[Contribution of connected prescriptions (NetSIG) for the management of molecular pathology exams]. / Apport de la prescription connectée (NetSIG) dans la prise en charge et la gestion des examens de pathologie moléculaire.

INTRODUCTION: This study describes our experience implementing a connected prescription software (NetSIG, Terascop) for molecular pathology exams. MATERIAL AND METHODS: NetSIG was set up for liquid biopsies and tissue testing. After registration and activation of regional pathology laboratories, NetSIG was implemented for external then internal prescriptions. RESULTS: NetSIG allows users to follow up on all prescriptions on the website, to interact through messages and to consult reports after validation. External set up was quick (3-4 months) and comprehensive (>70%). Prescriptions were made by physicians or more often by secretaries or referring pathologists. Internal prescriptions were made by pathologists then registered in NetSIG by our secretaries. This deployment strategy has resulted in very good completeness of prescriptions (>90%). DISCUSSION AND CONCLUSION: Connected prescriptions made this complex circuit more fluid and facilitated the redistribution of different administrative and technical tasks. The number of phone calls decreased sharply. Half of the prescriptions were made by pathologists and half by oncologists (physicians or secretaries). The mean dearchiving duration for blocks was one day. Mean forwarding of blocks was 2.5 days. Mean turnaround time was 8 days for targeted techniques and 13 days for Next Generation Sequencing. Physicians appreciated the interactivity of the software and the fact that they could consult it on a smartphone.

Evaluation of Next-Generation Sequencing Applied to Cryptosporidium parvum and Cryptosporidium hominis Epidemiological Study.

Background. Nowadays, most of the C. parvum and C. hominis epidemiological studies are based on gp60 gene subtyping using the Sanger sequencing (SgS) method. Unfortunately, SgS presents the limitation of being unable to detect mixed infections. Next-Generation Sequencing (NGS) seems to be an interesting solution to overcome SgS limits. Thus, the aim of our study was to (i) evaluate the reliability of NGS as a molecular typing tool for cryptosporidiosis, (ii) investigate the genetic diversity of the parasite and the frequency of mixed infections, (iii) assess NGS usefulness in Cryptosporidium sp. outbreak investigations, and (iv) assess an interpretation threshold of sequencing data. Methods. 108 DNA extracts from positive samples were sequenced by NGS. Among them, two samples were used to validate the reliability of the subtyping obtained by NGS and its capacity to detect DNA mixtures. In parallel, 106 samples from French outbreaks were used to expose NGS to epidemic samples. Results. NGS proved suitable for Cryptosporidium sp. subtyping at the gp60 gene locus, bringing more genetic information compared to SgS, especially by working on many samples simultaneously and detecting more diversity. Conclusions. This study confirms the usefulness of NGS applied to C. hominis and C. parvum epidemiological studies, especially aimed at detecting minority variants.

Diagnostic Accuracy of Four Commercial Triplex Immunochromatographic Tests for Rapid Detection of Rotavirus, Adenovirus, and Norovirus in Human Stool Samples.

Noroviruses (NoV), rotaviruses (RVA), and adenoviruses (AdV) are the main viral agents responsible for acute gastroenteritis (AGE) in humans. We aimed to determine the diagnostic accuracy of four commercial immunochromatographic tests (ICTs) intended for the rapid and simultaneous detection of these three pathogens. Diagnostic accuracy of bioNexia Noro/Rota-Adeno (bioMérieux), Immunoquick NoRotAdeno (Biosynex), Rota+Adeno+Noro combo card (CerTest Biotec), and Rida Quick Rota/Adeno/Noro Combi (R-Biopharm) ICTs was assessed retrospectively using a collection of 160 stool specimens (including 43 RVA-, 47 AdV-, and 42 NoV-positive samples) from French patients with AGE and using molecular methods as the reference standard. For RVA, the four ICTs demonstrated similar high sensitivity (93%) and excellent specificity (97.4 to 100%). For AdV, the four ICTs demonstrated similar poor sensitivity (54.3 to 58.7%) but excellent specificity (95.5 to 100%). They performed the best in AdV-F species (sensitivity, 80.8 to 84.6%) and worst in AdV non-F species (sensitivity, 22.2 to 27.8%). For NoV, the Rida Quick Rota/Adeno/Noro combi ICT exhibited high sensitivity (87.5%), but the sensitivity of the three others was poor (42.5 to 47.5%). The four ICTs exhibited high specificity (96.6 to 99.1%). Diagnostic accuracy was genogroup dependent. When we tested genogroup I NoV, the Rida Quick Rota/Adeno/Noro Combi ICT presented high sensitivity (90%), while the three other ICTs presented poor sensitivity (10 to 30%); when we tested genogroup II NoV, sensitivity was similar for the four ICTs (65 to 85%). In conclusion, the four ICTs are suitable first-line tests for the rapid diagnosis of RVA infections. The four ICTs are not suitable for the routine diagnosis of AdV infections but could provide a rapid response in case of positivity, notably in the context of AGE. Only the Rida Quick Rota/Adeno/Noro Combi ICT is suitable for the rapid detection of NoV, while the sensitivity for the detection of genogroup I NoV needs to be improved for the 3 other ICTs before being implemented in the routine diagnosis of NoV.

Role of AxyZ Transcriptional Regulator in Overproduction of AxyXY-OprZ Multidrug Efflux System in Achromobacter Species Mutants Selected by Tobramycin.

AxyXY-OprZ is an RND-type efflux system that confers innate aminoglycoside resistance to Achromobacter spp. We investigated here a putative TetR family transcriptional regulator encoded by the axyZ gene located upstream of axyXY-oprZ An in-frame axyZ gene deletion assay led to increased MICs of antibiotic substrates of the efflux system, including aminoglycosides, cefepime, fluoroquinolones, tetracyclines, and erythromycin, indicating that the product of axyZ negatively regulates expression of axyXY-oprZ Moreover, we identified an amino acid substitution at position 29 of AxyZ (V29G) in a clinical Achromobacter strain that occurred during the course of chronic respiratory tract colonization in a cystic fibrosis (CF) patient. This substitution, also detected in three other strains exposed in vitro to tobramycin, led to an increase in the axyY transcription level (5- to 17-fold) together with an increase in antibiotic resistance level. This overproduction of AxyXY-OprZ is the first description of antibiotic resistance acquisition due to modification of a chromosomally encoded mechanism in Achromobacter and might have an impact on the management of infected CF patients. Indeed, tobramycin is widely used for aerosol therapy within this population, and we have demonstrated that it easily selects mutants with increased MICs of not only aminoglycosides but also fluoroquinolones, cefepime, and tetracyclines.