According to the immunodepression status, the diagnosis of Pneumocystis jirovecii pneumonia (PjP) may be difficult. Molecular methods appear very sensitive, but they lack specificity because Pj DNA ...can be detected in Pneumocystis-colonized patients. The aim of this study was to evaluate the value of a serum ß-d-Glucan (BDG) assay for the diagnosis of PjP in a large cohort of HIV-negative and HIV-positive patients, either as a first-line diagnostic test for PjP or as a tool to distinguish between colonization and PjP in cases of low fungal load. Data of Pj qPCR performed on bronchopulmonary specimens over a 3-year period were retrieved retrospectively. For each result, we searched for a BDG serum assay performed within ±5 days. Among the 69 episodes that occurred in HIV-positive patients and the 609 episodes that occurred in immunocompromised HIV-negative patients, we find an equivalent sensitivity of BDG assays compared with molecular methods to diagnose probable/proven PjP, in a first-line strategy. Furthermore, BDG assay can be used confidently to distinguish between infected and colonized patients using a 80 pg/mL cut-off. Finally, it is necessary to search for causes of false positivity to increase BDG assay performance. BDG assay represents a valuable adjunctive tool to distinguish between colonization and infection.
Interest for the study of gut mycobiota in relation with human health and immune homeostasis has increased in the last years. From this perspective, new tools to study the immune/fungal interface are ...warranted. Systemic humoral immune responses could reflect the dynamic relationships between gut mycobiota and immunity. Using a novel flow cytometry technology (Fungi-Flow) to determine immunoglobulin (Ig) responses to fungi, we studied the relationships between gut mycobiota and systemic humoral anti-commensal immunity.
The Fungi-Flow method allows a sensitive and specific measurement of systemic IgG responses against 17 commensal and environmental fungi from the two main divisions; Ascomycota and Basidiomycota. IgG responses exhibited a high inter-individual variability. Anti-commensal IgG responses were contrasted with the relative abundance, alpha-diversity, and intra-genus richness of fungal species in gut mycobiota of twenty healthy donors. Categorization of gut mycobiota composition revealed two differentiated fungal ecosystems. Significant difference of anti-Saccharomyces systemic IgG responses were observed in healthy donors stratified according to the fungal ecosystem colonizing their gut. A positive and significant correlation was observed between the variety of IgG responses against fungal commensals and intestinal alpha-diversity. At the level of intra-genus species richness, intense IgG responses were associated with a low intra-genus richness for known pathobionts, but not commensals.
Fungi-Flow allows an easy and reliable measure of personalized humoral responses against commensal fungi. Combining sequencing technology with our novel Fungi-Flow immunological method, we propose that there are at least two defined ecosystems in the human gut mycobiome associated with systemic humoral responses. Fungi-Flow opens new opportunities to improve our knowledge about the impact of mycobiota in humoral anti-commensal immunity and homeostasis. Video Abstract.
Abstract
PCR-based methods applied to various body fluids emerged in recent years as a promising approach for the diagnosis of mucormycosis. In this study, we set up and assess the value of a qPCR to ...detect a wide variety of Mucorales species in a single tube. A pair of degenerated primers targeting the rDNA operon was used in a qPCR utilizing an intercalating fluorescent dye. Analytical assessment, using a wide variety of both Mucorales strains (8 genera, 11 species) and non-Mucorales strains (9 genera, 14 species), showed 100% sensitivity and specificity rates with a limit of detection at 3 rDNA copy/qPCR reaction. Subsequently, 364 clinical specimens from 166 at-risk patients were prospectively tested with the assay. All the seven patients classified as proven/probable mucormycosis using the EORTC-MSG criteria had a positive qPCR as well as a patient with a proven uncharacterized invasive mold infection. In addition, three out of seven patients with possible mold invasive infections had at least one positive qPCR test. Sensitivity was calculated between 73.33 and 100% and specificity between 98.10 and 100%. The qPCR method proposed showed excellent performances and would be an important adjunctive tool for the difficult diagnosis of mucormycosis diagnosis.
Lay abstract
qPCR-based diagnosis is the most reliable approach for mucormycosis. We set up a pan-Mucorales qPCR able to detect in a single reaction not less than 11 different species. Both analytical and clinical performances support its use in the clinical setting.
Summary Objectives The ß-D-glucan assay (BDG) has been added to the EORTC/MSG criteria for the diagnosis of invasive fungal infections (IFI), but data from pediatric populations is scarce. The aim of ...this study was to evaluate performance of BDG in a cohort of hemato-oncological children with hematological malignancy at risk for IFI. Methods 113 patients were included through an 18-month period. In addition to routine IFI screening, BDG was assayed once a week. IFIs were classified using EORTC/MSG criteria without including the BDG results. Performances were assessed after a ROC analysis for optimization and multivariate analysis to detect the causes of false positivity. Results 8 proven and 4 probable IFIs, and 7 possible IFIs were diagnosed in 9 and 7 patients, respectively. Sensitivity and specificity increased from 75% and 56% to 100% and 91.1%, respectively when considering the whole population and patients not having received any antifungals prior to the test. Multivariate analysis revealed that being younger than 7, severe colitis/mucositis, recent administration of polyvalent immunoglobulins and digestive colonization with Enterococcus sp were independent risk factors for false positivity. Conclusions BDG is a valuable test to detect IFI in pediatric patients not previously treated with antifungals and to detect the occurrence of chronic infection.
Abstract 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.
Les microsporidioses intestinales sont des infections sous-estimées affectant à la fois les patients immunodéprimés et immunocompétents. Le diagnostic microscopique en laboratoire médical est aujourd’hui supplanté par la PCR en temps réel. Cependant, peu de fabricants incluent les microsporidies dans leurs panels PCR pour le diagnostic des gastro-entérites infectieuses. Ici, nous avons évalué les performances des tests PCR en temps réel microsporidia generic et microsporidia typing (Bio-Evolution, France) sur le thermocycleur PCR en temps réel Rotor-Gene Q (Qiagen, France). Nous avons inclus 45 échantillons de selles négatifs et 44 échantillons positifs pour Enterocytozoon bieneusi ( n = 34, avec divers génotypes), Encephalitozoon intestinalis ( n = 4), Encephalitozoon hellem ( n = 4) et Encephalitozoon cuniculi ( n = 2). Nous avons également analysé les résultats sur 4 ans d’un programme de contrôle qualité inter-laboratoires dont 9 centres ont utilisé ces kits commerciaux. La sensibilité et la spécificité du kit microsporidia generic étaient respectivement de 86,4 % et 93,3 %. Encephalitozoon hellem et E. cuniculi ont été détectés par le kit microsporidia generic mais pas par le kit microsporidia typing. Ces résultats étaient cohérents avec ceux du programme de contrôle de qualité inter-laboratoires. En conclusion, les tests de PCR en temps réel Bio-Evolution sont des outils intéressants pour la microsporidiose intestinale, mais un résultat négatif pour le test de typage microsporidia nécessite une analyse supplémentaire pour confirmer les infections à E. hellem ou E. cuniculi .
Laboratory diagnosis of histoplasmosis is based on various methods, including microscopy, culture, antigen, and DNA detection of Histoplasma capsulatum var. capsulatum or Histoplasma capsulatum var. ...duboisii. To improve sensitivity of existing real-time quantitative PCR (qPCR) assays, we developed a new RT-qPCR assay that allows amplification of whole nucleic acids of Histoplasma spp. validated on suspected cases. The limit of detection was 20 copies, and the specificity against 114 fungal isolates/species was restricted to Histoplasma spp. Whole nucleic acids of 1319 prospectively collected consecutive samples from 907 patients suspected of having histoplasmosis were tested routinely between May 2015 and May 2019 in parallel with standard diagnostic procedures performed in parallel. Forty-four had proven histoplasmosis attributable to H. capsulatum var. capsulatum (n = 40) or H. capsulatum var. duboisii (n = 4) infections. The results of RT-qPCR were positive in 43 of 44 patients (97.7% sensitivity) in at least one specimen. Nine of 863 cases (99% specificity) were RT-qPCR positive and therefore classified as possible cases. RT-qPCR was positive in 13 of 30 (43.3%) blood samples tested in proven cases. A positive RT-qPCR result in blood was significantly associated with H. capsulatum var. capsulatum progressively disseminated histoplasmosis with a positive RT-qPCR result in 92.3% of the immunocompromised patients with disseminated disease. This new Histoplasma RT-qPCR assay enabling amplification of H. capsulatum var. capsulatum and H. capsulatum var. duboisii is highly sensitive and allows the diagnosis of histoplasmosis advantageously from blood and bronchoalveolar lavage fluid.
To identify the factors associated with the interindividual pharmacokinetic (PK) variability of micafungin and to evaluate the probability of reaching the previously determined PK/pharmacodynamic ...efficacy thresholds (AUC/MIC >5000 for non-parapsilosis Candida sp. and ≥285 for Candida parapsilosis) with the recommended 100 mg daily dose in ICU patients with sepsis and mechanical ventilation.
One hundred patients were included and 436 concentrations were available for PK analysis performed with NONMEM software. PTA was determined by Monte Carlo simulations.
Micafungin obeyed a two-compartment model with first-order elimination from the central compartment. Mean parameter estimates (percentage interindividual variability) were 1.34 L/h (34%) for clearance (CL), 11.80 L (38%) and 7.68 L (39%) for central (Vc) and peripheral (Vp) distribution volumes, respectively, and 4.67 L/h (37%) for distribution clearance. CL, Vc and Vp increased by 14% when the albumin level was ≤25 g/L and CL decreased by 25% when SOFA score was ≥10. Body weight was related to CL, Vc and Vp by allometric models. PTA was ≥90% in Candida albicans and Candida glabrata infections, except when the MIC was ≥0.015 mg/L, and ranged between 0% and 40% for C. parapsilosis infections with MIC ≥0.5 mg/L.
A possible increase in the dose should be evaluated for infections due to C. parapsilosis and for infections due to C. albicans and C. glabrata with MICs ≥0.015 mg/L.
Babesiosis is a tick-borne infectious disease, caused by an intraerythrocytic parasite of the genus Babesia. It has clinical, biological and microbiological similarities with Plasmodium related ...infections. In rare cases, babesiosis may be complicated by hemophagocytic lymphohistiocytosis, which occurs preferentially in the immunodeficient patient. We report here the case of a non-immunocompromised patient living in Manhattan, New York hospitalized for a complicated babesiosis of a hemophagocytic lymphohistiocytosis. After 7 days of hospitalization and treatment by azithromycin 500 mg/day and atovaquone 750 mg twice a day, the patient was discharged with an improvement in clinical symptoms and biological parameters.
Cryptococcal meningitis is a severe cause of central nervous system infections among immunocompromised solid organ transplant (SOT) patients. While new diagnostic methods as multiplex ...meningitis/encephalitis (ME) NAT (nucleic acid test) are increasingly used as a first‐line tool in hospital practice, data in HIV‐negative patients including SOT remain scarce. We report here false‐negative results of multiplex NAT among SOT patients with proven cryptococcal meningitis.
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