Detection of metallo-β-lactamase genes in clinical specimens by a commercial multiplex PCR system

https://doi.org/10.1016/j.mimet.2010.08.014Get rights and content

Abstract

hyplex®-MBL ID Multiplex PCR-ELISA, a novel method for identifying metallo-β-lactamase genes directly in clinical specimens, was evaluated using a consecutive collection of 326 samples from three hospitals in Greece characterized by high prevalence of VIM producers. The method exhibited high sensitivity (98.0%) and specificity (98.6%) and was proven reliable in detecting blaVIM genes in blood, urine, pus, and sputum samples that, as confirmed by conventional methods, contained various VIM-producing species. Future multicenter studies should be considered for the thorough evaluation of this method and its potential diagnostic utility.

Introduction

Dissemination of Gram-negative bacteria producing metallo-β-lactamases (MβLs) is of great concern. A variety of acquired MβLs, including IMP, VIM, SPM, GIM, and SIM have been described in species of clinical importance (Queenan and Bush, 2007). MβLs exhibit wide hydrolysis spectra affecting virtually all β-lactams including carbapenems. Moreover, MβL-encoding genes are commonly carried by multi-resistant integrons. Therapeutic choices in the respective infections are thus limited. MβLs, mainly of the VIM and IMP types, have been widely diffused among non-fermenters such as Pseudomonas aeruginosa and Acinetobacter baumannii and enterobacteria (mainly Klebsiella pneumoniae) (Walsh et al., 2005, Queenan and Bush, 2007) and in some countries, as in the case of Greece, have reached endemic proportions (Psichogiou et al., 2008). The early and accurate detection of MβL producers would facilitate administration of the appropriate therapy and the implementation of infection control measures, especially in high prevalence settings. Novel detection approaches utilizing molecular methods have been proposed for the timely detection of MβL producers (Cornaglia et al., 2007, Miriagou et al., 2010, Walsh et al., 2005). In this study, a new diagnostic method for the direct detection of MβL genes of the VIM and IMP types in clinical specimens, the hyplex® MBL ID Multiplex PCR-ELISA (AmplexDiagnostics, Gars, Germany), was evaluated. This system involves amplification of blaVIM/blaIMP bacterial DNA by multiplex PCR, reverse hybridisation of the PCR products to oligonucleotide probes specific for blaIMP (IMP-1 to IMP-22) and blaVIM (VIM-1 to VIM-13) genes that have been immobilized on polystyrene ELISA-plates, and detection of the hybridisation complexes by a peroxidase conjugated antibody.

Section snippets

Clinical samples

A total of 326 non-repetitive (one per patient) clinical samples received during September 2007 in the clinical microbiology laboratories of three tertiary care hospitals in Athens, known for their high prevalence of MβL producers (Giakkoupi et al., 2003a, Giakkoupi et al., 2003b, Psichogiou et al., 2008), were included in the study. Samples, including urine (n = 60), pus swabs (n = 91), bronchial secretions/sputum (n = 85), and positive blood cultures (n = 90), each containing at least one

Results and discussion

A total of 73 VIM-positive microorganisms were identified in 326 clinical samples confirming the high prevalence of VIM producers in this setting (Vatopoulos, 2008). Among VIM producers, P. aeruginosa was predominant (n = 42) followed by K. pneumoniae (n = 25) and Acinetobacter spp. (n = 6). VIM-positive isolates of various enterobacterial species were also identified in a sporadic fashion. The higher frequency of VIM producers was observed in respiratory samples (47%). The respective frequency in

References (10)

There are more references available in the full text version of this article.

Cited by (35)

  • Rapid detection of carbapenemase-producing Acinetobacter baumannii and carbapenem-resistant Enterobacteriaceae using a bioluminescence-based phenotypic method

    2018, Journal of Microbiological Methods
    Citation Excerpt :

    Phenotypic susceptibility testing of Enterobacteriaceae to carbapenems is sophisticated, and that is shown, for example, by the different breakpoints in meropenem minimal inhibition concentration (MIC) for Enterobacteriaceae (European Committee on Antimicrobial Susceptibility Testing vs. Clinical and Laboratory Standards Institute) to classify them as resistant or susceptible. PCR-based detection methods do exist (Avlami et al., 2010; Queenan and Bush, 2007; van der Zee et al., 2014) but are cost intensive and will not detect unknown resistance genes. Screening of patient samples based on culture is time consuming and the results are sometimes difficult to interpret.

  • The Changing Role of the Clinical Microbiology Laboratory in Defining Resistance in Gram-negatives

    2016, Infectious Disease Clinics of North America
    Citation Excerpt :

    In particular, hyplex ESBL ID, hyplex SuperBug ID, and hyplex CarbOXA ID detect, respectively: (i) TEM, SHV, CTX-M, and OXA; (ii) KPC, IMP, VIM, NDM, and OXA-48–like; and (iii) OXA-23, OXA-24/40, OXA-51–like genes. Hyplex SuperBug ID showed a 97% agreement with the standard PCR/sequencing.44,45 In another study, the combination of hyplex ESBL ID and hyplex SuperBug ID was able to detect all class A and B enzymes among Enterobacteriaceae with high sensitivity (100%) and specificity (98%).46

  • Diagnosis and antimicrobial treatment of invasive infections due to multidrug-resistant Enterobacteriaceae. Guidelines of the Spanish society of infectious diseases and clinical microbiology

    2015, Enfermedades Infecciosas y Microbiologia Clinica
    Citation Excerpt :

    There are many published studies of the application of multiplex PCR for detection of ESBLs and/or carbapenemases in Enterobacteriaceae29–35 (Table 3). With respect to MBL, a commercial multiplex PCR (Hyplex-MBL ID Multiplex PCR-ELISA®), which was proven reliable in detecting blaVIM genes in blood, urine, exudate, and sputum samples, is available.36 Overall, these methods lack clinical validation with multicentre studies.

View all citing articles on Scopus
View full text