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Emeline Scherer - One of the best experts on this subject based on the ideXlab platform.
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Molecular Strategies to Diagnose Mucormycosis
Journal of Fungi, 2019Co-Authors: Laurence Millon, Emeline Scherer, Steffi Rocchi, Anne-pauline BellangerAbstract:Molecular techniques have provided a new understanding of the epidemiology of mucormycosis and improved the diagnosis and therapeutic management of this life-threatening disease. PCR amplification and sequencing were first applied to better identify isolates that were grown from cultures of biopsies or bronchalveolar lavage samples that were collected in patients with Mucorales infection. Subsequently, molecular techniques were used to identify the fungus directly from the infected tissues or from bronchalveolar lavage, and they helped to accurately identify Mucorales fungi in tissue samples when the cultures were negative. However, these tools require invasive sampling (biospsy, bronchalveolar lavage), which is not feasible in patients in poor condition in Hematology or Intensive Care units. Very recently, PCR-based procedures to detect Mucorales DNA in non-invasive samples, such as plasma or serum, have proved successful in diagnosing mucormycosis early in all patients, whatever the clinical status, and these procedures are becoming essential to improving patient outcome.
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Quantitative PCR (qPCR) Detection of Mucorales DNA in Bronchoalveolar Lavage Fluid To Diagnose Pulmonary Mucormycosis.
Journal of clinical microbiology, 2018Co-Authors: Emeline Scherer, Xavier Iriart, Anne-pauline Bellanger, Damien Dupont, Juliette Guitard, Frédéric Gabriel, Sophie Cassaing, Elena Charpentier, Sarah Guenounou, Murielle CornetAbstract:Early 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 fluid samples (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, and Lichtheimia). Among these 374 patients, 24 patients had at least one bronchoalveolar lavage fluid sample with a positive PCR; 23/24 patients had radiological criteria for invasive fungal infections according to consensual criteria; 10 patients had probable or proven mucormycosis, and 13 additional patients had other invasive fungal infections (4 probable aspergillosis, 1 proven fusariosis, and 8 possible invasive fungal infections). Only 2/24 patients with a positive PCR result on a bronchoalveolar lavage fluid sample had a positive Mucorales culture. PCR was also positive on serum in 17/24 patients. In most cases, a positive PCR result was first detected using sera (15/17). However, a positive PCR on bronchoalveolar lavage fluid 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 fluid could provide additional support for earlier administration of Mucorales-directed antifungal therapy, thus improving the outcome of lung mucormycosis cases.
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Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients.
Clinical Infectious Diseases, 2013Co-Authors: Laurence Millon, Emeline Scherer, Anne-pauline Bellanger, Steffi Rocchi, Fabrice Larosa, Quentin Lepiller, Faezeh Legrand, Etienne Daguindau, Joel Leroy, Frédéric GrenouilletAbstract:BACKGROUND: The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. METHODS: We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. RESULTS: No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. CONCLUSION: Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.
Frédéric Grenouillet - One of the best experts on this subject based on the ideXlab platform.
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Identification of Mucorales by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry.
Journal of fungi (Basel Switzerland), 2019Co-Authors: Patrick Schwarz, Houssem Guedouar, Farah Laouiti, Frédéric Grenouillet, Eric DannaouiAbstract:More than 20 different species of Mucorales can be responsible for human mucormycosis. Accurate identification to the species level is important. The morphological identification of Mucorales is not reliable, and the currently recommended identification standard is the molecular technique of sequencing the internal transcribed spacer regions. Nevertheless, matrix-assisted laser desorption ionization time-of-flight mass spectrometry has been shown to be an accurate alternative for the identification of bacteria, yeasts, and even filamentous fungi. Therefore, 38 Mucorales isolates, belonging to 12 different species or varieties, mainly from international collections, including 10 type or neo-type strains previously identified by molecular methods, were used to evaluate the usefulness of matrix-assisted laser desorption ionization time-of-flight mass spectrometry for the identification of human pathogenic Mucorales to the species level. One to three reference strains for each species were used to create a database of main spectrum profiles, and the remaining isolates were used as test isolates. A minimum of 10 spectra was used to build the main spectrum profile of each database strain. Interspecies discrimination for all the isolates, including species belonging to the same genus, was possible. Twenty isolates belonging to five species were used to test the database accuracy, and were correctly identified to the species level with a log-score >2. In summary, matrix-assisted laser desorption ionization time-of-flight mass spectrometry is a reliable and rapid method for the identification of most of the human pathogenic Mucorales to the species level.
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Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients.
Clinical Infectious Diseases, 2013Co-Authors: Laurence Millon, Emeline Scherer, Anne-pauline Bellanger, Steffi Rocchi, Fabrice Larosa, Quentin Lepiller, Faezeh Legrand, Etienne Daguindau, Joel Leroy, Frédéric GrenouilletAbstract:BACKGROUND: The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. METHODS: We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. RESULTS: No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. CONCLUSION: Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.
Hermann Einsele - One of the best experts on this subject based on the ideXlab platform.
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evaluation of aspergillus and Mucorales specific t cells and peripheral blood mononuclear cell cytokine signatures as biomarkers of environmental mold exposure
International Journal of Medical Microbiology, 2018Co-Authors: Lukas Page, Philipp Weis, Ana Maria Waagagasser, Mariola Dragan, Thomas Dandekar, Tobias Muller, Marcus Dittrich, Maria Lazariotou, Johanna Helm, Hermann EinseleAbstract:Abstract Mold specific T-cells have been described as a supportive biomarker to monitor invasive mycoses and mold exposure. This study comparatively evaluated frequencies and cytokine profiles of Aspergillus fumigatus and Mucorales reactive T-cells depending on environmental mold exposure. Peripheral blood mononuclear cells (PBMCs) obtained from 35 healthy donors were stimulated with mycelial lysates of A. fumigatus and three human pathogenic Mucorales species. CD154+ specific T-cells were quantified by flow cytometry. In a second cohort of 20 additional donors, flow cytometry was complemented by 13-plex cytokine assays. Mold exposure of the subjects was determined using a previously established questionnaire. Highly exposed subjects exhibited significantly greater CD154+ A. fumigatus and Mucorales specific naive and memory T-helper cell frequencies. Significant correlation (r = 0.48 – 0.79) was found between A. fumigatus and Mucorales specific T-cell numbers. Logistic regression analyses revealed that combined analysis of mold specific T-cell frequencies and selected cytokine markers (A. fumigatus: IL-5 and TNF-α, R. arrhizus: IL-17A and IL-13) significantly improves classification performance, resulting in 75–90 % predictive power using 10-fold cross-validation. In conclusion, mold specific T-cell frequencies and their cytokine signatures offer promising potential in the assessment of environmental mold exposure. The cytokines identified in this pilot study should be validated in the clinical setting, e. g. in patients with hypersensitivity pneumonitis.
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Mucorales spores induce a proinflammatory cytokine response in human mononuclear phagocytes and harbor no rodlet hydrophobins
Virulence, 2017Co-Authors: Sebastian Wurster, Vanessa Thielen, Philipp Weis, Paul Walther, Johannes Elias, Ana Maria Waagagasser, Mariola Dragan, Thomas Dandekar, Hermann Einsele, Jurgen LofflerAbstract:Mucormycoses are life-threatening infections in immunocompromised patients. This study characterizes the response of human mononuclear cells to different Mucorales and Ascomycota. PBMC, monocytes, and monocyte derived dendritic cells (moDCs) from healthy donors were stimulated with resting and germinated stages of Mucorales and Ascomycota. Cytokine response and expression of activation markers were studied. Both inactivated germ tubes and resting spores of Rhizopus arrhizus and other human pathogenic Mucorales species significantly stimulated mRNA synthesis and secretion of proinflammatory cytokines. Moreover, R. arrhizus spores induced the upregulation of co-stimulatory molecules on moDCs and a specific T-helper cell response. Removal of rodlet hydrophobins by hydrofluoric acid treatment of A. fumigatus conidia resulted in enhanced immunogenicity, whereas the cytokine response of PBMCs to dormant R. arrhizus spores was not influenced by hydrofluoric acid. Scanning electron micrographs of Mucorales spores did not exhibit any morphological correlates of rodlet hydrophobins. Taken together, this study revealed striking differences in the response of human mononuclear cells to resting stages of Ascomycota and Mucorales, which may be explained by absence of an immunoprotective hydrophobin layer in Mucorales spores.
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clinical evaluation of a Mucorales specific real time pcr assay in tissue and serum samples
Journal of Medical Microbiology, 2016Co-Authors: Jan Springer, Michaela Lackner, Hermann Einsele, Cornelia Lassflorl, Christian Ensinger, Brigitte Risslegger, Charles Oliver Morton, David Nachbaur, Werner J Heinz, Juergen LoefflerAbstract:Molecular diagnostic assays can accelerate the diagnosis of fungal infections and subsequently improve patient outcomes. In particular, the detection of infections due to Mucorales is still challenging for laboratories and physicians. The aim of this study was to evaluate a probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples from patients suffering from invasive mucormycosis (IMM). This assay can detect a broad range of clinically relevant Mucorales species and can be used to complement existing diagnostic tests or to screen high-risk patients. An advantage of the Muc18S assay is that it exclusively detects Mucorales species allowing the diagnosis of Mucorales DNA without sequencing within a few hours. In paraffin-embedded tissue samples this PCR-based method allowed rapid identification of Mucorales in comparison with standard methods and showed 91 % sensitivity in the IMM tissue samples. We also evaluated serum samples, an easily accessible material, from patients at risk from IMM. Mucorales DNA was detected in all patients with probable/proven IMM (100 %) and in 29 % of the possible cases. Detection of IMM in serum could enable an earlier diagnosis (up to 21 days) than current methods including tissue samples, which were gained mainly post-mortem. A screening strategy for high-risk patients, which would enable targeted treatment to improve patient outcomes, is therefore possible.
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development and application of two independent real time pcr assays to detect clinically relevant Mucorales species
Journal of Medical Microbiology, 2016Co-Authors: Jan Springer, Hermann Einsele, Daniel Goldenberger, Friderike Schmidt, Maja Weisser, Elisabeth Wehrlewieland, Reno Frei, Jurgen LofflerAbstract:PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.
Jan Springer - One of the best experts on this subject based on the ideXlab platform.
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antifungal resistant Mucorales in different indoor environments
Mycology, 2019Co-Authors: Liliana Aranha Caetano, Tiago Faria, Jan Springer, Juergen Loeffler, Carla ViegasAbstract:This paper sought to address the prevalence of Mucorales in different indoor environments in Portugal. Environmental samples (183 in total) were collected at dwellings (n = 79) and workplaces (bake...
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Antifungal-resistant Mucorales in different indoor environments
Taylor & Francis Group, 2019Co-Authors: Liliana Aranha Caetano, Tiago Faria, Jan Springer, Juergen Loeffler, Carla ViegasAbstract:This paper sought to address the prevalence of Mucorales in different indoor environments in Portugal. Environmental samples (183 in total) were collected at dwellings (n = 79) and workplaces (bakeries, swine farms, taxis, waste-sorting plants) (n = 93) by passive sampling using electrostatic dust collector (EDC), air-conditioning filters, litter, and/or raw materials. Samples were inoculated onto non-selective MEA and DG18 media and were screened for antifungal drug-resistance in azole-supplemented agar Sabouraud media. A probe-based Mucorales-specific real-time PCR assay (Muc18S) was used to detect Mucorales in complement to conventional culture-based methods. Mucorales order was found as more prevalent in air-conditioning filters from waste-sorting fork lifters (35.7%). Amongst Mucorales isolates able to grow in azole-supplemented media, 16 isolates of Mucor sp., Rhizopus sp. or Rhizomucor sp. were not susceptible to 1 mg/L voriconazole, and four isolates of Mucor sp. or Rhizopus sp. were not susceptible to 4 mg/L itraconazole. In conclusion, combination of the culture-based and molecular methods proved to be reliable for Mucorales order identification in complex environmental samples
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clinical evaluation of a Mucorales specific real time pcr assay in tissue and serum samples
Journal of Medical Microbiology, 2016Co-Authors: Jan Springer, Michaela Lackner, Hermann Einsele, Cornelia Lassflorl, Christian Ensinger, Brigitte Risslegger, Charles Oliver Morton, David Nachbaur, Werner J Heinz, Juergen LoefflerAbstract:Molecular diagnostic assays can accelerate the diagnosis of fungal infections and subsequently improve patient outcomes. In particular, the detection of infections due to Mucorales is still challenging for laboratories and physicians. The aim of this study was to evaluate a probe-based Mucorales-specific real-time PCR assay (Muc18S) using tissue and serum samples from patients suffering from invasive mucormycosis (IMM). This assay can detect a broad range of clinically relevant Mucorales species and can be used to complement existing diagnostic tests or to screen high-risk patients. An advantage of the Muc18S assay is that it exclusively detects Mucorales species allowing the diagnosis of Mucorales DNA without sequencing within a few hours. In paraffin-embedded tissue samples this PCR-based method allowed rapid identification of Mucorales in comparison with standard methods and showed 91 % sensitivity in the IMM tissue samples. We also evaluated serum samples, an easily accessible material, from patients at risk from IMM. Mucorales DNA was detected in all patients with probable/proven IMM (100 %) and in 29 % of the possible cases. Detection of IMM in serum could enable an earlier diagnosis (up to 21 days) than current methods including tissue samples, which were gained mainly post-mortem. A screening strategy for high-risk patients, which would enable targeted treatment to improve patient outcomes, is therefore possible.
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development and application of two independent real time pcr assays to detect clinically relevant Mucorales species
Journal of Medical Microbiology, 2016Co-Authors: Jan Springer, Hermann Einsele, Daniel Goldenberger, Friderike Schmidt, Maja Weisser, Elisabeth Wehrlewieland, Reno Frei, Jurgen LofflerAbstract:PCR-based detection of Mucorales species could improve diagnosis of suspected invasive fungal infection, leading to a better patient outcome. This study describes two independent probe-based real-time PCR tests for detection of clinically relevant Mucorales, targeting specific fragments of the 18S and the 28S rRNA genes. Both assays have a short turnaround time, allow fast, specific and very sensitive detection of clinically relevant Mucorales and have the potential to be used as quantitative tests. They were validated on various clinical samples (fresh and formalin-fixed paraffin-embedded specimens, mainly biopsies, n = 17). The assays should be used as add-on tools to complement standard techniques; a combined approach of both real-time PCR assays has 100 % sensitivity. Genus identification by subsequent sequencing is possible for amplicons of the 18S PCR assay. In conclusion, combination of the two independent Mucorales assays described in this study, 18S and 28S, detected all clinical samples associated with proven Mucorales infection (n = 10). Reliable and specific identification of Mucorales is a prerequisite for successful antifungal therapy as these fungi show intrinsic resistance to voriconazole and caspofungin.
Anne-pauline Bellanger - One of the best experts on this subject based on the ideXlab platform.
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Molecular Strategies to Diagnose Mucormycosis
Journal of Fungi, 2019Co-Authors: Laurence Millon, Emeline Scherer, Steffi Rocchi, Anne-pauline BellangerAbstract:Molecular techniques have provided a new understanding of the epidemiology of mucormycosis and improved the diagnosis and therapeutic management of this life-threatening disease. PCR amplification and sequencing were first applied to better identify isolates that were grown from cultures of biopsies or bronchalveolar lavage samples that were collected in patients with Mucorales infection. Subsequently, molecular techniques were used to identify the fungus directly from the infected tissues or from bronchalveolar lavage, and they helped to accurately identify Mucorales fungi in tissue samples when the cultures were negative. However, these tools require invasive sampling (biospsy, bronchalveolar lavage), which is not feasible in patients in poor condition in Hematology or Intensive Care units. Very recently, PCR-based procedures to detect Mucorales DNA in non-invasive samples, such as plasma or serum, have proved successful in diagnosing mucormycosis early in all patients, whatever the clinical status, and these procedures are becoming essential to improving patient outcome.
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Quantitative PCR (qPCR) Detection of Mucorales DNA in Bronchoalveolar Lavage Fluid To Diagnose Pulmonary Mucormycosis.
Journal of clinical microbiology, 2018Co-Authors: Emeline Scherer, Xavier Iriart, Anne-pauline Bellanger, Damien Dupont, Juliette Guitard, Frédéric Gabriel, Sophie Cassaing, Elena Charpentier, Sarah Guenounou, Murielle CornetAbstract:Early 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 fluid samples (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, and Lichtheimia). Among these 374 patients, 24 patients had at least one bronchoalveolar lavage fluid sample with a positive PCR; 23/24 patients had radiological criteria for invasive fungal infections according to consensual criteria; 10 patients had probable or proven mucormycosis, and 13 additional patients had other invasive fungal infections (4 probable aspergillosis, 1 proven fusariosis, and 8 possible invasive fungal infections). Only 2/24 patients with a positive PCR result on a bronchoalveolar lavage fluid sample had a positive Mucorales culture. PCR was also positive on serum in 17/24 patients. In most cases, a positive PCR result was first detected using sera (15/17). However, a positive PCR on bronchoalveolar lavage fluid 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 fluid could provide additional support for earlier administration of Mucorales-directed antifungal therapy, thus improving the outcome of lung mucormycosis cases.
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detection d adn de Mucorales par pcr quantitative dans le liquide de lavage broncho alveolaire interet pour le diagnostic des mucormycoses
Journal De Mycologie Medicale, 2017Co-Authors: Scherer Emeline, Xavier Iriart, Anne-pauline Bellanger, Damien Dupont, Frédéric Gabriel, J Guitard, Muriel Cornet, Francoise Botterel, Laurence MillonAbstract:Objectif Plusieurs etudes retrospectives ont montre que la detection d’ADN de Mucorales par PCR quantitative dans le serum permettait un diagnostic precoce des mucormycoses [1–4] . La PCR est desormais de plus en plus souvent appliquee pour la detection d’ADN fongique dans le liquide de lavage broncho-alveolaire (LBA). L’objectif de ce travail etait d’evaluer l’interet de la PCR sur le LBA pour le diagnostic des mucormycoses par une analyse retrospective des resultats des PCR Mucorales sur LBA realisees dans notre laboratoire ces 3 dernieres annees. Methodes Au total, 415 LBA (360 patients) ont ete recus dans notre laboratoire entre janvier 2013 et janvier 2017 avec une demande de PCR Mucorales. L’extraction d’ADN a ete realisee a partir du LBA a l’aide du kit NucleoSpin ® Tissue (Macherey Nagel, D’uren, Allemagne). La detection d’ADN a ete realisee par une combinaison de 3 qPCR ciblant les 4 genres principalement retrouves en France ( Lichtheimia , Rhizomucor , Rhizopus , Mucor ), comme decrit precedemment [3] . Resultats La PCR Mucorales sur LBA a ete negative chez 344/360 patients. Des resultats positifs de la PCR Mucorales sur LBA ont ete retrouves chez 16/360 patients, avec un cycle seuil (Cq) median de 34,5 cycles, [ range : 27–41]. Pour 4/16 patients, le diagnostic de mucormycose prouvee a ete retenu (Cq median : 35,5, [32–41]). Pour 5/16 patients, le diagnostic de mucormycose probable a ete retenu (Cq median : 29, [27–39]) ; ces 5 patients avaient egalement des PCR Mucorales positives, pour la meme cible, sur le serum. Pour 2/16 patients, une autre infection fongique invasive (IFI) a ete diagnostiquee (1 aspergillose invasive probable [Cq = 35], 1 fusariose prouvee [Cq = 39]) ; ces 2 patients avaient egalement des PCR Mucorales positive sur le serum dans la meme periode, et la possibilite d’infection mixte a ete evoquee. Pour 4/16 patients, le diagnostic d’IFI possible avec PCR Mucorales positives a ete retenu (Cq median : 36,5, [33–40]). Pour le dernier patient qui presentait une sarcoidose traitee par corticoides au long cours, le diagnostic de mucormycose n’a pas ete retenu (Cq = 41). Conclusion Ce premier travail, retrospectif, realise sur la totalite des prelevements « issus des soins » adresses a notre laboratoire, montre que la detection d’ADN de Mucorales par qPCR sur le LBA permet d’obtenir un argument supplementaire en faveur du diagnostic de mucormycose. Les performances de cette technique sur le LBA pourront etre evaluees plus precisement dans le cadre du PHRC national ModiMucor.
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Quantitative polymerase chain reaction detection of circulating DNA in serum for early diagnosis of mucormycosis in immunocompromised patients.
Clinical Infectious Diseases, 2013Co-Authors: Laurence Millon, Emeline Scherer, Anne-pauline Bellanger, Steffi Rocchi, Fabrice Larosa, Quentin Lepiller, Faezeh Legrand, Etienne Daguindau, Joel Leroy, Frédéric GrenouilletAbstract:BACKGROUND: The aim of our study was to assess the detection of circulating DNA from the most common species of Mucorales for early diagnosis of mucormycosis in at-risk patients. METHODS: We retrospectively evaluated a combination of 3 quantitative polymerase chain reaction (qPCR) assays using hydrolysis probes targeting Mucor/Rhizopus, Lichtheimia (formerly Absidia), and Rhizomucor for circulating Mucorales detection. Serial serum samples from 10 patients diagnosed with proven mucormycosis (2-9 samples per patient) were analyzed. RESULTS: No cross-reactivity was detected in the 3 qPCR assays using 19 reference strains of opportunistic fungi, and the limit of detection ranged from 3.7 to 15 femtograms/10 µL, depending on the species. DNA from Mucorales was detected in the serum of 9 of 10 patients between 68 and 3 days before mucormycosis diagnosis was confirmed by histopathological examination and/or positive culture. All the qPCR results were concordant with culture and/or PCR-based identification of the causing agents in tissue (Lichtheimia species, Rhizomucor species, and Mucor/Rhizopus species in 4, 3, and 2 patients, respectively). Quantitative PCR was negative in only 1 patient with proven disseminated mucormycosis caused by Lichtheimia species. CONCLUSION: Our study suggests that using specific qPCR targeting several species of Mucorales according to local ecology to screen at-risk patients could be useful in a clinical setting. The cost and efficacy of this strategy should be evaluated. However, given the human and economic cost of mucormycosis and the need for rapid diagnosis to initiate prompt directed antifungal therapy, this strategy could be highly attractive.