Diagnostic Use of PCR in Carbapenamase-producing Enterobacteriaceae (CPE): An Improved Method to Overcome the presence of inhibitors for DNA Extraction from Blood Cultures
- Author:
Zeti Norfidiyati Salmuna
1
Author Information
1. Medical Microbiology and Parasitology, School of Medical Sciences, University Sains Malaysia Health Campus, 16150, Kubang Kerian, Kelantan, Malaysia
- Collective Name:Zeti Norfidiyati Salmuna; Murnihayati Hassan; Habsah Hasan; Zakuan Zainy Deris
- Publication Type:Journal Article
- Keywords:
Carbapenamase-producing Enterobacteriaceae;
PCR inhibitors;
Sensitivity;
Specificity
- MeSH:
PCR inhibitors
- From:Malaysian Journal of Medicine and Health Sciences
2019;15(1):1-4
- CountryMalaysia
- Language:English
-
Abstract:
Introduction: Carpanenamase-producing Enterobacteriaceae (CPE) has emerged as a threat to hospitalized patients. Phenotypic test such as Modified hodge test was less sensitive and specific especially to detect blaNDM-1 which is the most predominant genotype in this region. Nucleic acid amplification technology offers improved specificity and sensitivity. Failed amplification due to the presence of inhibitors is a limitation. In this study, we tried to use previous method described by Villumseen et al with some modification using another DNA extraction kit. Methods: Ten mls of sterile whole blood taken from nearly expired blood bag from blood bank was spiked with 200 µl of 0.5mcFarland bacterial suspension from thirty-six confirmed isolates of blaNDM-1 carbapenamase-producing Klebsiella pneumoniae in an aerobic Bactec Plus and incubated until the growth was detected. The blood specimen was subjected to DNA extraction method using Macherey-Nachel, Nucleospin® Blood QuickPure followed with multiplex PCR. Results: Out of the 36 isolates, 12 isolates revealed blaNDM-1 , 9 isolates revealed blaNDM-1 and blaOXA-48, 7 isolates revealed blaNDM-1, blaVIM and blaKPC genotypes that were amplified at cycle threshold of less than 30. Another 8 isolates could not pick up any genotypes possibly due to pipetting error as all the internal control were amplified. Eight true negative gram negative isolates underwent same procedure and none amplified at a cycle threshold less than 30. Conclusion: This modified method was proved to give a high yield of CPE genotypes with the cycle threshold was set at less than or equal to 30 and able to overcome the presence of PCR inhibitors
- Full text:11.2019my0340.pdf
