species to the species level. A database including the reference spectra of 28 clinically relevant species from seven Aspergillus sections (five common and 23 unusual species) was engineered. The profiles of young and mature colonies were analysed for each reference strain, and species-specific spectral fingerprints were identified. The performance of the database was then tested on 124 clinical and 16 environmental isolates previously characterized by partial sequencing of the ß-tubulin and calmodulin genes. One hundred and thirty-eight isolates of 140 (98.6%) were correctly identified. Two atypical isolates could not be identified, but no isolate was misidentified (specificity: 100%). The database, including species-specific spectral fingerprints of young and mature colonies of the reference strains, allowed identification regardless of the maturity of the clinical isolate. These results indicate that MALDI-TOF MS is a powerful tool for rapid and accurate identification of both common and unusual species of Aspergillus. It can give better results than morphological identification in clinical laboratories.