Médiateurs lipidiques de l'inflammation et populations lymphocytaires au cours des infections à Pneumocystis : études cliniques et modèle murin

La pneumocystose (PCP) est une pneumonie interstitielle opportuniste due au champignon Pneumocystis jirovecii. Au cours de cette maladie, les lésions pulmonaires sont davantage liées à une réponse inflammatoire excessive de l'hôte, qu'à une action cytopathogène directe du champignon. Les lymphocytes dont les T CD4 sont des cellules clés de la réponse immune contre P. jirovecii. Néanmoins, les connaissances restent limitées sur le rôle spécifique des sous-populations lymphocytaires au cours de la PCP. Par ailleurs, les lipides pro-inflammatoires et pro-résolutifs de l'inflammation (SPM) ont été très peu étudiés dans le cadre de la PCP. Ce travail de thèse avait pour objectif d'étudier les réponses lymphocytaires et les réponses lipidiques pro-inflammatoires et pro-résolutives au cours de la PCP grâce à deux études cliniques et une étude expérimentale sur modèle murin. La première étude clinique, réalisée chez des patients immunodéprimés atteints ou non de PCP, a montré un taux de lymphocytes B (LB) sanguins plus fréquemment abaissé chez les sujets infectés. Chez les patients atteints de PCP, le ratio Th1/Th2 était significativement abaissé (Th1 sanguins diminués et Th2 augmentés) chez les patients décédés. Ceci était associé à une diminution des T CD8 cytotoxiques et des cellules NK chez les patients décédés. La 2ème étude sur modèle murin a mis en évidence une réponse immune différente à P. murina en fonction du statut immunitaire de l'hôte, en particulier la formation de structures iBALT (Induced Bronchus-Associated Lymphoid Tissue) regroupant des macrophages, des LB et lymphocytes T probablement CD4 Tfh chez les souris immunocompétentes mais absentes chez les souris immunodéprimées. En cas d'immunodépression, les différents profils de T CD4 helper étaient tous augmentés au pic de l'infection. Le dosage des lipides a montré l'implication de la prostaglandine E2 (PGE2) dans la physiopathologie de l'infection à P. murina, notamment pour les souris immunodéprimées qui avaient une concentration plus élevée en PGE2 au pic de l'infection. Ceci était accompagné d'une augmentation globale des SPMs, qui étaient au contraire peu modifiés chez les souris immunocompétentes. Un enrichissement du régime lipidique en oméga 6 améliorait l'évolution de la maladie, en association avec une diminution de la PGE2, une diminution globale des SPMs et une augmentation du ratio Th1/Th2. La PGE2 est connue pour inhiber la réponse Th1 et induire la production de SPMs, eux même inhibiteurs de la réponse Th1. Elle pourrait ainsi représenter un élément important dans la physiopathologie de la PCP. L'étude des lipides chez l'Homme a confirmé l'intérêt de la PGE2 dans la maladie car cet eicosanoïde était plus élevé chez les patients évoluant défavorablement, en lien avec une réponse Th1 diminuée et certains SPM plus élevés. Les patients évoluant défavorablement avaient aussi des acides gras oméga 6 totaux diminués et des acides gras oméga 9 augmentés par rapport aux patients évoluant favorablement. En conclusion, l'ensemble des données obtenues à partir des études cliniques et du modèle murin est en faveur de la coexistence de deux réponses à une infection par Pneumocystis, selon le statut immunitaire de l'hôte. Les réponses Tfh et LB semblent être majoritaires chez un hôte immunocompétent au sein de formations iBALT. A l'inverse, chez les hôtes immunodéprimés, en l'absence de T CD4 et/ou de LB, une réponse Th1/Tc1 pourrait être bénéfique pour l'élimination du champignon. La PGE2 qui oriente l'immunité de l'hôte vers une réponse Th2 et la résolution de l'inflammation pourrait s'opposer à la bonne élimination de Pneumocystis dans un contexte d'immunodépression. Des études complémentaires seront nécessaires afin de comprendre ces mécanismes et l'effet de l'apport nutritionnel en lipides, notamment en oméga 6. L'intérêt de la PGE2, des T CD4 Th1 et des T CD8 cytotoxiques en tant que marqueurs pronostiques de la PCP devra être confirmé à plus large échelle.Pneumocystis pneumonia (PCP) is an opportunistic interstitial pneumonia caused by the fungus Pneumocystis jirovecii. In this disease, lung lesions are more related to an excessive inflammatory response of the host than to a direct cytopathogenic action of the fungus. Lymphocytes including CD4 T cells are key cells in the immune response against P. jirovecii. Nevertheless, knowledge remains limited on the specific role of lymphocyte subpopulations during PCP. In addition, pro-inflammatory and pro-resolving lipid mediators (SPMs) have been very little studied in PCP. The objective of this thesis work was to study lymphocyte responses, pro-inflammatory and pro-resolving lipid mediators during PCP through two clinical studies and an experimental mouse model study. The first clinical study, conducted in immunocompromised patients with and without PCP, showed that the levels of peripheral blood B cells (LB) were more frequently lowered in infected subjects. On the other hand, in patients diagnosed with PCP, the Th1/Th2 ratio was significantly reduced (Th1 decreased and Th2 increased) in deceased patients. This was associated with a decrease in cytotoxic CD8 T cells and in NK cells in the deceased patients. The second study, on a mouse model, demonstrated a different immune response to P. murina depending on the immune status of the host. Notably, the formation of an iBALT (induced Bronchus-associated lymphoid tissue) that included macrophages, LB and probably CD4+ follicular helper (Tfh) cells was observed in immunocompetent mice but was absent in immunocompromised mice. In immunosuppressed mice, the different CD4 T helper profiles were all increased at the peak of infection. Lipid assay showed the involvement of prostaglandin E2 (PGE2) in the pathophysiology of P. murina infection, particularly in immunosupressed mice that had a higher PGE2 concentration at the peak of infection. This was accompanied by an overall increase in SPMs, which were poorly modified in immunocompetent mice. An enrichment of the lipid diet with omega-6 fatty acids improved the disease progression, in association with a decrease in PGE2, an overall decrease in SPMs and an increase in the Th1/Th2 ratio. PGE2 is known to inhibit the Th1 response and induce the production of SPMs, which themselves inhibit the Th1 response. PGE2 may thus represent an important element in the pathophysiology of PCP. The study of lipids in humans has confirmed the interest of PGE2 in the disease because this eicosanoid was higher in patients with an unfavorable progression, in connection with a decreased Th1 response and some higher SPMs concentrations. Patients with an unfavorable evolution also had decreased total omega-6 fatty acids and increased omega-9 fatty acids compared to patients with a favorable evolution. In conclusion, all the data obtained from the clinical studies and the mouse model support the coexistence of two responses to Pneumocystis infection, depending on the immune status of the host. LB and Tfh cells appear to be the two major types of lymphocytes in the lungs of infected immunocompetent hosts, organized within iBALT formations. Conversely, in immunocompromised hosts, in the absence of CD4 T and/or LB, a Th1/Tc1 response may be beneficial for the elimination of the fungus. PGE2, which directs host immunity towards a Th2 response and resolution of inflammation, could thus hinder the elimination of Pneumocystis in an immunocompromised context. Further studies will be necessary to understand these mechanisms and the effect of lipid dietary intake, particularly of omega 6 fatty acids. The interest of PGE2, Th1 CD4 Tcells and cytotoxic CD8 T cells as prognostic markers of PCP will need to be confirmed on a wider scale

Lipid mediators of inflammation and lymphocyte populations during pneumocystis infections : clinical studies and mouse model

La pneumocystose (PCP) est une pneumonie interstitielle opportuniste due au champignon Pneumocystis jirovecii. Au cours de cette maladie, les lésions pulmonaires sont davantage liées à une réponse inflammatoire excessive de l'hôte, qu'à une action cytopathogène directe du champignon. Les lymphocytes dont les T CD4 sont des cellules clés de la réponse immune contre P. jirovecii. Néanmoins, les connaissances restent limitées sur le rôle spécifique des sous-populations lymphocytaires au cours de la PCP. Par ailleurs, les lipides pro-inflammatoires et pro-résolutifs de l'inflammation (SPM) ont été très peu étudiés dans le cadre de la PCP. Ce travail de thèse avait pour objectif d'étudier les réponses lymphocytaires et les réponses lipidiques pro-inflammatoires et pro-résolutives au cours de la PCP grâce à deux études cliniques et une étude expérimentale sur modèle murin. La première étude clinique, réalisée chez des patients immunodéprimés atteints ou non de PCP, a montré un taux de lymphocytes B (LB) sanguins plus fréquemment abaissé chez les sujets infectés. Chez les patients atteints de PCP, le ratio Th1/Th2 était significativement abaissé (Th1 sanguins diminués et Th2 augmentés) chez les patients décédés. Ceci était associé à une diminution des T CD8 cytotoxiques et des cellules NK chez les patients décédés. La 2ème étude sur modèle murin a mis en évidence une réponse immune différente à P. murina en fonction du statut immunitaire de l'hôte, en particulier la formation de structures iBALT (Induced Bronchus-Associated Lymphoid Tissue) regroupant des macrophages, des LB et lymphocytes T probablement CD4 Tfh chez les souris immunocompétentes mais absentes chez les souris immunodéprimées. En cas d'immunodépression, les différents profils de T CD4 helper étaient tous augmentés au pic de l'infection. Le dosage des lipides a montré l'implication de la prostaglandine E2 (PGE2) dans la physiopathologie de l'infection à P. murina, notamment pour les souris immunodéprimées qui avaient une concentration plus élevée en PGE2 au pic de l'infection. Ceci était accompagné d'une augmentation globale des SPMs, qui étaient au contraire peu modifiés chez les souris immunocompétentes. Un enrichissement du régime lipidique en oméga 6 améliorait l'évolution de la maladie, en association avec une diminution de la PGE2, une diminution globale des SPMs et une augmentation du ratio Th1/Th2. La PGE2 est connue pour inhiber la réponse Th1 et induire la production de SPMs, eux même inhibiteurs de la réponse Th1. Elle pourrait ainsi représenter un élément important dans la physiopathologie de la PCP. L'étude des lipides chez l'Homme a confirmé l'intérêt de la PGE2 dans la maladie car cet eicosanoïde était plus élevé chez les patients évoluant défavorablement, en lien avec une réponse Th1 diminuée et certains SPM plus élevés. Les patients évoluant défavorablement avaient aussi des acides gras oméga 6 totaux diminués et des acides gras oméga 9 augmentés par rapport aux patients évoluant favorablement. En conclusion, l'ensemble des données obtenues à partir des études cliniques et du modèle murin est en faveur de la coexistence de deux réponses à une infection par Pneumocystis, selon le statut immunitaire de l'hôte. Les réponses Tfh et LB semblent être majoritaires chez un hôte immunocompétent au sein de formations iBALT. A l'inverse, chez les hôtes immunodéprimés, en l'absence de T CD4 et/ou de LB, une réponse Th1/Tc1 pourrait être bénéfique pour l'élimination du champignon. La PGE2 qui oriente l'immunité de l'hôte vers une réponse Th2 et la résolution de l'inflammation pourrait s'opposer à la bonne élimination de Pneumocystis dans un contexte d'immunodépression. Des études complémentaires seront nécessaires afin de comprendre ces mécanismes et l'effet de l'apport nutritionnel en lipides, notamment en oméga 6. L'intérêt de la PGE2, des T CD4 Th1 et des T CD8 cytotoxiques en tant que marqueurs pronostiques de la PCP devra être confirmé à plus large échelle.Pneumocystis pneumonia (PCP) is an opportunistic interstitial pneumonia caused by the fungus Pneumocystis jirovecii. In this disease, lung lesions are more related to an excessive inflammatory response of the host than to a direct cytopathogenic action of the fungus. Lymphocytes including CD4 T cells are key cells in the immune response against P. jirovecii. Nevertheless, knowledge remains limited on the specific role of lymphocyte subpopulations during PCP. In addition, pro-inflammatory and pro-resolving lipid mediators (SPMs) have been very little studied in PCP. The objective of this thesis work was to study lymphocyte responses, pro-inflammatory and pro-resolving lipid mediators during PCP through two clinical studies and an experimental mouse model study. The first clinical study, conducted in immunocompromised patients with and without PCP, showed that the levels of peripheral blood B cells (LB) were more frequently lowered in infected subjects. On the other hand, in patients diagnosed with PCP, the Th1/Th2 ratio was significantly reduced (Th1 decreased and Th2 increased) in deceased patients. This was associated with a decrease in cytotoxic CD8 T cells and in NK cells in the deceased patients. The second study, on a mouse model, demonstrated a different immune response to P. murina depending on the immune status of the host. Notably, the formation of an iBALT (induced Bronchus-associated lymphoid tissue) that included macrophages, LB and probably CD4+ follicular helper (Tfh) cells was observed in immunocompetent mice but was absent in immunocompromised mice. In immunosuppressed mice, the different CD4 T helper profiles were all increased at the peak of infection. Lipid assay showed the involvement of prostaglandin E2 (PGE2) in the pathophysiology of P. murina infection, particularly in immunosupressed mice that had a higher PGE2 concentration at the peak of infection. This was accompanied by an overall increase in SPMs, which were poorly modified in immunocompetent mice. An enrichment of the lipid diet with omega-6 fatty acids improved the disease progression, in association with a decrease in PGE2, an overall decrease in SPMs and an increase in the Th1/Th2 ratio. PGE2 is known to inhibit the Th1 response and induce the production of SPMs, which themselves inhibit the Th1 response. PGE2 may thus represent an important element in the pathophysiology of PCP. The study of lipids in humans has confirmed the interest of PGE2 in the disease because this eicosanoid was higher in patients with an unfavorable progression, in connection with a decreased Th1 response and some higher SPMs concentrations. Patients with an unfavorable evolution also had decreased total omega-6 fatty acids and increased omega-9 fatty acids compared to patients with a favorable evolution. In conclusion, all the data obtained from the clinical studies and the mouse model support the coexistence of two responses to Pneumocystis infection, depending on the immune status of the host. LB and Tfh cells appear to be the two major types of lymphocytes in the lungs of infected immunocompetent hosts, organized within iBALT formations. Conversely, in immunocompromised hosts, in the absence of CD4 T and/or LB, a Th1/Tc1 response may be beneficial for the elimination of the fungus. PGE2, which directs host immunity towards a Th2 response and resolution of inflammation, could thus hinder the elimination of Pneumocystis in an immunocompromised context. Further studies will be necessary to understand these mechanisms and the effect of lipid dietary intake, particularly of omega 6 fatty acids. The interest of PGE2, Th1 CD4 Tcells and cytotoxic CD8 T cells as prognostic markers of PCP will need to be confirmed on a wider scale

New Insights into Blood Circulating Lymphocytes in Human Pneumocystis Pneumonia

The host lymphocyte response is decisive in Pneumocystis pneumonia (PCP) pathophysiology but little is known of the specific roles of lymphocyte subpopulations in this fungal infection. Peripheral NK, NKT, B, TCD4+ and TCD8+ subpopulations were compared by immunophenotyping between 20 patients diagnosed with PCP (PCP(+)] and 20 uninfected immunosuppressed patients (PCP(−)). Among PCP(+) subjects, the lymphocyte populations were also compared between surviving and deceased patients. Low B cell count (<40 cells/µL) was more frequent in PCP(+) than in PCP(−) patients (p = 0.03), while there was no difference for the TCD4 count. Among the PCP(+) group, the 7 deceased patients had lower Th1 (p = 0.02) and Tc1 (p = 0.03) populations, higher Th2 response (p = 0.03), higher effector TCD8 (p < 0.01), lower central memory TCD8 (p = 0.04) and reduced NK cells (p = 0.02) compared with the 13 survivors. Th1/Th2 ratio < 17, CD8 Tc1 < 44%, effector TCD8 < 25%, central memory TCD8 < 4%, NK cells < 50 cells/µL and total lymphocytes < 0.75 G/L were associated with a higher risk of mortality (p = 0.003, p = 0.007, p = 0.0007, p = 0.004, p = 0.02 and p = 0.019, respectively). The traditional analysis of TCD4 and TCD8 populations may be insufficient in the context of PCP. It could be completed by using B cells to predict the risk of PCP, and by using lymphocyte subpopulations or total lymphocyte count, which are easy to obtain in all health care facilities, to evaluate PCP prognosis

Apport du séquençage nouvelle génération au typage MLST de Pneumocystis jirovecii dans le cadre d'une épidémie chez des patients transplantés d'organe solide

Mémoire de Diplôme d'Etudes Spécialisées (DES) tenant lieu de thèse d'exercice.Multilocus Sequence Typing (MLST) allows accurate strain characterization, but Sanger sequencing presents limitations for subpopulations determination. Yet, mixed strains coinfections are frequent in Pneumocystis jirovecii Pneumonia (PCP). Next Generation Sequencing (NGS) allows both majority and minority variant studies. We evaluated the added value of NGS in an epidemiological MLST study of a PCP cluster in solid organ transplant (SOT) recipients. Thirty-two patients (12 grouped cases SOT and 20 controls) presenting with PCP at Grenoble Hospital, France, were included between 2012 and 2015. A three loci scheme was used for MLST analysis (MIT26S, CYTB, SOD). New primers were designed to obtain ~ 750 bp amplicons (GS +). Sequencing was achieved on 32 samples with a NGS technology (GS junior +, Roche), and with Sanger technology for 12 of them. Among the 12 SOT, 5 shared a major genotype C2a. Proportion of mixed genotypes infections was significantly higher with NGS analysis (65%) compared to Sanger (25%). NGS approach revealed infections with more than 2 subpopulations (53%) and the presence of ultra-minority strains (1%) among which the C2a genotype. Transmission map confirmed the probable nosocomial PCP acquisition. The 732bp MIT26S sequence revealed three new SNPs and a new MNP which increased significantly the discriminatory power of MIT26S locus. As expected, in this context of PCP, NGS outperforms Sanger approach for sub-population determination and revealed new discriminating SNPs. These findings bring new insight for future epidemiological studies on this non cultivable opportunistic fungus.Le typage par Multilocus Sequence Typing (MLST) offre une caractérisation précise des souches, mais les performances du séquençage Sanger (Sseq) dans la détection de variants minoritaires sont limitées. Or, les infections mixtes sont fréquentes dans les pneumonies à Pneumocystis jirovecii (PCP). Le séquençage nouvelle génération (NGS) permet l’étude des populations minoritaires et majoritaires. Nous avons étudié l’apport du NGS pour le typage MLST d’un cluster de PCP chez des patients transplantés d’organe solide (TOS). Trente-deux patients (12 TOS du cluster et 20 patients contrôle) présentant une PCP au CHU Grenoble-Alpes entre 2012 et 2015 ont été inclus. Une stratégie MLST à trois loci (MIT26S, CYTB, SOD) a été choisie, avec de nouvelles amorces afin d’obtenir des amplicons d'environ 750pb. Les souches de 12 des 32 patients ont aussi été analysées par Sseq. Parmi les 12 TOS, 5 partageaient un même génotype C2a, non retrouvé dans le groupe contrôle. La proportion d’infections mixtes était plus élevée avec le NGS (65%) qu’avec le Sseq (25%). Le NGS a révélé des infections avec plus de deux souches (53%), et la présence de souches ultra-minoritaires, parmi lesquelles le génotype C2a. Une carte de transmission a confirmé la probable acquisition nosocomiale de PCP. Les séquences étendues de MIT26S ont révélé 4 nouveaux polymorphismes augmentant significativement le pouvoir discriminant du locus. Comme attendu, dans ce contexte de PCP, le NGS surpasse les capacités du Sseq pour la détection d’infections mixtes et a mis en évidence de nouveaux polymorphismes. Ce travail ouvre de nouvelles perspectives pour les futures études épidémiologiques de ce champignon non cultivable

Immune Response in Pneumocystis Infections According to the Host Immune System Status

International audienceThe host immune response is critical in Pneumocystis pneumonia (PCP). Immunocompetent hosts can eliminate the fungus without symptoms, while immunodeficient hosts develop PCP with an unsuitable excessive inflammatory response leading to lung damage. From studies based on rodent models or clinical studies, this review aimed to better understand the pathophysiology of Pneumocystis infection by analysing the role of immune cells, mostly lymphocytes, according to the immune status of the infected host. Hence, this review first describes the immune physiological response in infected immunocompetent hosts that are able to eliminate the fungus. The objective of the second part is to identify the immune elements required for the control of the fungus, focusing on specific immune deficiencies. Finally, the third part concentrates on the effect of the different immune elements in immunocompromised subjects during PCP, to better understand which cells are detrimental, and which, on the contrary, are beneficial once the disease has started. This work highlights that the immune response associated with a favourable outcome of the infection may differ according to the immune status of the host. In the case of immunocompetency, a close communication between B cells and TCD4 within tertiary lymphocyte structures appears critical to activate M2 macrophages without much inflammation. Conversely, in the case of immunodeficiency, a pro-inflammatory response including Th1 CD4, cytotoxic CD8, NK cells, and IFNγ release seems beneficial for M1 macrophage activation, despite the impact of inflammation on lung tissue

Evaluation of the Performance of the Novodiag® Stool Parasites Assay for the Detection of Intestinal Protozoa and Microsporidia

International audienceObjectives: We aimed to assess the performance of the Novodiag® Stool Parasites (NSP) assay in the diagnosis of the most common intestinal protozoan and microsporidia infections. Methods: A panel of 167 selected stool samples was retrospectively analysed with the NSP assay and compared to routine microscopy and qPCR methods for the detection of pathogenic protozoa and microsporidia. Results: Whereas specificity was high for all protozoa and microsporidia, NSP sensitivity was strongly dependent on the comparative method used as reference. When compared to microscopic methods, NSP sensitivity was high (96.7 to 100%) for Blastocystis hominis, Entamoeba histolytica and Cyclospora cayetanensis but was lower for Giardia intestinalis (85.2%) and ≤50% for Cystoisospora belli and Dientamoeba fragilis. In comparison to conventional qPCR, the NSP assay demonstrated lower sensitivity characteristics dependent on parasite loads, reaching 60 to 70% for G. intestinalis, D. fragilis, Cryptosporidium spp. and E. histolytica. Sensitivity was 100% for Enterocytozoon bieneusi, but none of the five samples containing Encephalitozoon spp. were detected. Conclusions: The overall performance of the NSP assay in the diagnosis of gastrointestinal protozoa and microsporidia seems to be better than or equivalent to that observed with microscopic methods but inferior to that obtainable with classical targeted qPCR

New Insights into Blood Circulating Lymphocytes in Human Pneumocystis Pneumonia

International audienceThe host lymphocyte response is decisive in Pneumocystis pneumonia (PCP) pathophysiology but little is known of the specific roles of lymphocyte subpopulations in this fungal infection. Peripheral NK, NKT, B, TCD4+ and TCD8+ subpopulations were compared by immunophenotyping between 20 patients diagnosed with PCP (PCP(+)] and 20 uninfected immunosuppressed patients (PCP(−)). Among PCP(+) subjects, the lymphocyte populations were also compared between surviving and deceased patients. Low B cell count (<40 cells/µL) was more frequent in PCP(+) than in PCP(−) patients (p = 0.03), while there was no difference for the TCD4 count. Among the PCP(+) group, the 7 deceased patients had lower Th1 (p = 0.02) and Tc1 (p = 0.03) populations, higher Th2 response (p = 0.03), higher effector TCD8 (p < 0.01), lower central memory TCD8 (p = 0.04) and reduced NK cells (p = 0.02) compared with the 13 survivors. Th1/Th2 ratio < 17, CD8 Tc1 < 44%, effector TCD8 < 25%, central memory TCD8 < 4%, NK cells < 50 cells/µL and total lymphocytes < 0.75 G/L were associated with a higher risk of mortality (p = 0.003, p = 0.007, p = 0.0007, p = 0.004, p = 0.02 and p = 0.019, respectively). The traditional analysis of TCD4 and TCD8 populations may be insufficient in the context of PCP. It could be completed by using B cells to predict the risk of PCP, and by using lymphocyte subpopulations or total lymphocyte count, which are easy to obtain in all health care facilities, to evaluate PCP prognosis

Real-time PCR for diagnosis of imported schistosomiasis

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QPCR detection of Mucorales DNA in bronchoalveolar lavage fluid to diagnose pulmonary mucormycosis

International audienceEarly diagnosis and treatment are essential to improving the outcome of mucormycosis. The aim of this retrospective study was to assess the contribution of quantitative PCR detection of Mucorales DNA in bronchoalveolar lavage fluids for early diagnosis of pulmonary mucormycosis.Bronchoalveolar lavage fluids (n=450) from 374 patients with pneumonia and immunosuppressive conditions were analyzed using a combination of 3 quantitative PCR assays targeting the main genera involved in mucormycosis in France (Rhizomucor, Mucor/Rhizopus, Lichtheimia).Among these 374 patients, 24 had at least one bronchoalveolar lavage with a positive PCR; 23/24 patients had radiological criteria for invasive fungal infections according to consensual criteria : 10 patients with probable or proven mucormycosis, and 13 additional patients with other invasive fungal infections (4 probable aspergillosis, 1 proven fusariosis, and 8 possible invasive fungal infections). Only 2/24 patients with a positive PCR on bronchoalveolar lavage had a positive Mucorales culture.PCR was also positive on serum in 17/24 patients. In most cases, PCR was first detected positive on sera (15/17). However, a positive PCR on bronchoalveolar lavage was the earliest and/or the only biological test revealing mucormycosis in 4 patients with a final diagnosis of probable or proven mucormycosis, 3 patients with probable aspergillosis and one patient with a possible invasive fungal infection.Mucorales PCR performed on bronchoalveolar lavage could provide additional arguments for earlier administration of Mucorales-directed antifungal therapy, thus improving the outcome of lung mucormycosis

Invasive aspergillosis due to Aspergillus section Usti: a multicenter retrospective study

BACKGROUND Aspergillus spp. of section Usti (A.ustus) represent a rare cause of invasive aspergillosis (IA). This multicenter study describes the epidemiology and outcome of A. ustus infections. METHODS Patients with A. ustus isolated from any clinical specimen were retrospectively identified in 22 hospitals from 8 countries. When available, isolates were sent for species identification (BenA/CaM sequencing) and antifungal susceptibility testing. Additional cases were identified by review of the literature. Cases were classified as proven/probable IA or no infection, according to standard international criteria. RESULTS Clinical report forms were obtained for 90 patients, of which 27 had proven/probable IA. Additional 45 cases were identified from literature review for a total of 72 cases of proven/probable IA. Hematopoietic cell and solid organ transplant recipients accounted for 47% and 33% cases, respectively. Only 8% patients were neutropenic at time of diagnosis. Ongoing anti-mold prophylaxis was present in 47% cases. Pulmonary IA represented 67% cases. Primary or secondary extra-pulmonary sites of infection were observed in 46% cases, with skin being affected in 28% cases. Multiple antifungal drugs were used (consecutively or in combination) in 67% cases. The 24-week mortality rate was 58%. A. calidoustus was the most frequent causal agent. Minimal inhibitory concentrations encompassing 90% isolates (MIC90) were 1, 8, >16 and 4 µg/mL for amphotericin B, voriconazole, posaconazole and isavuconazole, respectively. CONCLUSIONS Aspergillus ustus IA mainly occurred in non-neutropenic transplant patients and was frequently associated with extra-pulmonary sites of infection. Mortality rate was high and optimal antifungal therapy remains to be defined