Rapid and simple identification of carbapenemase genes, bla NDM, bla OXA-48, bla VIM, bla IMP-14 and bla KPC groups, in Gram-negative bacilli by in-house loop-mediated isothermal amplification with hydroxynaphthol blue dye

Abstract

Carbapenem-resistant Enterobacteriaceae isolates by carbapenemase production are being reported globally with increasing frequency, leading to limited therapeutic options. We therefore developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB) for rapid confirmation of bla , bla, bla , bla and bla groups. Sixty-two Enterobacteriaceae and Pseudomonas spp. isolates carrying various carbapenemase genes (28 bla, 9 bla , 2 bla, 1 bla , 1 bla, 1 bla , 1 bla, 4 bla , 1 bla, 1 bla & bla , 7 bla, 3 bla and 3 bla) and 37 non-carbapenemase-producing Enterobacteriaceae isolates as confirmed by the PCR methods were included. Bacterial DNA was extracted by a simple boiling method. The LAMP-HNB method for each target gene was carried out using a set of six primers under isothermal condition at 65 °C in an ordinary water bath within 60 min and visual measurement of reaction by the change from violet to sky blue. This method had high efficiency (100% sensitivity and specificity) for identifying the bla , bla, bla , bla and bla groups compared with the PCR method. The HNB is easy to prepare, inexpensive and provides reliable results. Therefore, this method could be used as a confirmatory carbapenemase test in routine laboratory or for epidemiological purposes.

Profile of Enterobacteria Resistant to Beta-Lactams

“…A literature review was carried out to determine the potential of resistance by molecular methodology found in this study, which has genes described in the literature that encode the β-lactamase enzyme. The result of the literary survey is shown in Table 3 [ 3 , 6 , 16 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 ...…”

NDM Metallo-β-Lactamases and Their Bacterial Producers in Health Care Settings

“…In addition, LAMP does not require gel electrophoresis, making it convenient in clinical laboratories. A commercially available LAMP system called Eazyplex (AmplexDiagnostics) has been evaluated for detecting carbapenemase genes in Acinetobacter (226,227), Enterobacteriaceae (216,227,228), and P. aeruginosa (216,227,228), with 100% sensitivity and specificity for bla NDM . LAMP has been evaluated only for detecting bla NDM-1 , and since only 4 or 6 primers can be used in LAMP, it may not be able to detect all bla NDM variants due to the possibility of nucleotide mutations occurring in primer binding regions.…”

Carbapenemases in Enterobacteriaceae: Detection and Antimicrobial Therapy

“…A PCR-based method in a cartridge format developed to detect CPE in rectal swabs, which is run on the GeneXpert platform, displayed high sensitivity (96.6%) and specificity (98.6%) within a short time (32–48 min) (Tato et al, 2016). Srisrattakarn et al (2017) developed a loop-mediated isothermal amplification method with hydroxynaphthol blue dye (LAMP-HNB), which was highly efficient (100% sensitivity and specificity). In 2018, the microfluidic chip technology which allows the rapid detection of pathogens and their resistance genes (Kim et al, 2017) was used to detect carbapenem-resistance genes, with high sensitivity and specificity (both >90.0%), and fully met the requirements for clinical diagnoses (Zhang G. et al, 2018).…”