Rapid Typing of Transmissible Spongiform Encephalopathy Strains with Differential ELISA

A strain-typing ELISA distinguishes bovine spongiform encephalopathy from other scrapie strains in small ruminants

The Revolution of Lateral Flow Assay in the Field of AMR Detection

International audienceThe global spread of antimicrobial resistant (AMR) bacteria represents a considerable public health concern, yet their detection and identification of their resistance mechanisms remain challenging. Optimal diagnostic tests should provide rapid results at low cost to enable implementation in any microbiology laboratory. Lateral flow assays (LFA) meet these requirements and have become essential tools to combat AMR. This review presents the versatility of LFA developed for the AMR detection field, with particular attention to those directly triggering β-lactamases, their performances, and specific limitations. It considers how LFA can be modified by detecting not only the enzyme, but also its β-lactamase activity for a broader clinical sensitivity. Moreover, although LFA allow a short time-to-result, they are generally only implemented after fastidious and time-consuming techniques. We present a sample processing device that shortens and simplifies the handling of clinical samples before the use of LFA. Finally, the capacity of LFA to detect amplified genetic determinants of AMR by isothermal PCR will be discussed. LFA are inexpensive, rapid, and efficient tools that are easy to implement in the routine workflow of laboratories as new first-line tests against AMR with bacterial colonies, and in the near future directly with biological media

Development and validation of a lateral flow immunoassay for rapid detection of NDM-producing <em>Enterobacteriaceae</em>

International audienceThe global spread of carbapenemase-producing Enterobacteriaceae (CPE) that are often resistant to most, if not all, classes of antibiotics is a major public health concern. The NDM-1 carbapenemase is among the most worrisome carbapenemases given its rapid worldwide spread. We have developed and evaluated a lateral flow immunoassay (LFIA) (called the NDM LFIA) for the rapid and reliable detection of NDM-like carbapenemase-producing Enterobacteriaceae from culture colonies. We evaluated the NDM LFIA using 175 reference enterobacterial isolates with characterized β-lactamase gene content and 74 nonduplicate consecutive carbapenem-resistant clinical isolates referred for expertise to the French National Reference Center (NRC) for Antibiotic Resistance during a 1-week period (in June 2016). The reference collection included 55 non-carbapenemase producers and 120 carbapenemase producers, including 27 NDM producers. All 27 NDM-like carbapenemase producers of the reference collection were correctly detected in less than 15 min by the NDM LFIA, including 22 strains producing NDM-1, 2 producing NDM-4, 1 producing NDM-5, 1 producing NDM-7, and 1 producing NDM-9. All non-NDM-1 producers gave a negative result with the NDM LFIA. No cross-reaction was observed with carbapenemases (VIM, IMP, NDM, KPC, and OXA-48-like), extended-spectrum β-lactamases (ESBLs) (TEM, SHV, and CTX-M), AmpCs (CMY-2, DHA-2, and ACC-1), and oxacillinases (OXA-1, -2, -9, and -10). Similarly, among the 74 referred nonduplicate consecutive clinical isolates, all 7 NDM-like producers were identified. Overall, the sensitivity and specificity of the assay were 100% for NDM-like carbapenemase detection with strains cultured on agar. The NDM LFIA was efficient, rapid, and easy to implement in the routine workflow of a clinical microbiology laboratory for the confirmation of NDM-like carbapenemase-producing Enterobacteriaceae

Development and Multicentric Validation of a Lateral Flow Immunoassay for Rapid Detection of MCR-1-Producing Enterobacteriaceae

International audienceColistin has become a last-resort antibiotic for the treatment of infections caused by highly drug-resistant Gram-negative bacteria. Moreover, it has been widely used in the livestock sector

Duplex lateral flow immunoassay (LFIA) for detection of Yersinia pestis in environmental samples and suspicious substances

International audienceYersinia pestis (Y. pestis), the causative agent of the plague disease, has long been considered as a potent weapon of biological warfare. The rapid detection of the bioterrorist agent in environment or suspicious substances is essential to protect the civil population in case of malicious act. In this context, several dipsticks for rapid detection of Y. pestis have been developed and are now available on the market. These tests target almost exclusively the fraction capsular antigen 1 (F1) expressed mostly at 37°C (in course of infection or in-vitro culture), and are thus not always adapted for detection of Y. pestis from environmental source.For this reason, we developed a duplex lateral flow immunoassay (LFIA) combining the detection of F1 and an antigen expressed independently of the temperature. An industrial pre-batch was produced and its analytical sensitivity was assessed using bacteria grown at temperature in the range of 20°C to 37°C. The specificity was checked using various Y. pestis strains, strains of closely related species and more distantly related species. All ten Y. pestis strains grown at 30°C or 37°C were detected positive, as well as some Y. pseudotuberculosis strains but with a lower signal intensity. Other species were tested negative. Moreover, we investigated the impact of various environmental matrices and suspicious substances on the field. Most of them had no effect on the observed results

A multiplex lateral flow immunoassay for the rapid identification of NDM-, KPC-, IMP- and VIM-type and OXA-48-like carbapenemase-producing enterobacteriaceae

Objectives: The global spread of carbapenemase-producing Enterobacteriaceae represents a substantial challenge in clinical practice and rapid and reliable detection of these organisms is essential. The aim of this study was to develop and validate a lateral flow immunoassay (Carba5) for the detection of the five main carbapenemases (KPC-, NDM-, VIM-and IMP-type and OXA-48-like). Methods: Carba5 was retrospectively and prospectively evaluated using 296 enterobacterial isolates from agar culture. An isolated colony was suspended in extraction buffer and then loaded on the manufactured Carba5. Results: All 185 isolates expressing a carbapenemase related to one of the Carba5 targets were correctly and unambiguously detected in <15 min. All other isolates gave negative results except those producing OXA-163 and OXA-405, which are considered low-activity carbapenemases. No cross-reaction was observed with non-targeted carbapenemases, ESBLs, AmpCs or oxacillinases (OXA-1, -2, -9 and -10). Overall, this assay reached 100% sensitivity and 95.3%(retrospectively) to 100% (prospectively) specificity. Conclusions: Carba5 is efficient, rapid and easy to implement in the routine workflow of a clinical microbiology laboratory for confirmation of the five main carbapenemases encountered in Enterobacteriaceae

A Lateral Flow Immunoassay for the Rapid Identification of CTX-M-Producing Enterobacterales from Culture Plates and Positive Blood Cultures

International audienceWe have developed a lateral flow immunoassay (LFIA), named NG-Test CTX-M MULTI (NG-Test), to detect group 1, 2, 8, 9, 25 CTX-M producers from agar plates and from positive blood cultures in less than 15 min. The NG-Test was validated retrospectively on 113 well-characterized enterobacterial isolates, prospectively on 102 consecutively isolated ESBL-producers from the Bicêtre hospital and on 100 consecutive blood cultures positive with a gram-negative bacilli (GNB). The NG-Test was able to detect all CTX-M producers grown on the different agar plates used in clinical microbiology laboratories. No false positive nor negative results were observed. Among the 102 consecutive ESBL isolates, three hyper mucous isolates showed an incorrect migration leading to invalid results (no control band). Using an adapted protocol, the results could be validated. The NG-Test detected 99/102 ESBLs as being CTX-Ms. Three SHV producers were not detected. Among the 100 positive blood cultures with GNB tested 10/11 ESBL-producers were detected (8 CTX-M-15, 2 CTX-M-27). One SHV-2-producing-E. cloacae was missed. The NG-Test CTX-M MULTI showed 100% sensitivity and specificity with isolates cultured on agar plates and was able to detect 98% of the ESBL-producers identified in our clinical setting either from colonies or from positive blood cultures

Assessment of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Rapid Detection of Pathogenic Bacteria from Respiratory Samples in Patients with Hospital-Acquired Pneumonia

Rapid identification of the causative agent of hospital-acquired pneumonia (HAP) will allowan earlier administration of a more appropriate antibiotic and could improve the outcome of thesepatients. The aim of this study was to develop a rapid protocol to identify the main microorganismsinvolved in HAP by loop-mediated isothermal amplification (LAMP) directly from respiratory samples.First of all, a rapid procedure (<30 min) to extract the DNA from bronchoalveolar lavage (BAL),endotracheal aspirate (EA) or bronchoaspirate (BAS) was set up. A specific LAMP forStaphylococcusaureus,Escherichia coli,Klebsiella pneumoniae, Pseudomonas aeruginosa,Stenotrophomonas maltophiliaandAcinetobacter baumanniiwas performed with the extracted solution at 65◦C for 30-40 min. Overall,58 positive BAL and 83 EA/BAS samples were tested. The limits of detection varied according to themicroorganism detected. Validation of the LAMP assay with BAL samples showed that the assay was 100% specific and 86.3% sensitive (positive predictive value of 100% and a negative predictive valueof 50%) compared with culture. Meanwhile for BAS/EA samples, the assay rendered the followingstatistical parameters: 100% specificity, 94.6% sensitivity, 100% positive predictive value and 69.2%negative predictive value. The turnaround time including sample preparation and LAMP was circa1 h. LAMP method may be used to detect the most frequent bacteria causing HAP. It is a simple,cheap, sensitive, specific and rapid assay