Evaluation of loop-mediated isothermal amplification assays for rapid detection of blaKPC producing Serratia spp. in clinical specimens: A prospective diagnostic accuracy study.
ABSTRACT: The prevalence of carbapenem-resistant Serratia spp. is increasing owing to the propagation of ? lactamase Klebsiella pneumoniae carbapenemase (blaKPC) and it has become one of the major global health concerns. As effective therapies for such resistant pathogens are limited, there is a great need for the rapid and sensitive characterization of the pathogen. In the present study, a loop-mediated isothermal amplification (LAMP) method for the rapid detection of Serratia spp. with blaKPC in pure cultures and clinical specimens was developed. A calcein indicator and real-time turbidity recording system were used to assess the LAMP reaction. The LAMP assay was compared with conventional PCR and real-time PCR kits for the target pathogen. The desired amplification was achieved using selected primers and detection was possible using both the calcein indicator method and the real-time turbity recording system at 65?C for 60 min. The sensitivity of the detection system for blaKPC-producing Serratia spp. reached a detection limit of 3.92 pg/µl DNA, which was 10 times more sensitive than conventional PCR. Specificity testing indicated that the primers were highly specific. Compared with conventional culture methods and real-time PCR, the LAMP assay was more sensitive, easier for laboratory staff to master and less influenced by the clinical specimen matrix. In conclusion, a LAMP assay for blaKPC-producing Serratia spp. that permitted rapid, sensitive and economical detection for this pathogen was successfully developed. Comparisons with alternative methods indicated that the LAMP assay was more feasible in a clinical setting.
Project description:Klebsiella pneumoniae is a wide-spread nosocomial pathogen. A rapid and sensitive molecular method for the detection of K. pneumoniae in clinical samples is needed to guide therapeutic treatment. In this study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of capsular polysaccharide synthesis regulating gene rcsA from K. pneumoniaein clinical samples by using two methods including real-time turbidity monitoring and fluorescence detection to assess the reaction. Then dissemination of K. pneumoniae strains was investigated from ICU patients in three top hospitals in Beijing, China. The results showed that the detection limit of the LAMP method was 0.115 pg/μl DNA within 60 min under isothermal conditions (61°C), a 100-fold increase in sensitivity compared with conventional PCR. All 30 non- K. pneumoniae strains tested were negative for LAMP detection, indicating the high specificity of the LAMP reaction. To evaluate the application of the LAMP assay to clinical diagnosis, of 110 clinical sputum samples collected from ICU patients with clinically suspected multi-resistant infections in China, a total of 32 K. pneumoniae isolates were identified for LAMP-based surveillance of rcsA. All isolates belonged to nine different K. pneumoniae multilocus sequence typing (MLST) groups. Strikingly, of the 32 K. pneumoniae strains, 18 contained the Klebsiella pneumoniae Carbapenemase (KPC)-encoding gene bla KPC-2 and had high resistance to β-lactam antibiotics. Moreover, K. pneumoniae WJ-64 was discovered to contain bla KPC-2 and bla NDM-1genes simultaneously in the isolate. Our data showed the high prevalence of bla KPC-2 among K. pneumoniae and co-occurrence of many resistant genes in the clinical strains signal a rapid and continuing evolution of K. pneumoniae. In conclusion, we have developed a rapid and sensitive visual K. pneumoniae detection LAMP assay, which could be a useful tool for clinical screening, on-site diagnosis and primary quarantine purposes.
Project description:New Delhi metallo-?-lactamase 1 (NDM-1), which is associated with resistance to carbapenem, was first reported in 2008. A sensitive and rapid molecular assay to detect the plasmid bla(NDM-1) in clinical isolates is needed to control its spread. We describe a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of bla(NDM-1) from pure culture and sputum, urine, and fecal samples. Eight sets of primers were designed to recognize six or eight distinct sequences on target bla(NDM-1), and one set was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for bla(NDM-1) detection were determined. The sensitivity of the LAMP assay for bla(NDM-1) detection in sputum, urine, and fecal samples was also tested. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 70 min at an isothermal temperature of 65°C. The sensitivity of LAMP, with a detection limit of 10.70 pg/?l DNA, was 100-fold greater than that of PCR. Thirteen infection bacterial strains without bla(NDM-1) were selected for testing of specificity, and the results of the amplification were negative, which showed that the primers had good levels of specificity. The LAMP method reported here is demonstrated to be a potentially valuable means for the detection of bla(NDM-1) and rapid clinical diagnosis, being fast, simple, and low in cost.
Project description:Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants (bla(KPC)) have been reported, with KPC-2 (bla(KPC-2)) and KPC-3 (bla(KPC-3)) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of bla(KPC) variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla(KPC) variants (bla(KPC-2) to bla(KPC-11)). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla(KPC) variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with bla(KPC-3) and bla(KPC-2) identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel bla(KPC) variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.
Project description:We evaluated the performance of the Verigene Gram-Negative Blood Culture Nucleic Acid Test (BC-GN; Nanosphere, Northbrook, IL, USA), an automated multiplex assay for rapid identification of positive blood cultures caused by 9 Gram-negative bacteria (GNB) and for detection of 9 genes associated with ?-lactam resistance. The BC-GN assay can be performed directly from positive blood cultures with 5 minutes of hands-on and 2 hours of run time per sample. A total of 397 GNB positive blood cultures were analyzed using the BC-GN assay. Of the 397 samples, 295 were simulated samples prepared by inoculating GNB into blood culture bottles, and the remaining were clinical samples from 102 patients with positive blood cultures. Aliquots of the positive blood cultures were tested by the BC-GN assay. The results of bacterial identification between the BC-GN assay and standard laboratory methods were as follows: Acinetobacter spp. (39 isolates for the BC-GN assay/39 for the standard methods), Citrobacter spp. (7/7), Escherichia coli (87/87), Klebsiella oxytoca (13/13), and Proteus spp. (11/11); Enterobacter spp. (29/30); Klebsiella pneumoniae (62/72); Pseudomonas aeruginosa (124/125); and Serratia marcescens (18/21); respectively. From the 102 clinical samples, 104 bacterial species were identified with the BC-GN assay, whereas 110 were identified with the standard methods. The BC-GN assay also detected all ?-lactam resistance genes tested (233 genes), including 54 bla(CTX-M), 119 bla(IMP), 8 bla(KPC), 16 bla(NDM), 24 bla(OXA-23), 1 bla(OXA-24/40), 1 bla(OXA-48), 4 bla(OXA-58), and 6 blaVIM. The data shows that the BC-GN assay provides rapid detection of GNB and ?-lactam resistance genes in positive blood cultures and has the potential to contributing to optimal patient management by earlier detection of major antimicrobial resistance genes.
Project description:We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of bla(KPC) genes. The TaqMan real-time PCR assay amplified a 399-bp product from the bla(KPC) gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum beta-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the bla(KPC) PCR assay was used to confirm the presence of bla(KPC) genes in any meropenem-resistant Klebsiella spp. The PCR assay detected bla(KPC) in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of bla(KPC) in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two bla(KPC) gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with bla(KPC-2) and bla(KPC-3), respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.
Project description:Rapid diagnosis of infections caused by carbapenem-resistant <i>Enterobacteriaceae</i> (CRE) is crucial for proper treatment and infection control. The Xpert Carba-R assay is a qualitative multiplex real-time PCR method that qualitatively detects and differentiates five common carbapenemase genes (<i>bla</i> <sub>KPC</sub>, <i>bla</i> <sub>NDM</sub>, <i>bla</i> <sub>VIM</sub>, <i>bla</i> <sub>OXA-48</sub>, and <i>bla</i> <sub>IMP</sub>) directly from rectal swabs or purified colonies within approximately 1 h. We performed a multicenter evaluation of the investigational use of the Carba-R assay for detection and differentiation of carbapenemase genes from sputum specimens in patients with a clinical diagnosis of pneumonia. The intra- and interassay coefficients of variation values for the Carba-R assay were 0.2% to 2.0% and 1.4% to 2.3%, respectively. A total of 301 sputum specimens were collected and tested. Compared to bacterial culture followed by PCR identification of resistance genes from colonies, the Carba-R assay reduced turnaround time from 56 to 84 h to less than 2 h. Carbapenemase genes were detected by the Carba-R assay in <i>Klebsiella pneumoniae</i> (<i>n</i> = 236), <i>Escherichia coli</i> (<i>n</i> = 22), <i>Enterobacter cloacae</i> (<i>n</i> = 23), <i>Klebsiella oxytoca</i> (<i>n</i> = 8), <i>Serratia marcescens</i> (<i>n</i> = 6), <i>Citrobacter freundii</i> (<i>n</i> = 4), and <i>Klebsiella aerogenes</i> (<i>n</i> = 2). The Carba-R assay detected 112 <i>bla</i> <sub>KPC</sub> (33.5%), 70 <i>bla</i> <sub>NDM</sub> (21.0%), 8 <i>bla</i> <sub>IMP</sub> (2.4%), and 2 <i>bla</i> <sub>VIM</sub> (0.6%) genes, with positive percent agreement, negative percent agreement, and concordance rates of 92.9%, 86.7%, and 88.3%, respectively, for the dominant <i>bla</i> <sub>KPC</sub> and 85.0%, 87.8%, and 87.4%, respectively, for the <i>bla</i> <sub>NDM</sub> genes. Neither method detected the <i>bla</i> <sub>OXA-48</sub> carbapenemase gene. The convenient, rapid, and simple characteristics of the Xpert Carba-R assay make it a potential tool for CRE detection and identification directly in sputum specimens.
Project description:Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) ?-lactamase-producing strains are of growing concern. Several genotypes of the GES ?-lactamase gene (bla GES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of bla GES and another LAMP method to discriminate carbapenemase genotypes of bla GES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting bla GES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting bla GES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected bla GES with high sensitivity in all DNA-spiked samples; PCR did not detect bla GES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing bla GES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two bla GES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES ?-lactamase gene detection assay using the LAMP method. Our new assays effectively detect bla GES and critical unique mutations.
Project description:<h4>Purpose</h4>Carbapenemase-producing <i>Enterobacteriaceae</i> (CPE) infection constitutes a public health threat. Timely and efficient diagnosis is of paramount importance for prompt and effective therapy. In order to quickly and comprehensively detect the five major families of carbapenemases (<i>bla</i> <sub>KPC</sub>, <i>bla</i> <sub>NDM</sub>, <i>bla</i> <sub>VIM</sub>, <i>bla</i> <sub>IMP</sub>, and <i>bla</i> <sub>OXA-48-like</sub>), colorimetric loop-mediated isothermal amplification (LAMP) was employed.<h4>Materials and methods</h4>Five sets of LAMP primers were designed, each of which can, respectively, amplify all the carbapenemase subtypes described in this work. Twenty whole genome sequencing-verified-"standard strains", including 1 <i>bla</i> <sub>NDM-1</sub>, 1 <i>bla</i> <sub>NDM-5</sub>, 1 <i>bla</i> <sub>NDM-6</sub>, 1 <i>bla</i> <sub>NDM-7</sub>, 2 <i>bla</i> <sub>IMP-4</sub>, 1 <i>bla</i> <sub>IMP-8</sub>, 2 <i>bla</i> <sub>KPC-2</sub>, 1 <i>bla</i> <sub>KPC-3</sub>, 1 <i>bla</i> <sub>KPC-4</sub>, 1 <i>bla</i> <sub>KPC-5</sub>, 1 <i>bla</i> <sub>KPC-6</sub>, 1 <i>bla</i> <sub>KPC-7</sub>, 1 <i>bla</i> <sub>OXA-48</sub> and 1 <i>bla</i> <sub>OXA-181</sub> carrier, and 1 <i>bla</i> <sub>VIM</sub> and <i>bla</i> <sub>OXA-244</sub>, 1 <i>bla</i> <sub>KPC-2</sub> and <i>bla</i> <sub>IMP-4</sub>, 1 <i>bla</i> <sub>KPC-2</sub> and <i>bla</i> <sub>VIM-1</sub> and 1 <i>bla</i> <sub>KPC-2</sub> and <i>bla</i> <sub>NDM-1</sub>-co-carriers, were used to establish a 25-microliter visual LAMP reaction system (kept at 65°C for 30 minutes in water bath). Color change from bright pink to yellow indicated positive amplification. In addition, 126 pre-verified clinical carbapenem-resistant <i>Enterobacteriaceae</i> (CRE) isolates, including 65 CPE (23 <i>bla</i> <sub>NDM</sub>, 2 <i>bla</i> <sub>OXA-48-like</sub>, 1 <i>bla</i> <sub>KPC</sub> and <i>bla</i> <sub>VIM</sub>, 2 <i>bla</i> <sub>IMP</sub>, and 37 <i>bla</i> <sub>KPC</sub> carriers) and 61 non-CPE, were also detected.<h4>Results</h4>With the lowest detection limit of 10 colony forming units (CFU) per reaction for LAMP and 10<sup>3</sup> CFU per reaction for PCR, the LAMP system demonstrated dramatically higher sensitivity while retaining the same specificity. Furthermore, we demonstrated concordant results between the two methods for the 126 clinical isolates.<h4>Conclusion</h4>Therefore, LAMP could be used for rapid identification of the five major carbapenemase gene families in routine clinical laboratories.
Project description:Intrinsic ?-lactam resistance in Stenotrophomonas maltophilia is caused by bla L1 and/or bla L2, a kind of metallo-?-lactamase with a broad substrate spectrum including carbapenems. A rapid and sensitive molecular method for the detection of bla L1 in clinical samples is needed to guide therapeutic treatment. In present study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of bla L1 in clinical samples by using two methods including a chromogenic method using calcein/Mn(2+) complex and the real-time turbidity monitoring to assess the reaction. Then dissemination of L1-producing S. maltophilia was investigated from ICU patients in three top hospital in Beijing, China. The results showed that both methods detected the target DNA within 60 min under isothermal conditions (65°C). The detection limit of LAMP was 3.79 pg/?l DNA, and its sensitivity 100-fold greater than that of conventional PCR. All 21 test strains except for S. maltophilia were negative for bla L1, indicative of the high-specificity of the primers for the bla L1. A total of 22 L1-positive isolates were identified for LAMP-based surveillance of bla L1 from 105 ICU patients with clinically suspected multi-resistant infections. The sequences of these bla L1 genes were conservative with only a few sites mutated, and the strains had highly resistant to ?-lactam antibiotics. The MLST recovered that 22 strains belonged to seven different S. maltophilia sequence types (STs). Furthermore, co-occurrence of bla L1 and bla L2 genes were detected in all of isolates. Strikingly, S. maltophilia DCPS-01 was recovered to contain bla L1, bla L2, and bla NDM-1 genes, possessing an ability to hydrolyse all ?-lactams antibiotics. Our data showed the diversity types of S. maltophilia carrying bla L1 and co-occurrence of many resistant genes in the clinical strains signal an ongoing and fast evolution of S. maltophilia resulting from their wide spread in the respiratory infections, and therefore will be difficult to control.
Project description:Zika virus (ZIKV) is a mosquito-borne flavivirus, which is a pathogen affecting humans in Africa, Asia, and America. It is necessary to detect ZIKV with a rapid and sensitive molecular method to guide timely treatment. In this study, a loop-mediated isothermal amplification (LAMP) assay was described, which is an attractive option as a fast, sensitive, and specific method for ZIKV detection using the NS5 protein coding region and the envelope protein (EP) coding region as target sequences. Two different techniques, a calcein/Mn2+ complex chromogenic method and real-time turbidity monitoring, were employed. The specificity and sensitivity of the LAMP assay were determined. The assay's detection limit was 0.5?×?10-9 pmol/µl DNA for NS5 protein coding region and 1.12?×?10-11 pmol/µl DNA for E coding region, respectively, which is a 100-fold increase in sensitivity compared with real-time reverse transcription-polymerase chain reaction (RT-PCR) and conventional PCR. All 12 non-ZIKA respiratory pathogens tested were negative for LAMP detection, indicating the high specificity of the primers for ZIKV. In conclusion, a visual detection LAMP assay was developed, which could be a useful tool for primary quarantine purposes and clinical screening, especially in situations where resources are poor and in point-of-care tests.