The Application Value Of MALDI-TOF MS In Rapid Detection Of Carbapenem-resistant Enterobacterales

Objectives1.To analyze the prevalence of carbapenem-resistant Enterobacterales（CRE）strains isolated from December 2018 to October 2020 in the Affiliated Hospital of Chengde Medical University（our hospital）.2.To evaluate the application of enzymatic hydrolysis assay in the rapid detection of carbapenemase-producing carbapenem-resistant Enterobacterales（CP-CRE）by a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry（MALDI-TOF MS）method.3.To compare the diagnostic performance of MALDI-TOF MS with that of the PCR,the enzymatic immunochromatography assay,and the modified carbapenem inactivation method of for detecting carbapenemase.Methods1.52 CRE strains and 36 carbapenem-sensitive Enterobacterales（CSE）strains were collected from a variety of clinical specimens in the Affiliated Hospital of Chengde Medical University,and 40 CRE strains were collected from in the Chengde Central Hospital,during December 2018 and October2020.These strains were identified by Vitek 2.0 and VITEK MS（VITEK Mass Spectrometry）.The drug susceptibility results of Vitek 2.0 were reviewed by the Kirby-Bauer test.To analyze the prevalence of CRE strains in our hospital.2.Taking 82 Enterobacterales（52 CREs and 30 CSEs）in our hospital as research objects of retrospective study,using the PCR and the enzymatic immunochromatography assay to detect the bla_KPC、bla_NDM、bla_VIM、bla_IMP and bla_OXA-48 carbapenemase gene,and the modified carbapenem inactivation method（m CIM and e CIM）to detect the carbapenemase phenotype.3.Preliminary experiments for enzymatic hydrolysis:incubate 0.5 McF bacterial suspension with imipenem solutions of different concentrations（0.5mg/mL,1.0 mg/mL,2.0 mg/mL,4.0 mg/mL）at 35°C for 30 min,60 min,120min,240 min,and the characteristic peaks of imipenem were observed by VITEK MS.Select the optimal conditions of bacterial suspension concentration,imipenem concentration and incubation time.To help formally test the establishment of MALDI-TOF MS detection standard for CP-CRE.4.First,using PCR as the reference method,to study whether 82Enterobacterales（52 CREs and 30 CSEs）in our hospital produce carbapenemase,bacteria were suspended in imipenem solution,and to assess whether the tested strain produces carbapenemase by VITEK MS.Define log RQ=log peak intensity of imipenem hydrolysate/peak intensity of imipenem.Draw receiver operating characteristic curve（ROC curve）,and use ROC curve to compare the hydrolysis rate of imipenem after bacteria co-incubation with imipenem in different times（5 min,10 min,20 min,30 min）.To calculate the Cutoff value by the largest value of the area under the curve（AUC）.When the log RQ value≥cutoff value,the bacterium produces carbapenemase,which is CP-CRE.On the contrary,the bacterium does not produce carbapenemase.Finally,a prospective study was conducted to verify the detection effect of this method with 40 CREs isolated from Chengde Central Hospital and 6 CSEs from our hospital during the same period.5.To compare the diagnostic performance among MALDI-TOF MS,the PCR,the enzymatic immunochromatography assay,and modified carbapenem inactivation method through the Kappa test.Results1.The prevalence of CRE strains in our hospitalThe identification results of Vitek 2.0 and VITEK MS are consistent.The 52 CRE strains in our hospital consisted of 37 Klebsiella pneumoniae,9Escherichia coli,4 Enterobacter cloacae,2 Klebsiella oxytoca.All stored CRE strains were resistant to imipenem and/or meropenem.The CREs in our hospital mainly isolated from sputum（33.9%）,urine（25%）and blood（23.1%）.Among them,CREs in our hospital are mainly distributed in the Department of Geriatrics（30.8%）,the Department of Intensive Medicine（23.1%）and the Department of Hematology（15.4%）.2.Carbapenemase test resultsThe PCR and enzymatic immunochromatography assay showed the same results for detecting carbapenemase.In the 52 CREs from our hospital,100%of them were carbapenemase gene positive.The resistance determinants present in these isolates were 61.5%（32/52）bla_KPC,34.6%（18/52）bla_NDM,1.9%（1/52）bla_IMP,and1.9%（1/52）were both positive for bla_KPC and bla_NDM;30 strains of CSE in our hospital did not detect carbapenemase resistance gene;Moreover,86.5%（32/37）of carbapenem-resistant Klebsiella pneumoniae（CR-KPN）in our hospital are bla_KPC positive strains,8.1%（3/37）are bla_NDMpositive strains,2.7%（1/37）are both bla_KPC and bla_NDM positive strains,2.7%（1/37）are bla_IMP positive strains;9 carbapenem-resistant Escherichia coli（CR-ECO）are all bla_NDM positive strains（9/9）.3.Pre-experimental results of enzymatic hydrolysisWhen 0.5 McF bacterial suspension was suspended in 0.5 mg/ml imipenem solution,incubated 30 min,the characteristic peak of imipenem（300±0.55 m/z）basically disappeared.After extending the incubation time,the test results did not change.After increasing the drug concentration,the imipenem peak still exists.The optimal conditions for enzymatic hydrolysis are 0.5 McF bacterial suspension and 0.5 mg/mL imipenem incubation at 30min.4.Selection and verification of the best incubation time and Cutoff value for enzymatic hydrolysisThe mean log RQ value was 0.996±0.184 of 52 CRE strains,while the log RQ value of 30 CSE strains was-1.419±0.187.The ROC curve after 30min incubation had the largest AUC,which was 1.000（p<0.001）.Thus,the Cutoff value of log RQ was set at-0.678 with sensitivity of 100%and specificity of 100%.The 52 CRE strains are all CP-CRE,while the 30 CSE strains are noncarbapenemase producers.The enzymatic hydrolysis method showed that the 40 CREs of Chengde Central Hospital were all CP-CRE,and the 6 CSEs of our hospital did not produce carbapenemase..Prospective studies have verified that the enzymatic hydrolysis assay was extremely consistent with the PCR results（Kappa=1.0）.5.Comparison of various carbapenemase detection methodsBased on comparison,the sensitivity and specificity of MALDI-TOF MS,PCR and enzymatic immunochromatography assay are 100%,while the sensitivity and specificity of modified carbapenem inactivation method are96.0%and 93.8%.Conclusions1.The CRE strains in our hospital are mainly Klebsiella pneumoniae and Escherichia coli,both can produce carbapenemase,of which bla_KPC and bla_NDM carbapenemase are the dominant ones.Simultaneously,The CRE are mainly distributed in the Department of Geriatrics,Critical Care Medicine and Hematology.The above departments should pay attention to more reasonable experience in medication.2.When the enzymatic hydrolysis assay is incubated for 30 min,and the log RQ value is greater than or equal to-0.678,the tested strain is CP-CRE.The sensitivity and specificity are both 100%.This easy-to-perform assay is time-saving,cost-efficient,and highly reliable and might be used to detect CP-CRE.3.Among multifarious carbapenem enzyme detection methods,the MALDI-TOF MS based on enzymatic hydrolysis has 100%sensitivity and 100%specificity,which can detect carbapenemase more quickly,more accurately and less costly,it is beneficial to determine the source of infection earlier,guide clinical medication more precise and block the spread of CRE timelier.