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HIV-associated cryptococcal meningitis is a leading cause of AIDS-related mortality. The AMBITION-cm trial showed that a regimen based on a single high dose of liposomal amphotericin B deoxycholate (AmBisome group) was non-inferior to the WHO-recommended treatment of seven daily doses of amphotericin B deoxycholate (control group) and was associated with fewer adverse events. We present a five-country cost-effectiveness analysis.The AMBITION-cm trial enrolled patients with HIV-associated cryptococcal meningitis from eight hospitals in Botswana, Malawi, South Africa, Uganda, and Zimbabwe. Taking a health service perspective, we collected country-specific unit costs and individual resource-use data per participant over the 10-week trial period, calculating mean cost per participant by group, mean cost-difference between groups, and incremental cost-effectiveness ratio per life-year saved. Non-parametric bootstrapping and scenarios analyses were performed including hypothetical real-world resource use. The trial registration number is ISRCTN72509687, and the trial has been completed.The AMBITION-cm trial enrolled 844 participants, and 814 were included in the intention-to-treat analysis (327 from Uganda, 225 from Malawi, 107 from South Africa, 84 from Botswana, and 71 from Zimbabwe) with 407 in each group, between Jan 31, 2018, and Feb 17, 2021. Using Malawi as a representative example, mean total costs per participant were US$1369 (95% CI 1314-1424) in the AmBisome group and $1237 (1181-1293) in the control group. The incremental cost-effectiveness ratio was $128 (59-257) per life-year saved. Excluding study protocol-driven cost, using a real-world toxicity monitoring schedule, the cost per life-year saved reduced to $80 (15-275). Changes in the duration of the hospital stay and antifungal medication cost showed the greatest effect in sensitivity analyses. Results were similar across countries, with the cost per life-year saved in the real-world scenario ranging from $71 in Botswana to $121 in Uganda.The AmBisome regimen was cost-effective at a low incremental cost-effectiveness ratio. The regimen might be even less costly and potentially cost-saving in real-world implementation given the lower drug-related toxicity and the potential for shorter hospital stays.European Developing Countries Clinical Trials Partnership, Swedish International Development Cooperation Agency, Wellcome Trust and Medical Research Council, UKAID Joint Global Health Trials, and the National Institute for Health Research.For the Chichewa, Isixhosa, Luganda, Setswana and Shona translations of the abstract see Supplementary Materials section.

A diabetes é considerada uma das maiores crises de saúde do século 21, e tem mostrado um crescimento significativo nos últimos anos, de acordo com a Federação Internacional de Diabetes. O diagnóstico em estágios iniciais é de grande importância no controle da doença, mas este se mostra um desafio devido à sutileza com que os sintomas são apresentados no início. O presente trabalho teve como objetivo validar um método de análise automatizada dos sintomas para auxiliar na detecção do risco de diabetes em estágios iniciais. Uma rede neural ELM foi utilizada com o auxílio de seleção de features realizada com algoritmo genético e os resultados foram comparados com os de algoritmos como MLP, SVM e RBF. A acurácia média obtida com a ELM foi de 98,64%

Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC (NCT03214250). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P = 0.006 compared to historical 1-year OS of 35%, n = 34) but was not met for sotiga/chemo (48.1%, P = 0.062, n = 36) or sotiga/nivo/chemo (41.3%, P = 0.223, n = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials.

International audience; Africa, although not unique in this context, is a favourable environment for fungal infections, given the high burden of risk factors. An online survey was developed asking about laboratory infrastructure and antifungal drug availability. We received 40 responses (24.4% response rate) of 164 researchers contacted from 21 African countries. Only five institutions (12.5%) of 40 located in Cameroon, Kenya, Nigeria, Sudan, and Uganda potentially fulfilled the minimum laboratory requirements for European Confederation of Medical Mycology Excellence Centre blue status. Difficulties included low access to susceptibility testing for both yeasts and moulds (available in only 30% of institutions) and Aspergillus spp antigen detection (available in only 47.5% of institutions as an in-house or outsourced test), as well as access to mould-active antifungal drugs such as amphotericin B deoxycholate (available for 52.5% of institutions), itraconazole (52.5%), voriconazole (35.0%), and posaconazole (5.0%). United and targeted efforts are crucial to face the growing challenges in clinical mycology.

CD19-directed chimeric antigen receptor–modified (CAR T) T cells achieve durable remissions in about 30% to 40% of relapsed/refractory large B-cell lymphomas. T-cell exhaustion and/or an immunosuppressive tumor microenvironment may contribute to CAR T-cell failure. Pembrolizumab, an anti-PD1 immune checkpoint inhibitor, may reverse T-cell exhaustion after CAR T-cell therapy. We treated 12 patients with B-cell lymphomas who were either refractory to (n = 9) or relapsed after (n = 3) CD19-directed CAR T-cell (4-1BB–costimulated) therapy with pembrolizumab 200 mg IV every 3 weeks. Median time from CAR T-cell infusion to first pembrolizumab dose was 3.3 months (range, 0.4-42.8 months). Pembrolizumab was well tolerated, and the only grade ≥3 adverse events related to pembrolizumab were neutropenia (n = 3; 25%). Best overall response rate after pembrolizumab was 25% (3 of 12 patients; 1 complete response; 2 partial responses). One (8%) patient had stable disease; thus, 4 of 12 (33%) patients had clinical benefit. After pembrolizumab, 4 patients with clinical benefit had an increase in percentage of CAR T cells by mass cytometry by time of flight (CyTOF); 3 of 4 of these patients also had increases in CAR19 transgene levels by quantitative polymerase chain reaction. Deep immune profiling using CyTOF revealed increased CAR T-cell activation and proliferation and less T-cell exhaustion in clinical responders. Together, PD1 blockade with pembrolizumab after CD19-directed CAR T-cell therapy appears safe and may achieve clinical responses in some patients with B-cell lymphomas refractory to or relapsed after CAR T-cell therapy. This trial was registered at www.clinicaltrials.gove as #NCT02650999.

Some risk factors for severe coronavirus disease 2019 (COVID-19) have been identified, including age, race, and obesity. However, 20%–50% of severe cases occur in the absence of these factors. Cytomegalovirus (CMV) is a herpesvirus that infects about 50% of all individuals worldwide and is among the most significant nongenetic determinants of immune system. We hypothesized that latent CMV infection might influence the severity of COVID-19. Our analyses demonstrate that CMV seropositivity is associated with more than twice the risk of hospitalization due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Immune profiling of blood and CMV DNA quantitative polymerase chain reaction in a subset of patients for whom respiratory tract samples were available revealed altered T-cell activation profiles in absence of extensive CMV replication in the upper respiratory tract. These data suggest a potential role for CMV-driven immune perturbations in affecting the outcome of SARS-CoV-2 infection and may have implications for the discrepancies in COVID-19 severity between different human populations.

Supplementary Figure from Immunologic Features in De Novo and Recurrent Glioblastoma Are Associated with Survival Outcomes

Glioblastoma (GBM) is an immunologically “cold” tumor characterized by poor responsiveness to immunotherapy. Standard of care for GBM is surgical resection followed by chemoradiotherapy and maintenance chemotherapy. However, tumor recurrence is the norm, and recurring tumors are found frequently to have acquired molecular changes (e.g., mutations) that may influence their immunobiology. Here, we compared the immune contexture of de novo GBM and recurrent GBM (rGBM) using high-dimensional cytometry and multiplex IHC. Although myeloid and T cells were similarly abundant in de novo and rGBM, their spatial organization within tumors differed and was linked to outcomes. In rGBM, T cells were enriched and activated in perivascular regions and clustered with activated macrophages and fewer regulatory T cells. Moreover, a higher expression of phosphorylated STAT1 by T cells in these regions at recurrence was associated with a favorable prognosis. Together, our data identify differences in the immunobiology of de novo GBM and rGBM and identify perivascular T cells as potential therapeutic targets.See related Spotlight by Bayik et al., p. 787

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PDF file - 54K, Patient characteristics

PDF file - 2.6MB, Characterization of PD1+CD45+ cells

PDF file - 2.6MB, Relationship between tumor infiltration with Foxp3 regulatory T cells and survival

PDF file - 3.4MB, Relationship between PDL-1 expression and survival

PDF file - 64K, List of antibodies used for immunofluorescence staining

PDF file - 2.7MB, Correlation between PDL-1 expression and PD-1 positive infiltrating T cells

PDF file - 4.8MB, Colocalization between PD1 positive cells and Foxp3- or Tim3- positive cells

PDF file - 4.8MB, Synergy between anti-HPV vaccine and anti-PDL-1 to inhibit tumor growth

Treatment efficacy with chimeric antigen receptor (CAR) T cell therapy in glioblastoma (GBM) is undermined by an immunosuppressive tumor microenvironment (TME). We previously showed that CAR T cell therapy targeting epidermal growth factor receptor variant III (EGFRvIII) produces anti-tumor activity against recurrent GBM and causes upregulation of programmed death-ligand 1 (PD-L1) in the TME. Here, we conducted a phase I trial to study the impact of CART-EGFRvIII cells administered concomitantly with the PD-1 inhibitor pembrolizumab in patients (n = 7) with newly diagnosed, EGFRvIII + GBM. Treatment was well tolerated without incidence of dose-limiting toxicity. However, no signal of efficacy was detected with a median progression-free survival of 5.2 months (90% CI, 2.9–6.0 months) and median overall survival of 11.8 months (90% CI, 9.2–14.2 months). In addition, PD-1 expression in the CART-EGFRvIII infusion product did not correlate with outcomes, and peripheral CAR T cell engraftment was relatively short-lived. Together, these findings show the safety of combining CAR T cells and PD-1 inhibition in GBM but given the lack of efficacy, also indicate a need to consider alternative combinatorial strategies. ClinicalTrials.gov registration: NCT03726515.

Resistance to fluconazole (FLC), the most widely used antifungal drug, is typically achieved by altering the azole drug target and/or drug efflux pumps. Recent reports have suggested a link between vesicular trafficking and antifungal resistance. Here, we identified novelCryptococcus neoformansregulators of extracellular vesicle (EV) biogenesis that impact FLC resistance. In particular, the transcription factor Hap2 does not affect the expression of the drug target or efflux pumps, yet it impacts the cellular sterol profile. Subinhibitory FLC concentrations also downregulate EV production. Moreover,in vitrospontaneous FLC-resistant colonies showed altered EV production, and the acquisition of FLC resistance was associated with decreased EV production in clinical isolates. Finally, the reversion of FLC resistance was associated with increased EV production. These data suggest a model in which fungal cells can regulate EV production in place of regulating the drug target gene expression as a first line of defense against antifungal assault in this fungal pathogen.IMPORTANCEExtracellular vesicles (EVs) are membrane-enveloped particles that are released by cells into the extracellular space. Fungal EVs can mediate community interactions and biofilm formation but thier functions remain poorly understood. Here, we report the identification of the first regulators of EV production in the major fungal pathogenCryptococcus neoformans.Surprisingly, we uncover a novel role of EVs in modulating antifungal drug resistance. Disruption of EV production was associated with altered lipid composition and changes in fluconazole susceptibility. Spontaneous azole-resistant mutants were deficient in EV production, while loss of resistance restored initial EV production levels. These findings were recapitulated inC. neoformansclinical isolates, indicating that azole resistance and EV production are coregulated in diverse strains. Our study reveals a new mechanism of drug resistance in which cells adapt to azole stress by modulating EV production.

Cryptococcus neoformans is an opportunistic fungal pathogen known for its remarkable ability to infect and subvert phagocytes. This ability provides survival and persistence within the host and relies on phenotypic plasticity. The viable but nonculturable (VBNC) phenotype was recently described in C. neoformans, whose study is promising in understanding the pathophysiology of cryptococcosis. The use of fluorescent strains is improving host interaction research, but it is still underexploited. Here, we fused histone H3 or the poly(A) binding protein (Pab) to enhanced green fluorescent protein (eGFP) or mCherry, obtaining a set of C. neoformans transformants with different colors, patterns of fluorescence, and selective markers (hygromycin B resistance [Hyg

While large outbreaks of C. auris in hospital settings have been described, few clear cases of direct transmission have been documented. We here investigated the transmission of C. auris clade I between two patients with a 41- to 61-day delay between exposure and the development of colonization. This may lead to changes in the recommendations concerning treatment of C. auris cases, as an incubation period of this length is one of the first to be reported.

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.

Patients with cancer have high mortality from coronavirus disease 2019 (COVID-19), and the immune parameters that dictate clinical outcomes remain unknown. In a cohort of 100 patients with cancer who were hospitalized for COVID-19, patients with hematologic cancer had higher mortality relative to patients with solid cancer. In two additional cohorts, flow cytometric and serologic analyses demonstrated that patients with solid cancer and patients without cancer had a similar immune phenotype during acute COVID-19, whereas patients with hematologic cancer had impairment of B cells and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody responses. Despite the impaired humoral immunity and high mortality in patients with hematologic cancer who also have COVID-19, those with a greater number of CD8 T cells had improved survival, including those treated with anti-CD20 therapy. Furthermore, 77% of patients with hematologic cancer had detectable SARS-CoV-2-specific T cell responses. Thus, CD8 T cells might influence recovery from COVID-19 when humoral immunity is deficient. These observations suggest that CD8 T cell responses to vaccination might provide protection in patients with hematologic cancer even in the setting of limited humoral responses.

Although invasive pulmonary aspergillosis (IPA) is typically described in immunocompromised host, patient with severe influenzae can develop IPA. Similarly, patients with severe COVID-19 complicated with IPA are increasingly reported. Here, we describe a case of invasive aspergillosis with triazole-resistant A. fumigatus (TR34/L98H mutation) in a 56-year-old patient with COVID-19 in intensive care unit. This report highlights the need to define the available tools for diagnosis of invasive aspergillosis in severe COVID-19 patients., Highlights • Association between COVID-19 and invasive aspergillosis. • Early screening for aspergillosis should be performed in respiratory specimen. • This case emphasizes the need to screen isolates for pan-azole resistance.

Standardized, reproducible and validated Mucorales quantitative PCR (qPCR) assays are needed in the context of routine testing in diagnostic labs. We, therefore, compared the commercial MucorGenius

Chemotherapy combined with immunotherapy has improved the treatment of certain solid tumors, but effective regimens remain elusive for pancreatic ductal adenocarcinoma (PDAC). We conducted a randomized phase 2 trial evaluating the efficacy of nivolumab (nivo; anti-PD-1) and/or sotigalimab (sotiga; CD40 agonistic antibody) with gemcitabine/nab-paclitaxel (chemotherapy) in patients with first-line metastatic PDAC ( NCT03214250 ). In 105 patients analyzed for efficacy, the primary endpoint of 1-year overall survival (OS) was met for nivo/chemo (57.7%, P = 0.006 compared to historical 1-year OS of 35%, n = 34) but was not met for sotiga/chemo (48.1%, P = 0.062, n = 36) or sotiga/nivo/chemo (41.3%, P = 0.223, n = 35). Secondary endpoints were progression-free survival, objective response rate, disease control rate, duration of response and safety. Treatment-related adverse event rates were similar across arms. Multi-omic circulating and tumor biomarker analyses identified distinct immune signatures associated with survival for nivo/chemo and sotiga/chemo. Survival after nivo/chemo correlated with a less suppressive tumor microenvironment and higher numbers of activated, antigen-experienced circulating T cells at baseline. Survival after sotiga/chemo correlated with greater intratumoral CD4 T cell infiltration and circulating differentiated CD4 T cells and antigen-presenting cells. A patient subset benefitting from sotiga/nivo/chemo was not identified. Collectively, these analyses suggest potential treatment-specific correlates of efficacy and may enable biomarker-selected patient populations in subsequent PDAC chemoimmunotherapy trials.

Malignant external otitis is an aggressive and potentially life-threatening infection. This rare disorder is typically caused by Pseudomonas aeruginosa and affects almost exclusively elderly diabetic patients. However, fungal malignant external otitis have been identified, especially in immunocompromised hosts. We report a rare case of invasive malignant external otitis caused by Aspergillus flavus in a diabetic patient without other underlying immunosuppression. A review of Aspergillus spp. malignant external otitis since voriconazole became the first line for invasive aspergillosis was performed. A 72-year-old man with diabetes mellitus developed invasive malignant external otitis with a vascular involvement. The patient was treated with empiric courses of antibiotics until a fungal infection was diagnosed. Proven Apsergillus infection was based on histopathological examination and isolation of A. flavus from culture of osteo-meningeal biopsies. Despite optimal antimicrobial therapy with voriconazole, the patient presented with cerebral infarction in the setting of an angioinvasive fungal infection leading to a fatal outcome. From a review of the literature, we found 39 previously published cases of proven Aspergillus spp. malignant external otitis treated with new triazoles. Given our experience and the literature review, a fungal etiology should be considered early in the course of malignant external otitis unresponsive to a conventional broad spectrum antibiotic therapy, with the need for a tissue biopsy to confirm the diagnosis.

Background Hepatosplenic candidiasis (HSC) used to be reported in patients with acute myeloid leukemia (AML) without antifungal prophylaxis. The aim was to describe the clinical features and outcomes of HSC over the last 13 years in a single French hematology center. Methods All patients diagnosed with HSC between 2008 and 2020 were included in a single-center retrospective cohort study. Data were collected from patient charts, and HSC was classified according to the 2020 European Organisation for Research and Treatment of Cancer/Mycoses Study Group definitions. Results Sixty patients were included, with 18.3% proven, 3.3% probable, and 78.3% possible HSC according to the 2020 European Organization for Research and Treatment of Cancer Mycoses Study Group classification. Among them, 19 patients were treated for acute myeloid leukemia (AML), 21 for lymphomas, and 14 for acute lymphoblastic leukemia. HSC occurred in 13 patients after autologous stem cell transplantation for lymphoma. At HSC diagnosis, 13 patients were receiving antifungal prophylaxis. Candida colonization was present in 84.2%, with prior candidemia in 36.7% of cases. β-D-glucans was positive in 55.8%, and 45.8% of tissue biopsies were contributive. First-line antifungal therapy was azoles in 61.7%, and steroids were associated in 45% of cases. At 3 months of follow-up, partial response to antifungal therapy was 94.2%. At last follow-up (mean, 22.6 months), 41 patients (68.3%) presented a complete hematological remission and 22 patients were deceased, none because of HSC. Conclusions The epidemiology of HSC has changed in the last decade, with fewer cases occurring in the AML setting. A better identification of patients at risk could lead to specific prophylaxis and improved diagnosis.

Phaeohyphomycoses comprise a heterogeneous group of fungal infections caused by dematiaceous fungi and have primarily been reported in patients with underlying acquired immunodeficiencies, such as hematological malignancies or solid-organ transplants. Over the past decade, a growing number of patients with phaeohyphomycosis but otherwise healthy were reported with autosomal recessive (AR) CARD9 deficiency. We report a 28-year-old woman who presented with invasive rhinosinusitis caused by

Scheduling residential loads for financial savings and user comfort may be performed by smart home controllers (SHCs). For this purpose, the electricity utility’s tariff variation costs, the lowest tariff cost schedules, the user’s preferences, and the level of comfort that each load may add to the household user are examined. However, the user’s comfort modeling, found in the literature, does not take into account the user’s comfort perceptions, and only uses the user-defined preferences for load on-time when it is registered in the SHC. The user’s comfort perceptions are dynamic and fluctuating, while the comfort preferences are fixed. Therefore, this paper proposes the modeling of a comfort function that takes into account the user’s perceptions using fuzzy logic. The proposed function is integrated into an SHC that uses PSO for scheduling residential loads, and aims at economy and user comfort as multiple objectives. The analysis and validation of the proposed function includes different scenarios related to economy–comfort, load shifting, consideration of energy tariffs, user preferences, and user perceptions. The results show that it is more beneficial to use the proposed comfort function method only when the user requires SHC to prioritize comfort at the expense of financial savings. Otherwise, it is more beneficial to use a comfort function that only considers the user’s comfort preferences and not their perceptions.

We describe 7 cases of extensive tinea corporis since 2018 in a hospital in Paris, France, after failure to cure with terbinafine. Molecular analysis indicated Trichophyton mentagrophytes internal transcribed spacer type VIII (T. indotineae). This strain, which has mutations in the squalene epoxidase gene, is spreading on the Indian subcontinent.

Background Early diagnosis and prompt initiation of specific antifungal treatment are essential for improving the prognosis of mucormycosis. We aimed to assess the performance of serum Mucorales quantitative polymerase chain reaction (qPCR) for the early diagnosis and follow-up of mucormycosis. Methods We prospectively enrolled 232 patients with suspicion of invasive mold disease, evaluated using standard imaging and mycological procedures. Thirteen additional patients with proven or probable mucormycosis were included to analyze DNA load kinetics. Serum samples were collected twice-a-week for Mucorales qPCR tests targeting the Mucorales genera Lichtheimia, Rhizomucor, and Mucor/Rhizopus. Results The sensitivity was 85.2%, specificity 89.8%, and positive and negative likelihood ratios 8.3 and 0.17, respectively in this prospective study. The first Mucorales qPCR-positive serum was observed a median of 4 days (interquartile range [IQR], 0–9) before sampling of the first mycological or histological positive specimen and a median of one day (IQR, −2 to 6) before the first imaging was performed. Negativity of Mucorales qPCR within seven days after liposomal-amphotericin B initiation was associated with an 85% lower 30-day mortality rate (adjusted hazard ratio = 0·15, 95% confidence interval [.03–.73], P = .02). Conclusions Our study argues for the inclusion of qPCR for the detection of circulating Mucorales DNA for mucormycosis diagnosis and follow-up after treatment initiation. Positive results should be added to the criteria for the consensual definitions from the European Organization for the Research and Treatment of Cancer/Mycoses Study Group Education and Research Consortium (EORTC/MSGERC), as already done for Aspergillus PCR.

This review has incorporated the knowledge and experience of the leads of each of the laboratory working parties of the fungal PCR initiative in order to provide up-to-date information on the performance and developments of PCR methods for the detection of fungi that commonly cause invasive fungal disease (IFD). Molecular diagnosis of IFD enhances the current repertoire of mycological investigations. Providing superior sensitivity and turn-around-time over classical approaches, yet maintaining the benefits of classical tests (e.g. species level identification and identifying resistance). Standardization for Aspergillus PCR is almost complete; the recent release of commercial PCR assays for a wide range fungi (Aspergillus, Candida, Pneumocystis, Mucorales and Pan-fungal) and availability of external quality control schemes (e.g. Quality Control of Molecular Diagnostics for Aspergillus, Candida, Pneumocystis) means that fungal PCR testing is robust and ready for use, globally. Further work is needed to ascertain the utility of PCR in routine practice and to determine whether combining it with other biomarkers is an optimal strategy. PCR for detecting Mucorales sp. and on tissue, together with direct antifungal resistance detection in body fluids, may increase its diagnostic value across the board. This and the ability to diagnose Pneumocystis pneumonia and invasive candidiasis would go a long way towards attaining the long-held ambition of medical mycology to provide a comprehensive range of tests that can be relied upon to diagnose, at least, the common IFD. In short, PCR has a clear future and is close to achieving its full potential in our laboratories.

Annexins are multifunctional proteins that bind to phospholipid membranes in a calcium-dependent manner. Annexins play a myriad of critical and well-characterized roles in mammals, ranging from membrane repair to vesicular secretion. The role of annexins in the kingdoms of bacteria, protozoa and fungi have been largely overlooked. The fact that there is no known homologue of annexins in the yeast model organism Saccharomyces cerevisiae may contribute to this gap in knowledge. However, annexins are found in most medically important fungal pathogens, with the notable exception of Candida albicans. In this study we evaluated the function of the one annexin gene in Cryptococcus neoformans, a causative agent of cryptococcosis. This gene CNAG_02415, is annotated in the C. neoformans genome as a target of calcineurin through its transcription factor Crz1, and we propose to update its name to cryptococcal annexin, AnnexinC1. C. neoformans strains deleted for AnnexinC1 revealed no difference in survival after exposure to various chemical stressors relative to wild-type strain, as well as no major alteration in virulence or mating. The only alteration observed in strains deleted for AnnexinC1 was a small increase in the titan cells' formation in vitro. The preservation of annexins in many different fungal species suggests an important function, and therefore the lack of a strong phenotype for annexin-deficient C. neoformans indicates either the presence of redundant genes that can compensate for the absence of AnnexinC1 function or novel functions not revealed by standard assays of cell function and pathogenicity.

Bardet-Biedl syndrome (BBS) is an emblematic ciliopathy associated with retinal dystrophy, obesity, postaxial polydactyly, learning disabilities, hypogonadism and renal dysfunction. Before birth, enlarged/cystic kidneys as well as polydactyly are the hallmark signs of BBS to consider in absence of familial history. However, these findings are not specific to BBS, raising the problem of differential diagnoses and prognosis. Molecular diagnosis during pregnancies remains a timely challenge for this heterogeneous disease (22 known genes). We report here the largest cohort of BBS fetuses to better characterize the antenatal presentation. Prenatal ultrasound (US) and/or autopsy data from 74 fetuses with putative BBS diagnosis were collected out of which molecular diagnosis was established in 51 cases, mainly in BBS genes (45 cases) following the classical gene distribution, but also in other ciliopathy genes (6 cases). Based on this, an updated diagnostic decision tree is proposed. No genotype/phenotype correlation could be established but postaxial polydactyly (82%) and renal cysts (78%) were the most prevalent symptoms. However, autopsy revealed polydactyly that was missed by prenatal US in 55% of the cases. Polydactyly must be carefully looked for in pregnancies with apparently isolated renal anomalies in fetuses.

High-dimensional profiling approaches inform developmental relationships between tumor-infiltrating lymphocyte subpopulations.

Clinical metagenomics (CMg) is the process of sequencing nucleic acid of clinical samples to obtain clinically relevant information such as the identification of microorganisms and their susceptibility to antimicrobials. Over the last decades, sequencing and bioinformatic solutions supporting CMg have much evolved and an increasing number of case reports and series covering various infectious diseases have been published. Metagenomics is a new approach to infectious disease diagnosis that is currently being developed and is certainly one of the most promising for the coming years. However, most CMg studies are retrospective, and few address the potential impact CMg could have on patient management, including initiation, adaptation, or cessation of antimicrobials. In this narrative review, we have discussed the potential role of CMg in bacteriology, virology, mycology, and parasitology. Several reports and case-series confirm that CMg is an innovative tool with which one can (i) identify more microorganisms than with conventional methods in a single test, (ii) obtain results within hours, and (iii) tailor the antimicrobial regimen of patients. However, the cost-efficiency of CMg and its real impact on patient management are still to be determined.

The adoptive transfer of T lymphocytes reprogrammed to target tumor cells has demonstrated significant potential in various malignancies. However, little is known about the long-term potential and the clonal stability of the infused cells. Here, we studied the longest persisting CD19 redirected chimeric antigen receptor (CAR) T cells to date in two chronic lymphocytic leukemia (CLL) patients who achieved a complete remission in 2010. CAR T-cells were still detectable up to 10+ years post-infusion, with sustained remission in both patients. Surprisingly, a prominent, highly activated CD4+ population developed in both patients during the years post-infusion, dominating the CAR T-cell population at the late time points. This transition was reflected in the stabilization of the clonal make-up of CAR T-cells with a repertoire dominated by few clones. Single cell multi-omics profiling via Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-Seq) with TCR sequencing of CAR T-cells obtained 9.3 years post-infusion demonstrated that these long-persisting CD4+ CAR T-cells exhibited cytotoxic characteristics along with strong evidence of ongoing functional activation and proliferation. Our data provide novel insight into the CAR T-cell characteristics associated with long-term remission in leukemia.