A rapid low-cost real-time PCR for the detection of Klebsiella pneumonia carbapenemase genes.

Department of Laboratory Medicine, Beijing Tongren Hospital, Capital Medical University, No1, Dongjiaominxiang Road, Dongcheng District, Beijing, 100730, China.
Annals of Clinical Microbiology and Antimicrobials (Impact Factor: 2.19). 04/2012; 11(1):9. DOI: 10.1186/1476-0711-11-9
Source: PubMed


Klebsiella pneumonia carbapenemases (KPCs) are able to hydrolyze the carbapenems, which cause many bacteria resistance to multiple classes of antibiotics, so the rapid dissemination of KPCs is worrisome. Laboratory identification of KPCs-harboring clinical isolates would be a key to limit the spread of the bacteria. This study would evaluate a rapid low-cost real-time PCR assay to detect KPCs.
Real-time PCR assay based on SYBR GreenIwas designed to amplify a 106 bp product of the blaKPC gene from the 159 clinical Gram-negative isolates resistant to several classes of -lactam antibiotics through antimicrobial susceptibility testing. We confirmed the results of real-time PCR assay by the conventional PCR-sequencing. At the same time, KPCs of these clinical isolates were detected by the modified Hodge test (MHT). Then we compared the results of real-time PCR assay with those of MHT from the sensitivity and specificity. Moreover, we evaluated the sensitivity of the real-time PCR assay.
The sensitivity and specificity of the results of the real-time PCR assay compared with those of MHT was 29/29(100%) and 130/130(100%), respectively. The results of the real-time PCR and the MHT were strongly consistent (Exact Sig. (2-tailed) =1. 000; McNemar test). The real-time PCR detection limit was about 0.8 cfu using clinical isolates.
The real-time PCR assay could rapidly and accurately detect KPCs -harboring strains with high analytical sensitivity and specificity.

  • Source
    • "Delay in microbiology culture diagnostics for pneumonia-causing pathogens refractory to cultivation is a clinical problem which often results in empirical, sometimes inefficient, broad-spectrum antibiotic therapy until final microbiology results become available. Identification by molecular techniques might accelerate appropriate treatment of both ventilatorassociated pneumonia (VAP) and community-acquired pneumonia (CAP) [1] [2] which is of special importance for patients treated in intensive care untis (ICU) [3] . We investigated the Unyvero™ prototype with cartridge P50 covering 17 pneumonia-causing pathogens, among them the fastidious pneumonia-causing pathogens Legionella pneumophila [4] and Chlamydia pneumoniae [5] [6] , which are clinically important in immunocompromised patients [4] [7] . "

    Full-text · Article · Dec 2015
    • "al - time PCR to detect KPC from 120 stool and 128 nasal swabs samples with an LOD of 80 CFU ml À1 , and attained a 100% and 99% sensitivity and specificity respectively ( Table 1 ) ( Wang et al . 2012a , b ) . This assay could not differentiate between various KPC variants , although it was fast ( <2 h ) . Subsequent studies by the same authors ( Wang et al . 2012b ) with 159 clinical isolates ( Kl . pneumoniae , 9 K . oxytoca , E . coli , Enterobacter cloacae , Serratia marces - cens , Proteus mirabilis , Ac . baumannii and Ps . aeruginosa ) were , however , 100% sensitive and specific , with an LOD of 0Á8 CFU ml À1 and a turnaround time of ≤4 h . The SYBR Green is a cheaper and more sensitive ( "
    [Show abstract] [Hide abstract]
    ABSTRACT: Research articles describing carbapenemases and their genetic environments in Gram-negative bacteria were reviewed to determine the molecular epidemiology of carbapenemases in Africa. The emergence of resistance to the carbapenems, the last resort antibiotic for difficult to treat bacterial infections, affords clinicians few therapeutic options, with a resulting increase in morbidities, mortalities, and healthcare costs. However, the molecular epidemiology of carbapenemases throughout Africa is less described. Research articles and conference proceedings describing the carbapenemase genetic environment and molecular epidemiology in Africa were retrieved from Google Scholar, Scifinder, Pubmed, Web of Science, and Science Direct databases. Predominant carbapenemase genes so far described in Africa include the blaOXA-48 type, blaIMP, blaVIM, and blaNDM in Acinetobacter baumannii, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter spp., and Escherichia coli carried on various plasmid types and sizes, transposons, and integrons. Consequently, the true molecular epidemiology of carbapenemases and their genetic environment in Africa is still unknown. Class D and class B carbapenemases, mainly prevalent in A. baumannii, K. pneumoniae, E. cloacae, Citrobacter spp., and E. coli were the commonest carbapenemases. Carbapenemases are mainly reported in North and South Africa as under-resourced laboratories, lack of awareness and funding preclude the detection and reporting of carbapenemase-mediated resistance.
    No preview · Article · Jul 2015 · Microbial drug resistance (Larchmont, N.Y.)
  • [Show abstract] [Hide abstract]
    ABSTRACT: This review describes recent findings related to molecular-based methods of potential application in the diagnosis of bacterial hospital-acquired pneumonia (HAP)/ventilator-associated pneumonia (VAP). It focuses on methods capable of providing organism identification and keys of bacterial resistance necessary in clinical and epidemiological management of patients and on their ability to provide quantitative results. Significant advances have been made in recent years in the field of molecular diagnosis of bacterial pathogens. Real-time PCR, hybridization and mass spectrometry-based platforms dominate the scene. Some of the new technologies provide high sensitivity and specificity in the identification of single or multiple pathogens or a combination of etiological identification and antimicrobial resistance determinants in Staphylococcus aureus, nonfermenter Gram-negative bacilli and Enterobacteriaceae that are often associated with the cause of bacterial HAP/VAP in the late onset of the disease. In diagnosis made directly from clinical specimens and quantification of targets for bacterial load, some of them are promising. Despite some limitations, current molecular diagnostic methods have a great potential to include bacterial targets useful in the identification of microorganisms and antimicrobial resistance, to analyze directly unprocessed samples and to obtain quantitative results in bacterial HAP/VAP, an entity of complex microbiological diagnosis due to the features of the pathogens commonly implicated.
    No preview · Article · Aug 2012 · Current opinion in critical care
Show more