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Cryptococcal antigen (CrAg) is a capsule polysaccharide antigen that can be detected in the fluids of patients with cryptococcal infections. Cryptococcal Antigen Latex Agglutination System (CALAS), enzyme-linked immunosorbent assays (EIA), and lateral flow assay (LFA) are the main methods available. Two main commercial LFA kits are available: CryptoPS (Biosynex, Illkirch Graffenstaden, France) and CrAg LFA (IMMY, Inc. USA). In our lab, we prospectively used CryptoPS as a screening tool in serum for confirmed positive results with CALAS. We investigated the rigor of the CryptoPS test in serum in a multicentric evaluation over 3 years. To improve the specificity of CryptoPS in serum, we additionally implemented and evaluated a pretreatment protocol before CryptoPS testing. A total of 43 serum samples collected from 43 patients were investigated. We found that the CryptoPS assay is hampered by a high rate of false-positive results in serum with a high rate of CryptoPS-positive but CrAg LFA-negative and CALAS-negative sera in patients with no proof of Cryptococcus infection (n = 29). Using a simple pretreatment procedure (5 min incubation at 100°C and centrifugation) we were able to reverse false-positive results, suggesting that there could be interferent material present in the serum. Pretreatment also impacted the CryptoPS results (negative result) in two patients with the cryptococcal disease, one with isolated antigenemia and one with cryptococcal meningitis. Comparing the titers obtained with CALAS and CrAg LFA, we noticed that the titer obtained with CrAg LFA was almost 10-fold higher than those with CALAS. This study showed that Biosynex CryptoPS in serum could give false-positive results even in the absence of cryptococcal disease. These could be reduced by applying an easy pretreatment procedure to the serum before testing, with little but existing impact on the sensitivity.Lateral flow assays are useful to detect the cryptococcal antigen in human fluids. We investigated CryptoPS-positive results and observed that true false-positive results occurred. The false-positive results can be reduced by applying an easy pretreatment procedure.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Introduction In-house real-time PCR (qPCR) is increasingly used for the diagnosis of endemic mycoses and diverse assays are in use in specialized laboratories. External quality control is currently lacking. Objective To compare the performance of different molecular detection protocols for the detection of Histoplasma capsulatum and Coccidioides spp. in a multicenter study involving five European laboratories. Methods Two test sample panels were sent to each laboratory which performed the analysis with their in-house assays. Recipients were blinded to sample content. The Histoplasma-panel included 14 samples representing a range of concentrations of Histoplasma DNA (n = 7), as well as a negative control and DNA from other fungi to test for specificity (Paracoccidioides lutzii n = 1; Blastomyces dermatitidis n = 1; Aspergillus fumigatus n = 1; Emergomyces spp. n = 2, and Candida albicans n = 1). The Coccidioides-panel included 10 samples representing a range of DNA concentrations of Coccidioides posadasii (n = 6), as well as a negative control and DNA from other fungi to test specificity (Uncinocarpus reesii n = 1; Trichophyton violaceum n = 1; and Candida albicans n = 1). Regarding techniques used, four laboratories used Histoplasma qPCRs, and one laboratory a conventional PCR and a broad-range PCR (brPCR) for fungal DNA. Four laboratories used different Coccidioides qPCRs and one laboratory a brPCR to detect Coccidioides DNA. Results Concerning the Histoplasma panel, qPCR assays were the most sensitives and agreement in the lowest detected amount of Histoplasma DNA was very suitable, ranging from 1 pg to 4 pg [ Conclusion Specific protocols based on qPCR showed better sensitivity than conventional and brPCR. These methods are useful for the rapid and sensitive detection Histoplasma and Coccidioides. Application of these tests on clinical samples may speed up diagnosis and potentially limit laboratory exposure to these fungi. Comparisons of in-house tests are essential to assess the performance and detect potential cross-reactivities and achieve a consensus.

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Objectives To face and escape his environment and the host immune response, Candida neoformans is able to change his morphology (Titan cells) and his metabolism (dormancy, quiescence). Titan cells (TC) have been observed in lungs and brains in experimental mouse models of cryptococcosis and in patients. In 2018, three independent teams published three protocols allowing the reproducible generation of TC in vitro [Hommel B et al., (AA); Trevijano-Contador N, et al., (OZ), and Dambuza IM et al., (EB), in PLoS Pathogens 2018]. TC generated in these protocols exhibited the same features as the TC described in vivo. Our objective was to compare and describe the three protocols in parallel to highlight common and different features that can impact further study using those specific protocols. Methods A total of 22 h before starting the kinetics of the TC generation, the three protocols requested a pre-culture at 30°C in three different liquid media. The medium for TC production for the three labs was also different but a common factor is the addition of fetal calf serum (FBS) for OZ and EB. This kinetics was evaluated for size and quantity (%) of TC produced over a 72 h period (H0, H18, H24, H48, H72) at 30°C under shaking for AA while OZ and EB protocols incubate the cells at 37°C and 5% of CO2, while the whole transcriptome was analyzed at H0, H3, H7, and H18 in triplicates. Results OZ generated the highest percentage of TC, 63.1% and 58.2% at H18 and H24, and decreased drastically down to 6.7% at H48. EB reached a high percentage of TC at H24 for 46.7% and dropdown 1) differences at H0 which highlights the impact of the preculture on the TC process. The highest numbers of DGE are observed between H0 and H7 for the three protocols, where about two 450 DGE, two 000 DGE, and two 300 AA, OZ, and EB, respectively. After analysis of the PCA plot during the kinetics, EB and OZ are grouped while AA is not. That could be explained by the presence of FBS in OZ and EB protocols. Conclusion By running the three protocols in parallel, we showed here that the kinetics of TC generation differed between each other with a significant variation of the transcriptome. This is an important finding that paves the way to compare more deeply the transcriptome of C. neoformans during TC generation with the final goal is to identify the genes associated with TC generation.

S1.4 Fungal infections in Asia, bringing it out of the dark, September 21, 2022, 11:00 AM - 12:30 PM Background Routine laboratory testing for cryptococcal meningitis currently consists of Cryptococcal antigen (CrAg) testing in blood and cerebrospinal fluid (CSF), CSF India ink, and CSF fungal culture. Quantitative cryptococcal culture (QCC) is labor intensive and not feasible in most settings. Objectives We evaluated quantitative (qPCR) and reverse transcriptase qPCR (RT-qPCR) assays to quantify cryptococcal load in CSF, plasma, and blood. We also investigated the dynamics of fungal DNA and RNA detection during antifungal treatment. Methods We developed a qPCR assay that can differentiate serotypes A, D, and B/C of Cryptococcus neoformans and C. gattii based on the amplification of a unique nuclear Quorum sensing protein 1 (QSP1) and a multicopy 28S rRNA gene and evaluated the assays on 205-patient samples from the AMBITION-cm trial in Botswana and Malawi (2018-2021). CSF, plasma, and whole blood samples were stored per patient and were sampled at day 0 (baseline), day 7 and 14 for CSF and at day 1, 3 and 7 for plasma and whole blood post antifungal treatment initiation. A Roche LightCycler480 and Graph pad prism were used for data analysis. Results A total of 205/209 stored patient samples (85 from Botswana; 124 from Malawi), were used. For QSP1 qPCR tested in CSF at D0, 138 (81.7%) were serotype A, 28 (16.6%) were serotype B/C and 3 (1.8%) were a mixed infection of serotype A and B/C. There was no amplification with 36 (17.6%) samples. There was no difference in fungal loads at D0, D7, and D14 between serotype A and B/C with the QSP1 qPCR assay, and QCC. QCC showed a good correlation with qPCR quantification with QSP1 qPCR (slope = 0.797, R2 = 0.73) and with 28S rRNA qPCR (Slope = 0.771, R2 = 0.778) assays. The fungal load at D0 was significantly higher in patients who died at week 2 (w2) and at week 10 (w10) as compared with patients who survived post-week 10 (P .05). Detection of Cryptococcus DNA (28S rRNA qPCR) in plasma or whole blood within the first 24 h of treatment was significantly associated with early mortality at w2 and mortality at w10 (P Conclusion Quantification of C. neoformans and C. gattii load in CSF and plasma at D0 is useful in identifying patients at risk of death and may be a promising tool for monitoring treatment response in the future.

S2.2 Histoplasmosis and talaromycosis, September 21, 2022, 3:00 PM - 4:30 PM In sub-Saharan Africa (SSA) and West African countries, histoplasmosis is rarely diagnosed probably due to lack of epidemiological information, insufficient training and awareness of frontline healthcare workers, and clinical features very similar to those of tuberculosis that can be misleading. This fungal infection mainly affects immunocompromised patients and particularly advanced HIV patients, with a high case-fatality rate in the absence of treatment (from 40%). The classical diagnostic methods are microscopic observation of yeasts with suggestive morphology and a positive culture from a biological sample. However, direct examination requires regular practice, and the yeasts can be confused with other pathogens and culture takes prolonged incubation (often 2-6 weeks) and involves, when positive, handling in a level 3 security laboratory. Implementing non-invasive diagnostic tools will allow us to improve histoplasmosis diagnosis for the most exposed patients and also to evaluate the prevalence of this fungal infection in countries where data are still lacking. Rapid diagnostic tests (RDTs) such as the TB Lam for the diagnosis of tuberculosis or the Cryptococcal antigen (CrAg) lateral flow assay (LFA) for cryptococcosis have demonstrated their usefulness for the management of advanced HIV patients in similar contexts. Recently, two RDTs have been made commercially available for the diagnosis of histoplasmosis, based on urinary monoclonal antigen detection: (1) Histoplasma Capsulatum Urinary Antigen Rapid Test from Optimium Imaging Diagnostics (OIDx) and (2) Histoplasma Urine Antigen Lateral Flow Assay from MiraVista Diagnostics (MV). Objectives and Methods Our objective was to evaluate these new tools, by experimenting with their feasibility in low-and middle-income countries (LMICs) and by studying their diagnostic performances using different sample collections recovered from patients with disseminated histoplasmosis (culture proven), other HIV-related infections, and proven negative urines (culture and other Histoplasma antigen detections). Results Preliminary results were obtained using the EDIRAPHIS study frozen samples from hospitalized patients diagnosed with proven positive and negative histoplasmosis from French Guiana and Suriname (n = 43) tested with OIDx and MV tests. We calculated a Se = 74.2% and a Sp = 83.3% for OIDx and a Se = 77.4% and a Sp = 91,7% for MV. A low number of false positives for both tests, Conclusion These first results are very promising and will be completed with two other specimen collections to increase the total numbers of our sampling and get a whole picture of the performances of these two RDTs. The next step will be to implement these new tools at the bedside or in laboratories together with other tests in different settings across SSA. Diffusion of RDTs together with appropriate training of clinical and laboratory teams and accessibility to treatment may help reduce the burden of histoplasmosis in endemic areas of SSA where the prevalence of the advanced-HIV disease is high.

Fusarium spp. are plant pathogens and opportunistic pathogens in severely immunocompromised (hematological malignancy, neutropenia, solid organ transplantation, etc.) and severely burned patients. Invasive fusariosis often disseminates and mortality remains high partly due to delayed diagnosis in the absence of a positive culture. The aim of our study is to design a quantitative PCR (qPCR) assay and evaluate the detection of Fusarium spp. DNA for early diagnosis of invasive infection. A qPCR assay was designed and optimized to identify all Fusarium species complex and secondarily evaluated on patient samples. A total of 81 blood samples from 15 patients diagnosed with proven invasive fusariosis from 9 centers in France were retrospectively tested. Circulating DNA was detected in 14 patients out of 15 (sensitivity of 93% [95% Confidence Interval (CI95), 70.1-99.7]). Detection was possible up to 18 days (median 6 days) before the diagnosis was confirmed by positive blood culture or biopsy. By comparison serum galactomannan and ß-D-glucan were positive in 7.1 and 58.3% of patients respectively. qPCR was negative for all patients with other invasive fungal diseases (IFD) tested (n = 12) and IFD-free control patients (n = 40). No cross-reactions were detected using DNA extracted from 81 other opportunistic fungi. We developed and validated a pan-Fusarium qPCR assay in serum/plasma with high sensitivity, specificity, and reproducibility that could facilitate early diagnosis and treatment monitoring of invasive fusariosis. Lay Abstract Fusariosis ranks third among invasive mould infections. It is frequently diagnosed late due to the lack of specific tools. We designed and evaluated a new qPCR assay with high sensitivity and specificity allowing detection of Fusarium DNA in serum samples up to 18 days before conventional diagnosis.

Invasive fungal diseases are increasing issues in modern medicine, where the human immunodeficiency virus (HIV) pandemic and the wider use of immunosuppressive drugs generate an ever-growing number of immunocompromised patients with an increased susceptibility to uncommon fungal pathogens. In the past decade, new species have been reported as being responsible for disseminated and invasive fungal diseases in humans. Among them, the following genera are rare but seem emerging issues: Scopulariopsis, Hormographiella, Emergomyces, Westerdykella, Trametes, Actinomucor, Saksenaea, Apophysomyces, and Rhytidhysteron. Delay in diagnosis, which is often the case in these infections, jeopardizes patients’ prognosis and leads to increased mortality. Here we summarize the clinical and biological presentation and the key features to identify these emerging pathogens and we discuss the available antifungal classes to treat them. We focused on Pubmed to recover extensively reported human invasive cases and articles regarding the nine previously cited fungal organisms. Information concerning patient background, macroscopic and microscopic description and pictures of these fungal organisms, histological features in tissues, findings with commonly used antigen tests in practice, and hints on potential efficient antifungal classes were gathered. This review's purpose is to help clinical microbiologists and physicians to suspect, identify, diagnose, and treat newly encountered fungi in hospital settings.

Multiplex PCRs have been designed for including species other than Aspergillus fumigatus for the diagnosis of invasive aspergillosis, such as microarrays, liquid-phase array, and electrospray-ionization mass spectrometry (PCR/ESI MS). These methods are based on the selection of multiple primers to amplify different species with the specificity checked by hybridization to a probe or by base composition of the amplicon for the PCR/ESI MS. When testing complex samples such as respiratory specimens, some clinically relevant species can be missed. Indeed, it is impossible to design primers able to amplify all the known fungal species with the same efficiency. Therefore, the best amplified species may not be the most clinically relevant. Multiplex assays have also been proposed to detect A. fumigatus DNA and azole resistance. Since the gene responsible for azole resistance is single copy and the gene used for detection is multicopy, only the high fungal loads can be evaluated. Thus, although interesting for investigating mycobiome, the multiplex assays should be used with cautious for the diagnosis of IA or the detection of resistance. For the diagnosis of invasive aspergillosis, validated quantitative PCRs specifically targeting A. fumigatus or a limited set of species to increase sensitivity is a safer option.

International audience; Invasive aspergillosis (IA) incidence is increasing in several countries like France, and numerous cases are indeed missed and still only diagnosed at autopsy as evidenced by recently published data. Such missed diagnoses are obviously encountered when appropriate diagnostic tools are not available especially in low resource areas or when biologists have not been trained enough in medical mycology (i.e., microscopic examination and culture in most of those areas). Besides logistical issues, which are indeed critical, IA may not be recognized because clinicians failed to consider that risk factors are evolving with the IA burden now observed among patients with chronic lymphoid malignancies or receiving new bio-therapies, with diabetes mellitus or liver cirrhosis and/or acute alcoholic hepatitis, with patients from the intensive care unit (ICU) and among patients with some predisposing primary immune deficiencies now reaching the adult's age. This is also the case for human immunodeficiency virus (HIV)-infected patients who failed to meet the classical definitions of IA. From the radiology perspective, new entities of IA have also emerged which absolutely need to be recognized especially bronchial-based-IA among allogeneic stem cell transplant recipients. Finally, from the laboratory side, contribution and limits of indirect blood biomark-ers should be integrated to the clinical life in order not to miss IA cases. To conclude, several diagnostic tools should be combined and a constant dialog between laboratory and clinics is crucial to appropriately diagnose IA.

International audience; Detection of viral and bacterial DNA in nasopharyngeal aspirates (NPAs) is now a routine practice in emergency cases of febrile pneumonia. We investigated whether Pneumocystis jirovecii DNA could also be detected in these cases by conducting retrospective screening of 324 consecutive NPAs from 324 adult patients (198 or 61% were immunocompromised) admitted with suspected pulmonary infections during the 2012 influenza epidemic season, using a real-time quantitative polymerase chain reaction (PCR) assay (PjqPCR), which targets the P. jirovecii mitochondrial large subunit ribosomal RNA gene. These NPAs had already been tested for 22 respiratory pathogens (18 viruses and 4 bacteria), but we found that 16 NPAs (4.9%) were PjqPCR-positive, making P. jirovecii the fourth most prevalent of the 23 microorganisms in the screen. Eleven of the 16 PjqPCR-positive patients were immunocompromised, and five had underlying pulmonary conditions. Nine NPAs were also positive for another respiratory pathogen. Six had PjqPCR-positive induced sputa less than 3 days after the NPA procedure, and five were diagnosed with pneumocystis pneumonia (four with chronic lymphoproliferative disorders and one AIDS patient). In all six available pairs quantification of P. jirovecii DNA showed fewer copies in NPA than in induced sputum and three PjqPCR-negative NPAs corresponded to PjqPCR-positive bronchoalveolar lavage fluids, underscoring the fact that a negative PjqPCR screen does not exclude a diagnosis of pneumocystosis. Including P. jirovecii DNA detection to the panel of microorganisms included in screening tests used for febrile pneumonia may encourage additional investigations or support use of anti-pneumocystis pneumonia prophylaxis in immunocompromised patients.