Identification des Pseudomonas sp. par spectrométrie de masse type MALDI-TOF

International audienc

Validation de l’identification des Pseudomonas sp. par spectrométrie de masse type MALDI-TOF via la caractérisation d’une collection de souches environnementales et cliniques

Les bactéries appartenant au genre Pseudomonas sont décrites selon les espèces comme des dépolluants, pathogènes des végétaux, poissons, insectes, et humains potentiellement, mais aussi des contaminants des denrées alimentaires et des eaux hospitalières, voire comme des réservoirs de gènes de résistance aux antibiotiques. L’identification au rang d’espèce des Pseudomonas sp. constitue donc un défi pour le laboratoire d’hygiène hospitalière. Nous avons procédé à la validation de méthode en portée B flexible de l’identification bactérienne des Pseudomonas sp. par spectrométrie de masse (SM) MALDI-TOF (Bruker®) en utilisant comme méthode de référence l’identification moléculaire par séquençage des fragments de l’ADNr16S et rpoD à partir d’une collection de souches environnementales et cliniques. Le séquençage de l’ADNr 16S et de rpo a permis dans 65% des cas l’identification des souches au rang de l’espèce. Les essais de robustesse de la SM n’ont pas montré d’influence du type de milieu de culture (géloses au sang, gélose cétrimide, CFC et TSA) ni de l’âge des cultures sur la qualité de l’identification. La comparaison de méthodes a permis de mettre en évidence une bonne corrélation de l’identification au rang du genre (100%), du groupe d’espèces (100%) mais des résultats non satisfaisants de corrélation au rang de l’espèce (27,3%) pour les groupes putida, aeruginosa et fluorescens. Les souches cliniques étaient plus fréquemment résistantes aux antibiotiques que les souches environnementales, et des différences entre les groupes d’espèces ont été observées. Les souches environnementales appartenaient principalement au groupe oleovorans (espèces oleovorans et indoloxydans) tandis que les groupes putida et fluorescens étaient les plus représentés parmi les souches cliniques. L’identification des Pseudomonas sp. au rang de l’espèce reste difficile du fait de leur évolution taxonomique, la proximité phylogénétique entre les espèces et la faiblesse des bases de données existantes mais la spectrométrie de masse permet de les identifier en routine au rang de groupe d’espèces

Identification des Pseudomonas sp. par spectrométrie de masse type MALDI-TOF

International audienc

Additional file 1: of SOFIA®RSV: prospective laboratory evaluation and implementation of a rapid diagnostic test in a pediatric emergency ward

Study 2- SOFIA®RSV satisfaction survey. This survey was sent to the 13 pediatric consultants that were working in the ED during the study 2 period, 5 months after the end of the study. It includes 4 questions and one space for comments. (DOCX 11 kb

IgM triplet in neonatal diagnosis by immunoblotting and its potential use as a diagnostic marker for congenital toxoplasmosis

International audiencePrimary infection during pregnancy by the protozoan Toxoplasma gondii can be worrisome because transmission to the fetus may lead to congenital toxoplasmosis (CT). Neonatal diagnosis is usually performed by serological profile comparison of the mother and newborn. As previously reported in 2012 by C. L’Ollivier et al., three IgM bands at 75, 90 and 100 kDa called the “IgM triplet” has caught our attention and seems to be pathognomonic of CT. This retrospective multicenter study involved nine reference laboratories included in the French National Reference Center for Toxoplasmosis network and concerned determining the specificity and sensitivity of this IgM triplet. On this basis, we were able to propose a new read of the comparison of IgG and IgM immunoblot profiles of mother and infant to increase the sensitivity of this diagnostic marker. The effect of the trimester of pregnancy at the time of infection, but also of maternal treatment with pyrimethamine/sulfadiazine/folinic acid on the presence of this IgM triplet in the infant, could be studied. The presence of the triplet appears pathognomonic for the diagnosis of CT, and it increased the sensitivity of the immunoblot assay from 55.04% to 72.48%. As a result, it would be wise to enhance conventional immunoblot reading by adding the presence of the three IgM bands in the infant pattern for neonatal diagnosis of CT.La primo-infection pendant la grossesse par le protozoaire Toxoplasma gondii peut se révéler préoccupante car la transmission au fœtus peut conduire à une toxoplasmose congénitale (TC). Un diagnostic néonatal est généralement réalisé par comparaison des profils sérologiques de la mère et du nouveau-né. Comme précédemment rapporté en 2012 par C. L’Ollivier et al. , l’association de trois bandes d’IgM à 75, 90, et 100 kDa appelée la « triplette IgM » a retenu notre attention et semble être pathognomonique de la TC. Cette étude rétrospective multicentrique impliquant neuf laboratoires de référence inclus dans le réseau du Centre National de Référence pour la Toxoplasmose a permis de déterminer la spécificité et la sensibilité de cette triplette IgM. Ainsi, cela a permis de proposer une nouvelle lecture de la comparaison des profils d’immunoblot IgG et IgM de la mère et du nourrisson pour augmenter la sensibilité de ce marqueur diagnostique. L’effet du trimestre de la grossesse au moment de l’infection mais aussi du traitement maternel par pyriméthamine/sulfadiazine/acide folinique sur la présence de la triplette IgM chez l’enfant a pu être analysé. La présence de cette triplette semble pathognomonique pour le diagnostic de TC et elle permet d’augmenter la sensibilité du test immunoblot de 55,04 % à 72,48 %. Ainsi, il pourrait être judicieux d’améliorer la lecture conventionnelle de l’immunoblot en ajoutant la présence des trois bandes IgM dans le schéma du nourrisson pour le diagnostic néonatal de TC

Evaluation of two commercial kits and two laboratory-developed qPCR assays compared to LAMP for molecular diagnosis of malaria

International audienceAbstract Background Malaria is an infectious disease considered as one of the biggest causes of mortality in endemic areas. This life-threatening disease needs to be quickly diagnosed and treated. The standard diagnostic tools recommended by the World Health Organization are thick blood smears microscopy and immuno-chromatographic rapid diagnostic tests. However, these methods lack sensitivity especially in cases of low parasitaemia and non-falciparum infections. Therefore, the need for more accurate and reliable diagnostic tools, such as real-time polymerase chain reaction based methods which have proven greater sensitivity particularly in the screening of malaria, is prominent. This study was conducted at the French National Malaria Reference Centre to assess sensitivity and specificity of two commercial malaria qPCR kits and two in-house developed qPCRs compared to LAMP. Methods 183 blood samples received for expertise at the FNMRC were included in this study and were subjected to four different qPCR methods: the Biosynex Ampliquick ® Malaria test, the BioEvolution Plasmodium Typage test, the in-house HRM and the in-house TaqMan qPCRs. The specificity and sensitivity of each method and their confidence intervals were determined with the LAMP-based assay Alethia® Malaria as the reference for malaria diagnosis. The accuracy of species diagnosis of the Ampliquick ® Malaria test and the two in-house qPCRs was also evaluated using the BioEvolution Plasmodium Typage test as the reference method for species identification. Results The main results showed that when compared to LAMP, a test with excellent diagnostic performances, the two in-house developed qPCRs were the most sensitive (sensitivity at 100% for the in-house TaqMan qPCR and 98.1% for the in-house HRM qPCR), followed by the two commercial kits: the Biosynex Ampliquick ® Malaria test (sensitivity at 97.2%) and the BioEvolution Plasmodium Typage (sensitivity at 95.4%). Additionally, with the in-house qPCRs we were able to confirm a Plasmodium falciparum infection in microscopically negative samples that were not detected by commercial qPCR kits. This demonstrates that the var genes of P. falciparum used in these in-house qPCRs are more reliable targets than the 18S sRNA commonly used in most of the developed qPCR methods for malaria diagnosis. Conclusion Overall, these results accentuate the role molecular methods could play in the screening of malaria. This may represent a helpful tool for other laboratories looking to implement molecular diagnosis methods in their routine analysis, which could be essential for the detection and treatment of malaria carriers and even for the eradication of this disease

COVID-19-Associated Pulmonary Aspergillosis, Fungemia, and Pneumocystosis in the Intensive Care Unit: a Retrospective Multicenter Observational Cohort during the First French Pandemic Wave

International audienceThe aim of this study was to evaluate diagnostic means, host factors, delay of occurrence, and outcome of patients with COVID-19 pneumonia and fungal coinfections in the intensive care unit (ICU). From 1 February to 31 May 2020, we anonymously recorded COVID-19-associated pulmonary aspergillosis (CAPA), fungemia (CA-fungemia), and pneumocystosis (CA-PCP) from 36 centers, including results on fungal biomarkers in respiratory specimens and serum. We collected data from 154 episodes of CAPA, 81 of CA-fungemia, 17 of CA-PCP, and 5 of other mold infections from 244 patients (male/female [M/F] ratio = 3.5; mean age, 64.7 ± 10.8 years). CA-PCP occurred first after ICU admission (median, 1 day; interquartile range [IQR], 0 to 3 days), followed by CAPA (9 days; IQR, 5 to 13 days), and then CA-fungemia (16 days; IQR, 12 to 23 days) (P < 10-4). For CAPA, the presence of several mycological criteria was associated with death (P < 10-4). Serum galactomannan was rarely positive (<20%). The mortality rates were 76.7% (23/30) in patients with host factors for invasive fungal disease, 45.2% (14/31) in those with a preexisting pulmonary condition, and 36.6% (34/93) in the remaining patients (P = 0.001). Antimold treatment did not alter prognosis (P = 0.370). Candida albicans was responsible for 59.3% of CA-fungemias, with a global mortality of 45.7%. For CA-PCP, 58.8% of the episodes occurred in patients with known host factors of PCP, and the mortality rate was 29.5%. CAPA may be in part hospital acquired and could benefit from antifungal prescription at the first positive biomarker result. CA-fungemia appeared linked to ICU stay without COVID-19 specificity, while CA-PCP may not really be a concern in the ICU. Improved diagnostic strategy for fungal markers in ICU patients with COVID-19 should support these hypotheses. IMPORTANCE To diagnose fungal coinfections in patients with COVID-19 in the intensive care unit, it is necessary to implement the correct treatment and to prevent them if possible. For COVID-19-associated pulmonary aspergillosis (CAPA), respiratory specimens remain the best approach since serum biomarkers are rarely positive. Timing of occurrence suggests that CAPA could be hospital acquired. The associated mortality varies from 36.6% to 76.7% when no host factors or host factors of invasive fungal diseases are present, respectively. Fungemias occurred after 2 weeks in ICUs and are associated with a mortality rate of 45.7%. Candida albicans is the first yeast species recovered, with no specificity linked to COVID-19. Pneumocystosis was mainly found in patients with known immunodepression. The diagnosis occurred at the entry in ICUs and not afterwards, suggesting that if Pneumocystis jirovecii plays a role, it is upstream of the hospitalization in the ICU

Plasmodium ovale spp dhfr mutations associated with reduced susceptibility to pyrimethamine in sub-Saharan Africa: a retrospective genetic epidemiology and functional study

International audienc

French national epidemiology of bacterial superinfections in ventilator-associated pneumonia in patients infected with COVID-19: the COVAP study

International audienceAbstract Background Description and comparison of bacterial characteristics of ventilator-associated pneumonia (VAP) between critically ill intensive care unit (ICU) patients with COVID-19-positive, COVID + ; and non-COVID-19, COVID-. Methods Retrospective, observational, multicenter study that focused on French patients during the first wave of the pandemic (March–April 2020). Results 935 patients with identification of at least one bacteriologically proven VAP were included (including 802 COVID +). Among Gram-positive bacteria, S. aureus accounted for more than two-thirds of the bacteria involved, followed by Streptococcaceae and enterococci without difference between clinical groups regarding antibiotic resistance. Among Gram-negative bacteria, Klebsiella spp. was the most frequently observed bacterial genus in both groups, with K. oxytoca overrepresented in the COVID- group (14.3% vs . 5.3%; p &lt; 0.05). Cotrimoxazole-resistant bacteria were over-observed in the COVID + group (18.5% vs . 6.1%; p &lt;0.05), and after stratification for K. pneumoniae (39.6% vs . 0%; p &lt;0.05). In contrast, overrepresentation of aminoglycoside-resistant strains was observed in the COVID- group (20% vs . 13.9%; p &lt; 0.01). Pseudomonas sp. was more frequently isolated from COVID + VAPs (23.9% vs . 16.7%; p &lt;0.01) but in COVID- showed more carbapenem resistance (11.1% vs . 0.8%; p &lt;0.05) and greater resistance to at least two aminoglycosides (11.8% vs . 1.4%; p &lt; 0.05) and to quinolones (53.6% vs . 7.0%; p &lt;0.05). These patients were more frequently infected with multidrug-resistant bacteria than COVID + (40.1% vs . 13.8%; p &lt; 0.01). Conclusions The present study demonstrated that the bacterial epidemiology and antibiotic resistance of VAP in COVID + is different from that of COVID- patients. These features call for further study to tailor antibiotic therapies in VAP patients