Evaluation of LAMP assay using phenotypic tests and conventional PCR for detection of bla NDM-1 and bla KPC genes among carbapenem-resistant clinical Gram-negative isolates

2013 ◽  
Vol 62 (10) ◽  
pp. 1540-1544 ◽  
Author(s):  
Rachana Solanki ◽  
Lavanya Vanjari ◽  
Nagapriyanka Ede ◽  
Akhila Gungi ◽  
Amarendranath Soory ◽  
...  
Keyword(s):  
Point Of Care ◽  
Lamp Assay ◽  
Cost Effective ◽  
Turnaround Time ◽  
Conventional Pcr ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Phenotypic Methods ◽  
Sensitivity Specificity ◽  
Phenotypic Tests

Carbapenem-resistant pathogens cause infections associated with significant morbidity and mortality. This study evaluates the use of the loop-mediated isothermal amplification (LAMP) assay for rapid and cost-effective detection of bla NDM-1 and bla KPC genes among carbapenem-resistant Gram-negative bacteria in comparison with conventional PCR and existing phenotypic methods. A total of 60 carbapenem-resistant clinical isolates [Escherichia coli (15), Klebsiella pneumoniae (22), Acinetobacter baumannii (23)] were screened for the presence of carbapenemases (bla KPC and bla NDM-1) using phenotypic methods such as the modified Hodge test (MHT) and combined disc test (CDT) and molecular methods such as conventional PCR and LAMP assay. In all, 47/60 isolates (78.3 %) were MHT positive while 48 isolates were positive by CDT [46.6 % positive with EDTA, 30 % with 3′ aminophenylboronic acid (APB) plus EDTA and 1.6 % with APB alone]. Isolates showing CDT positivity with EDTA or APB contained bla NDM-1 and bla KPC genes, respectively. bla NDM-1 was present as a lone gene in 28 isolates (46.7 %) and present together with the bla KPC gene in 19 isolates (31.7 %). Only one E. coli isolate had a lone bla KPC gene. The LAMP assay detected either or both bla NDM-1 and bla KPC genes in four isolates that were missed by conventional PCR. Neither gene could be detected in 12 (20 %) isolates. The LAMP assay has greater sensitivity, specificity and rapidity compared to the phenotypic methods and PCR for the detection of bla NDM-1 and bla KPC. With a turnaround time of only 2–3 h, the LAMP assay can be considered a point-of-care assay.

scholarly journals Development of a LAMP method for detection of carbapenem-resistant Acinetobacter baumannii during a hospital outbreak

2020 ◽  
Vol 14 (05) ◽  
pp. 494-501
Author(s):  
Carolina Garciglia Mercado ◽  
Ramon Gaxiola Robles ◽  
Felipe Ascencio ◽  
Jesus Silva-Sanchez ◽  
Maria Teresa Estrada-Garcia ◽  
...  
Keyword(s):  
Predictive Value ◽  
Lamp Assay ◽  
Lamp Method ◽  
Conventional Pcr ◽  
Pcr Assay ◽  
Clinical Sensitivity ◽  
Hospital Facility ◽  
Hospital Outbreak ◽  
Carbapenem Resistant ◽  
Sensitivity Specificity

Introduction: Carbapenem-resistant A. baumannii (CRAB) represents a public health threat increasing worldwide. We assess the suitability of a loop-mediated isothermal amplification (LAMP) method for on-site screening of CRAB in a hospital facility. Methodology: A set of six primers were designed for recognizing eight distinct sequences on six targets: blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, and blaVIM. A LAMP method was developed, optimized and evaluated for the identification of CRAB in thirty-three environmental samples from an outbreak in an Intensive Care Unit (ICU) facility. Results: The sensitivity of the LAMP assay for the detection of A. baumannii was ten-fold higher than the PCR assay (1.0 ng.µL-1). The LAMP assays showed a higher detection rate for CRAB samples and robust diagnosis performance in comparison to a conventional PCR, with clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100% for blaOXA-23-like, blaOXA-51-like and blaVIM. Conclusions: The developed LAMP assays are powerful tools that can be useful in on-site screening of CRAB causing local outbreaks in clinics and hospitals facilities where costs and equipment restraints are imperative.

scholarly journals Simple, rapid, and cost-effective modified Carba NP test for carbapenemase detection among Gram-negative bacteria

2017 ◽  
Vol 9 (04) ◽  
pp. 303-307 ◽  
Author(s):  
Shoorashetty Manohara Rudresh ◽  
Giriyapur Siddappa Ravi ◽  
Lakshminarayanappa Sunitha ◽  
Sadiya Noor Hajira ◽  
Ellappan Kalaiarasan ◽  
...  
Keyword(s):  
Carbapenem Resistance ◽  
Cost Effective ◽  
Multidrug Resistant ◽  
Confirmatory Test ◽  
Resistant Bacteria ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Routine Basis ◽  
Phenotypic Tests

Abstract PURPOSE: Detection of carbapenemases among Gram-negative bacteria (GNB) is important for both clinicians and infection control practitioners. The Clinical and Laboratory Standards Institute recommends Carba NP (CNP) as confirmatory test for carbapenemase production. The reagents required for CNP test are costly and hence the test cannot be performed on a routine basis. The present study evaluates modifications of CNP test for rapid detection of carbapenemases among GNB. MATERIALS AND METHODS: The GNB were screened for carbapenemase production using CNP, CarbAcineto NP (CANP), and modified CNP (mCNP) test. A multiplex polymerase chain reaction (PCR) was performed on all the carbapenem-resistant bacteria for carbapenemase genes. The results of three phenotypic tests were compared with PCR. RESULTS: A total of 765 gram negative bacteria were screened for carbapenem resistance. Carbapenem resistance was found in 144 GNB. The metallo-β-lactamases were most common carbapenemases followed by OXA-48-like enzymes. The CANP test was most sensitive (80.6%) for carbapenemases detection. The mCNP test was 62.1% sensitive for detection of carbapenemases. The mCNP, CNP, and CANP tests were equally sensitive (95%) for detection of NDM enzymes among Enterobacteriaceae. The mCNP test had poor sensitivity for detection of OXA-48-like enzymes. CONCLUSION: The mCNP test was rapid, cost-effective, and easily adoptable on routine basis. The early detection of carbapenemases using mCNP test will help in preventing the spread of multidrug-resistant organisms in the hospital settings.

scholarly journals Comparison of Different Phenotypic Tests versus PCR in the Detection of Carbapenemase-Producing Pseudomonas aeruginosa Isolates in Hamadan, Iran

2021 ◽  
Vol 2021 ◽  
pp. 1-8
Author(s):  
Masoumeh Beig ◽  
Mohammad Taheri ◽  
Mohammad Reza Arabestani
Keyword(s):  
Pseudomonas Aeruginosa ◽  
High Sensitivity ◽  
Test Method ◽  
Clinical Samples ◽  
Predictive Values ◽  
Carbapenem Resistant ◽  
Accurate Detection ◽  
Phenotypic Methods ◽  
Sensitivity Specificity ◽  
Phenotypic Tests

In recent years, the prevalence of carbapenem-resistant Pseudomonas aeruginosa isolates has become a worldwide concern. Rapid and accurate detection of carbapenemase-producing P. aeruginosa isolates is so important. The aim of this study was to evaluate the performance of the phenotypic methods such as Modified Hodge test (MHT), CarbaNP (CNPt), combined double-disk synergy test (CDDT), and carbapenem inactivation method (CIM) for rapid and accurate detection of clinical carbapenemase production of P. aeruginosa isolates. This study was performed on 97 P. aeruginosa strains, which were isolated from clinical samples in Hamadan hospitals, western Iran in 2017-2018. Antibiotic susceptibility testing was performed using disk diffusion and minimum inhibitory concentration (MIC) by E-test method. We evaluated the performance of MHT, CarbaNP, CDDT, and CIM tests in comparison to polymerase chain reaction (PCR) for the detection of carbapenemase-producing isolates. Additionally, the presence of carbapenem-resistant genes was investigated using the PCR method. Our findings showed that the highest resistance was to cefoxitin (94.8%). Moreover, among the carbapenem antibiotics, the highest resistance was to imipenem (49.4%). Among the 49 carbapenem-resistant isolates, 42 (85.7%) isolates were MIC positive. The results of phenotypic tests showed that CarbaNP, CIM, CDDT, and MHT tests were positive in (48/49, 97.95%), (46/49, 93.87%), (27/49, 57.44%), and (25/49, 53.19%) of isolates, respectively. CarbaNP and CIM tests showed high sensitivity, specificity, positive predictive values (PPV), and negative predictive values (NPV) compared to PCR in P. aeruginosa isolates. CarbaNP and CIM tests are highly sensitive and specific tests for identifying carbapenemase-producing P. aeruginosa isolates.

scholarly journals Current and Future Pathotyping Platforms for Plasmodiophora brassicae in Canada

Plants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1446
Author(s):  
Heather H. Tso ◽  
Leonardo Galindo-González ◽  
Stephen E. Strelkov
Keyword(s):  
Crop Production ◽  
Plasmodiophora Brassicae ◽  
Cost Effective ◽  
Turnaround Time ◽  
Molecular Techniques ◽  
Differential Set ◽  
Management Plans ◽  
Increased Sensitivity ◽  
Phenotypic Methods ◽  
Important Disease

Clubroot, caused by Plasmodiophora brassicae, is one of the most detrimental threats to crucifers worldwide and has emerged as an important disease of canola (Brassica napus) in Canada. At present, pathotypes are distinguished phenotypically by their virulence patterns on host differential sets, including the systems of Williams, Somé et al., the European Clubroot Differential set, and most recently the Canadian Clubroot Differential set and the Sinitic Clubroot Differential set. Although these are frequently used because of their simplicity of application, they are time-consuming, labor-intensive, and can lack sensitivity. Early, preventative pathotype detection is imperative to maximize productivity and promote sustainable crop production. The decreased turnaround time and increased sensitivity and specificity of genotypic pathotyping will be valuable for the development of integrated clubroot management plans, and interest in molecular techniques to complement phenotypic methods is increasing. This review provides a synopsis of current and future molecular pathotyping platforms for P. brassicae and aims to provide information on techniques that may be most suitable for the development of rapid, reliable, and cost-effective pathotyping assays.

scholarly journals 2175. Rapid Detection of Carbapenemase Producing Organisms Directly from Blood Cultures Positive for Gram-Negative Bacilli

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S738-S738
Author(s):  
William Stokes ◽  
Johann Pitout ◽  
Lorraine Campbell ◽  
Deirdre Church ◽  
Dan Gregson
Keyword(s):  
Predictive Value ◽  
Rapid Detection ◽  
Blood Cultures ◽  
Gram Negative ◽  
Carbapenem Resistant ◽  
Direct Testing ◽  
Appropriate Treatment ◽  
Risk Patients ◽  
Health Zone ◽  
Sensitivity Specificity

Abstract Background The rapid detection of carbapenemase-producing organisms (CPOs) directly from blood cultures (BC) positive for Gram-negative bacilli (GNB) may accelerate the appropriate treatment of at-risk patients. Our objective was to evaluate the performance of two commercial assays in the rapid detection of CPOs directly from BC positive for GNB. Methods BC positive for GNB, taken from patients within the Calgary Health Zone over a 3 month period, were tested for the presence of CPOs with βCARBA® and NG-Test® CARBA 5. A subset of sterile BC samples was seeded with multi-drug-resistant (MDR) GNB. BC were incubated using the Bact-Alert™ system. Positive BC from clinical and seeded samples was tested directly with βCARBA and CARBA 5 from BC pellets processed for direct testing using an ammonium chloride lysis and wash method. Sensitivity, specificity, negative predictive value (NPV) and positive predictive value (PPV) were calculated with 95% confidence intervals for binomial proportions. Results 65 samples were tested (30 clinical, 35 seeded). Seeded samples included 1 GES, 4 IMP, 6 KPC, 1 co-producing KPC and NDM, 9 OXA, 4 VIM, 5 NDM, and 5 non-CPO carbapenem-resistant organisms. βCARBA had a sensitivity, specificity, NPV and PPV of 100% (88.4% - 100%), 65.7% (47.8–80.9%), 100%, and 71.4% (61.3%–79.8%), respectively. CARBA 5 had a sensitivity, specificity, NPV and PPV of 90.0% (73.5%–97.9%), 100% (90.0%–100%), 92.1% (80.0%–97.2%), and 100%. When excluding GES, which is known not to be detected by CARBA 5, sensitivity and NPV increased to 93.1% (77.2%–99.2%) and 93.1% (78.0%–98.1%), respectively. False negatives for βCARBA occurred with 1 VIM-1 and IMP-14. Conclusion This study demonstrates that the detection of CPOs directly from positive BC can be accurately achieved. βCARBA had excellent sensitivity but suffered from poor specificity. CARBA 5 had good sensitivity and specificity but is unable to detect certain CPOs. Testing positive BC directly using βCARBA and/or CARBA 5 may be useful in rapidly detecting CPOs. Results of direct testing from the CARBA5 assay would quickly identify patients amenable to treatment with avibactam combination compounds. Disclosures All authors: No reported disclosures.

scholarly journals Comparative Evaluation of Four Phenotypic Methods for Detection of Class A and B Carbapenemase-Producing Enterobacteriaceae in China

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Menglan Zhou ◽  
Danchen Wang ◽  
Timothy Kudinha ◽  
Qiwen Yang ◽  
Shuying Yu ◽  
...  
Keyword(s):  
Predictive Value ◽  
Comparative Evaluation ◽  
Detection Sensitivity ◽  
Detection Methods ◽  
Content Type ◽  
Class A ◽  
Carbapenem Resistant ◽  
Class B ◽  
Phenotypic Methods ◽  
Sensitivity Specificity

ABSTRACT The objective of this study was to evaluate the performance of four phenotypic methods in the detection of carbapenemase-producing Enterobacteriaceae (CPE) in China. We evaluated the performance of four carbapenemase detection methods, the modified Hodge test (MHT), the Carba NP test, the meropenem hydrolysis assay (MHA) with 1- and 2-h incubation, and the modified carbapenem inactivation method (mCIM) with meropenem, imipenem, and ertapenem, on 342 carbapenem-resistant Enterobacteriaceae isolates (CRE) in China. PCR was used as the gold standard. The 2-h-incubation MHA performed the best in carbapenemase detection (overall sensitivity, specificity, positive predictive value, and negative predictive value all 100%). Second was the Carba NP test, with a sensitivity of 99.6%. The 1-h-incubation MHA performed poorly in Klebsiella pneumoniae carbapenemase (KPC) detection (sensitivity, 71.3%). For mCIM, the best performance was observed with the meropenem disk. The MHT exhibited the worst performance, with a specificity of 88.8%. All assays except 1-h-incubation MHA, which failed to identify 68 KPC-2s, had a sensitivity of >98% in the detection of 172 KPCs. Likewise, all assays had a sensitivity of >95% in the detection of 70 class B carbapenemases, except for MHT (82.9%). The 2-h-incubation MHA significantly improved the accuracy in CPE detection compared with that for 1-h incubation and performed the best in the detection of class A and B carbapenemases. Our findings suggest that the MHA is the most practical assay for carbapenemase detection. For those who cannot afford the associated equipment, both the Carba NP test and mCIM are good alternatives with regard to the practical requirements of time and cost.

scholarly journals Carbapenemase Detection among Carbapenem-Resistant Glucose-Nonfermenting Gram-Negative Bacilli

2017 ◽  
Vol 55 (9) ◽  
pp. 2858-2864 ◽  
Author(s):  
Patricia J. Simner ◽  
Belita N. A. Opene ◽  
Krizia K. Chambers ◽  
Matthew E. Naumann ◽  
Karen C. Carroll ◽  
...  
Keyword(s):  
Method Comparison ◽  
Comparison Study ◽  
Gram Negative ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Class D ◽  
Class B ◽  
Synergy Test ◽  
Method Comparison Study ◽  
Phenotypic Tests

ABSTRACT Accurate detection of carbapenemase-producing glucose-nonfermenting Gram-negative bacilli (CPNFs), including Pseudomonas aeruginosa and Acinetobacter baumannii , is necessary to prevent their dissemination within health care settings. We performed a method comparison study of 11 phenotypic carbapenemase detection assays to evaluate their accuracy for the detection of CPNFs. A total of 96 carbapenem-resistant glucose-nonfermenting isolates were included, of which 29% produced carbapenemases. All CPNFs were molecularly characterized to identify β-lactamase genes. A total of 86% of the carbapenemase-producing P. aeruginosa isolates produced class B carbapenemases. Several assays performed with a sensitivity of >90% for the detection of carbapenemase-producing P. aeruginosa , including all rapid chromogenic assays and the modified carbapenem inactivation method. Most included assays, with the exception of the Manual Blue Carba assay, the Modified Carba NP assay, the boronic acid synergy test, and the metallo-β-lactamase Etest, had specificities of >90% for detecting carbapenemase-producing P. aeruginosa . Class D carbapenemases were the most prevalent carbapenemases among the carbapenemase-producing A. baumannii strains, with 60% of the carbapenemase-producing A. baumannii isolates producing acquired OXA-type carbapenemases. Although several assays achieved >90% specificity in identifying carbapenemase-producing A. baumannii , no assays achieved a sensitivity of greater than 90%. Our findings suggest that the available phenotypic tests generally appear to have excellent sensitivity and specificity for detecting carbapenemase-producing P. aeruginosa isolates. However, further modifications to existing assays or novel assays may be necessary to accurately detect carbapenemase-producing A. baumannii .

scholarly journals Rapid, reliable, and cheap point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP (Cap-iLAMP)

2020 ◽  
Author(s):  
Lukas Bokelmann ◽  
Olaf Nickel ◽  
Tomislav Maricic ◽  
Svante Paabo ◽  
Matthias Meyer ◽  
...  
Keyword(s):  
Point Of Care ◽  
Rna Extraction ◽  
Lamp Assay ◽  
Cost Effective ◽  
Diagnostic Methods ◽  
Current Standard ◽  
Laboratory Equipment ◽  
Hybridization Capture ◽  
Level Of Automation ◽  
High Level

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can easily be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation, sophisticated laboratory equipment and trained personnel to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification). This method combines a hybridization capture-based RNA extraction of non-invasive gargle lavage samples to concentrate samples and remove inhibitors with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP does not result in false positives and single infected samples can be detected in a pool among 25 uninfected samples, thus reducing the technical cost per test to ~1 Euro per individual.

scholarly journals Carba NP as a simpler, rapid, cost-effective, and a more sensitive alternative to other phenotypic tests for detection of carbapenem resistance in routine diagnostic laboratories

2017 ◽  
Vol 9 (02) ◽  
pp. 100-103 ◽  
Author(s):  
Shivani Shinde ◽  
Rajarshi Gupta ◽  
Shweta S. Raut ◽  
Gita Nataraj ◽  
Preeti R. Mehta
Keyword(s):  
Sensitivity And Specificity ◽  
Carbapenem Resistance ◽  
Cost Effective ◽  
Confirmatory Test ◽  
Reference Test ◽  
Resistant Strains ◽  
Low Sensitivity ◽  
Sensitivity Specificity ◽  
Phenotypic Tests ◽  
Accuracy Rates

Abstract PURPOSE: Resistance to carbapenems due to carbapenemases has been increasingly noticed in Enterobacteriaceae. Clinical and Laboratory Standards Institute (CLSI) has recommended the latest Carba NP (CNP) test as a confirmatory test for carbapenemase production in Enterobacteriaceae. Low sensitivity of disk diffusion (DD) and modified Hodge test (MHT) may result in missing out of resistant strains which can adversely affect clinical management. The present study compares three phenotypic tests - CNP test, DD, and MHT for detection of carbapenemase production. MATERIALS AND METHODS: Four hundred consecutive, nonduplicate Enterobacteriaceae isolates were tested for carbapenem resistance using ertapenem disc (10 μg) by Kirby–Bauer DD method, MHT, and CNP. These tests were performed and interpreted as per the CLSI standards. CNP was considered to be the reference test for comparison. Sensitivity, specificity, and accuracy rates for ertapenem DD and MHT were calculated. RESULTS: One hundred and six out of 400 strains were positive by CNP test. Of the 294 CNP-negative strains, 28 were resistant by DD and 18 were resistant by MHT. Of the 106 CNP-positive strains, 82 were resistant and 16 were intermediate by DD while 76 were positive by MHT ertapenem DD had a sensitivity and specificity of 66.04% and 90.48%, respectively. Sensitivity and specificity of MHT were 54.72% and 93.88%, respectively. There was considerable discordance between all the three tests. CONCLUSION: As a rapid, simple, and cost-effective test with a greater capability greater to detect carbapenemase producers, CNP can be implemented in routine diagnostic laboratories, thereby benefiting patient care and antimicrobial stewardship.

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