Molecular diagnosis of Pseudoterranova decipiens s.s in human, France

Background: Anisakis and Pseudoterranova are the main genera involved in human infections caused by nematodes of the Anisakidae family. Species identification is complicated due to the lack of differential morphological characteristics at the larval stage, thus requiring molecular differentiation. Pseudoterranova larvae ingested through raw fish are spontaneously eliminated in most cases, but mechanical removal by means of endoscopy might be required. To date, only very few cases of Pseudoterranova infection have been reported in France. Case presentation: A 19-year-old woman from Northeastern France detected, while brushing her teeth, a larva exiting through her mouth. The patient who presented with headache, diarrhea, and abdominal cramps reported having eaten baked cod. The worm was a fourth-stage larva with a size of 22 × 0.9 mm, and molecular biology identified it as Pseudoterranova decipiens sensu stricto (s. s.). In a second P. decipiens infection case, occurring a few months later, a worm exited through the patient’s nose after she had eaten raw sea bream. Conclusion: These two cases demonstrate that Pseudoterranova infection is not uncommon among French patients. Therefore, molecular techniques should be more widely applied for a better characterization of anisakidosis epidemiology in France

The Airway Microbiota in Cystic Fibrosis: A Complex Fungal and Bacterial Community—Implications for Therapeutic Management

International audienceBackground Given the polymicrobial nature of pulmonary infections in patients with cystic fibrosis (CF), it is essential to enhance our knowledge on the composition of the microbial community to improve patient management. In this study, we developed a pyrosequencing approach to extensively explore the diversity and dynamics of fungal and prokaryotic populations in CF lower airways. Methodology and Principal Findings Fungi and bacteria diversity in eight sputum samples collected from four adult CF patients was investigated using conventional microbiological culturing and high-throughput pyrosequencing approach targeting the ITS2 locus and the 16S rDNA gene. The unveiled microbial community structure was compared to the clinical profile of the CF patients. Pyrosequencing confirmed recently reported bacterial diversity and observed complex fungal communities, in which more than 60% of the species or genera were not detected by cultures. Strikingly, the diversity and species richness of fungal and bacterial communities was significantly lower in patients with decreased lung function and poor clinical status. Values of Chao1 richness estimator were statistically correlated with values of the Shwachman-Kulczycki score, body mass index, forced vital capacity, and forced expiratory volume in 1 s (p = 0.046, 0.047, 0.004, and 0.001, respectively for fungal Chao1 indices, and p = 0.010, 0.047, 0.002, and 0.0003, respectively for bacterial Chao1 values). Phylogenetic analysis showed high molecular diversities at the sub-species level for the main fungal and bacterial taxa identified in the present study. Anaerobes were isolated with Pseudomonas aeruginosa, which was more likely to be observed in association with Candida albicans than with Aspergillus fumigatus

Mucormycosis in Burn Patients

Patients with extensive burns are an important group at risk for cutaneous mucormycosis. This study aimed to perform a systematic review of all reported mucormycosis cases in burn patients from 1990 onward. A Medline search yielded identification of 7 case series, 3 outbreaks, and 25 individual cases reports. The prevalence reached 0.04%–0.6%. The median age was 42–48 in the case series and outbreaks, except for the studies from military centers (23.5–32.5) and in individual reports (29.5). The median total body surface area reached 42.5%–65%. Various skin lesions were described, none being pathognomonic: the diagnosis was mainly reached because of extensive necrotic lesions sometimes associated with sepsis. Most patients were treated with systemic amphotericin B or liposomal amphotericin B, and all underwent debridement and/or amputation. Mortality reached 33%–100% in the case series, 29%–62% during outbreaks, and 40% in individual cases. Most patients were diagnosed using histopathology and/or culture. Mucorales qPCR showed detection of circulating DNA 2–24 days before the standard diagnosis. Species included the main clinically relevant mucorales (i.e., Mucor, Rhizopus, Absidia/Lichtheimia, Rhizomucor) but also more uncommon mucorales such as Saksenaea or Apophysomyces. Contact with soil was reported in most individual cases. Bandages were identified as the source of contamination in two nosocomial outbreaks

Use of Fluorescence Resonance Energy Transfer Hybridization Probes To Evaluate Quantitative Real-Time PCR for Diagnosis of Ocular Toxoplasmosis

Toxoplasma gondii infection is an important cause of chorioretinitis in Europe and the United States. Ophthalmological examination and a good clinical response to adequate therapy mainly support ocular toxoplasmosis diagnosis. However, clinical diagnostic may be difficult in some atypical cases. In these cases, laboratory confirmation, based on detection of local specific antibodies and parasite DNA by conventional PCR, is therefore important to confirm the disease etiology. More recently, real-time PCR has been developed to improve prenatal congenital toxoplasmosis diagnosis. We therefore examined the diagnostic value of quantitative real-time PCR for the detection of T. gondii in aqueous humor samples, associated with quantification of human β-globin to control sample quantitative quality, by using a double fluorescence resonance energy transfer hybridization probes system with a double fluorescence reading. Of the 23 the clinically toxoplasmosis suspect patients, 22 showed serological evidence of exposure to Toxoplasma; one had a serological profile indicative of active infection. The analysis of paired aqueous humor and serum samples revealed an intraocular antibody production in 9 of 23 cases (39.1%). The quantitative real-time PCR revealed positive and high parasite numbers and high Toxoplasma/human genome ratios in three cases. Furthermore, PCR was the only positive confirmatory test in two cases (11.1%). None of the patients included in the control group (n = 7) had evidence of either local specific antibody production or T. gondii DNA detection, suggesting a good relative assay specificity. On the whole, quantitative real-time PCR appears to be useful for diagnosing atypical ocular toxoplasmosis presentations

Phylogenetic analysis of Trichophyton mentagrophytes human and animal isolates based on MnSOD and ITS sequence comparison

International audienceDermatophytes are keratinophilic fungi able to infect keratinized tissues of human or animal origin. Among them, Trichophyton mentagrophytes is known to be a species complex composed of several species or variants, which occur in both human and animals. Since the T mentagrophytes complex includes both anthropophilic and zoophilic pathogens, accurate molecular identification is a critical issue for comprehensive understanding of the clinical and epidemiological implications of the genetic heterogeneity of this complex. Here, 41 T mentagrophytes isolates from either human patients (114 isolates) or animals (27 isolates) with dermatophytosis were prospectively isolated by culture and identified on morphological bases at the University Hospital Centres of Lille and Poitiers, and the Veterinary School of Alfort, respectively. The isolates were differentiated by DNA sequencing of the variable internal transcribed spacer (ITS) regions flanking the 5.8S rDNA, and of the housekeeping gene encoding the manganese-containing superoxide dismutase (MnSOD), an enzyme which is involved in defence against oxidative stress and has previously provided interesting insight into both fungal taxonomy and phylogeny. ITS1-ITS2 regions and MnSOD sequences successfully differentiate between members of the T mentagrophytes complex and the related species Trichophyton rubrum. Whatever the phylogenetic marker used, members of this complex were classified into two major clades exhibiting a similar topology, with a higher variability when the ITS marker was used. Relationships between ITS/MnSOD sequences and host origin, clinical pattern and phenotypic characteristics (macroscopic and microscopic morphologies) were analysed

Acute Blastocystis-Associated Appendicular Peritonitis in a Child, Casablanca, Morocco

Despite increasing reports that Blastocystis infection is associated with digestive symptoms, its pathogenicity remains controversial. We report appendicular peritonitis in a 9-year-old girl returning to France from Morocco. Only Blastocystis parasites were detected in stools, appendix, peritoneal liquid, and recto-uterine pouch. Simultaneous gastroenteritis in 26 members of the child’s family suggested an outbreak

Actualités du diagnostic des infections parasitaires et fongiques

International audienceThe diagnosis of parasitic and fungal infections, historically based on the detection of these pathogens using direct diagnosis (macro/microscopic examination, culture) or serological methods, has considerably evolved in the last decades, especially with the development of molecular approaches and mass spectrometry. These techniques, as well as most analyses of parasitic and fungal serology, are mostly the preserve of Hospital University Centers Parasitology-Mycology laboratories. In 2016, the French association of medical parasitology and mycology teachers and hospital practitioners (Anofel) has provided a Catalogue of rare analyses, regularly updated and freely accessible on the Anofel website (https://anofel.net/). This tool, which hinges on 4 parts (parasitology, parasitic serology, mycology, and fungal serology), aims to provide information on all available analyses, and a list of hospital laboratories able to undertake them. It is complementary to the other reference works that were developed by our association, including the Guide of analyses and methods in parasitology and mycology, published in 2018, and the eANOFEL pictures and videos database, freely accessible online (http://www.eanofel.fr). In this article, we draw-up a state-of-the-art of the most specialized techniques available in the parasitology-mycology laboratories and presented in the Catalogue of rare analyses of the Anofel collegium, and their interest for the diagnosis of these infections.Le diagnostic des infections parasitaires et fongiques, historiquement basé sur la détection de ces pathogènes par diagnostic direct (examen macro/microscopique, culture) ou sérologique, a connu durant les dernières décennies des évolutions importantes, avec notamment le développement d’approches moléculaires et de la spectrométrie de masse. Ces techniques, de même que la plupart des analyses en sérologie parasitaire et fongique, restent le plus souvent l’apanage des laboratoires de centres hospitaliers universitaires spécialisés en parasitologie-mycologie. En 2016, l’Association française des enseignants et praticiens hospitaliers titulaires de parasitologie et mycologie médicales (Anofel) a mis à disposition un Catalogue des analyses rares en accès libre sur le site d’Anofel (https://anofel.net/), et régulièrement mis à jour. Cet outil, qui s’articule en 4 volets (parasitologie, sérologie parasitaire, mycologie et sérologie fongique) a pour objectif de faciliter l’accès aux informations sur les analyses disponibles et les laboratoires hospitaliers capables de les prendre en charge. Il complète les autres ouvrages de référence élaborés par la collégiale Anofel, dont le Guide des analyses et méthodes en parasitologie et mycologie, publié en 2018, et la base de données d’images et de vidéos, en accès libre sur internet (http://www.eanofel.fr). L’objectif de cet article est de dresser un état des lieux des techniques plus spécialisées disponibles dans les laboratoires de parasitologie-mycologie, présentées dans le Catalogue des analyses rares de la collégiale Anofel, et leur intérêt pour le diagnostic de ces infections

Evaluation des tests de PCR en temps reel Bio-Evolution Microsporidia generic et typing pour le diagnostic de la microsporidiose intestinale.

International audienceCases of intestinal microsporidiosis infection are underestimated and affect both immunocompromized and immunocompetent patients. Real-time PCR is superseding microscopic examination for its diagnosis in medical analysis laboratories. However, few manufacturers include microsporidia in their PCR panel for the diagnosis of infectious gastroenteritis. Here, we evaluated the performances of the real-time PCR assays microsporidia generic and microsporidia typing (Bio-Evolution, France) on the Rotor-Gene Q real-time PCR cycler (Qiagen, France). We included 45 negative and 44 positive stool samples for Enterocytozoon bieneusi ( n = 34, with various genotypes), Encephalitozoon intestinalis ( n = 4), Encephalitozoon hellem ( n = 4), and Encephalitozoon cuniculi ( n = 2). We also studied a four-year survey of an inter-laboratory quality control program including 9 centers that used this commercial assay. Sensitivity and specificity of the microsporidia generic assay were 86.4% and 93.3%, respectively. Encephalitozoon hellem and Encephalitozoon cuniculi were detected by the microsporidia generic PCR assay but not by the microsporidia typing PCR assay. These results were consistent with the results of the inter-laboratory quality control program. In conclusion, Bio-Evolution Real-time PCR assays are useful tools for intestinal microsporidiosis, but negative results for microsporidia typing assays require supplementary analyses to confirm E. hellem or E. cuniculi infections.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

Impact of domestic mould exposure on Aspergillus biomarkers and lung function in patients with chronic obstructive pulmonary disease

International audiencePatients with chronic obstructive pulmonary disease (COPD) are frequently colonised or sensitised by Aspergillus, but clinical significance remains unclear. Furthermore, little is known on the impact of indoor mould exposure during COPD. In this study, we assessed the relationship between domestic mould exposure, Aspergillus biomarkers and COPD severity during acute exacerbation and at stable state. Aspergillus section Fumigati culture in sputum and anti-Aspergillus antibodies detection (IgG and precipitins) were followed up in COPD patients that were prospectively recruited during exacerbation (n = 62), and underwent a visit at stable state after 18 months (n = 33). Clinical characteristics were collected at inclusion. Electrostatic dust collectors (EDCs) were used to measure domestic mould contamination. Aspergillus section Fumigati was more frequently detected during exacerbation (16.9%) than at stable state (4.0%), but the frequency of patients presenting with anti-Aspergillus antibodies was similar (32.2% and 33.3%, respectively). Aspergillus section Fumigati detection was associated with a higher body-mass index (BMI) during exacerbation, whereas patients with anti-Aspergillus antibodies presented a lower BMI and forced expiratory volume in 1 s, as well as a higher frequency of inhaled corticoids and higher total mould and Penicillium exposure at final visit (P 30 CFU/cm2 (P = 0.03). Aspergillosis was diagnosed in 2 patients (6.1%) who presented increased levels of antibodies. Our data suggest that anti-Aspergillus antibodies are associated with chronic lung function alteration and/or domestic mould exposure, thereby supporting the consideration of indoor mould contamination and anti-Aspergillus antibodies kinetics in COPD management

Factors associated with Pneumocystis colonization and circulating genotypes in chronic obstructive pulmonary disease patients with acute exacerbation or at stable state and their homes

International audienceAbstract Pneumocystis jirovecii colonization is frequent during chronic obstructive pulmonary disease (COPD) and patients constitute potential contributors to its interhuman circulation. However, the existence of an environmental reservoir cannot be excluded. We assessed the prevalence and factors associated with Pneumocystis colonization during COPD, and studied circulation between patients and their domestic environment. Pneumocystis molecular detection and mtLSU genotyping were performed in oro-pharyngeal washes (OPW) sampled in 58 patients with COPD acute exacerbation, and in indoor dust, sampled in patients’ homes using electrostatic dust collectors (EDCs). Lung and systemic inflammation was assessed. Pneumocystis carriage was evaluated in 28 patients after 18 months at stable state. Pneumocystis was detected in 11/58 OPWs during exacerbation (19.0%). Colonized patients presented a significantly lower body mass index, and higher serum IL-17 and CD62P. One patient presented positive detection of typable isolates in both OPW and EDC, with both isolates harboring mtLSU genotype 3. Pneumocystis genotype 1 was further detected in EDCs from three non-colonized patients and one colonized patient with non-typable isolate. Genotypes 1 and 2 were predominant in clinical isolates (both 42%), with genotype 3 representing 16% of isolates. Pneumocystis was detected in 3/28 patients at stable state (10.7%). These data suggest that Pneumocystis colonization could be facilitated by a lower BMI and be related to acute alteration of lung function during COPD exacerbation. It also suggests Th17 pathway and platelet activation could be involved in the anti-Pneumocystis response during colonization. Last, Pneumocystis detection in EDCs supports its potential persistence in indoor dust. Lay Summary Chronic obstructive pulmonary disease patients tend to be more frequently colonized by Pneumocystis during exacerbation (19.0%) than at stable state (10.7%). Factors associated with colonization include lower BMI, higher IL-17, and CD62P. Pneumocystis detection in patients’ dwellings suggests potential persistence in indoor dust