Real-time PCR as a Diagnostic Tool for Bacterial Diseases

Max Maurin

Disclosures

Expert Rev Mol Diagn. 2012;12(7):731-754. 

In This Article

qPCR for Detection of Antibiotic Resistances

In patients suffering from bacterial infections, quick determination of the antibiotic susceptibilities of the involved strain is of primary importance for rapid adaptation of the antibiotic therapy, especially in urgent situations. On the other hand, there are situations where acquired antibiotic resistances are difficult to detect by phenotypic methods because of poor expression of the resistance traits in vitro. In both cases, qPCR may allow rapid detection and identification of the antibiotic resistance genetic determinant (ARGD), including genetic alterations affecting structural or regulatory genes involved in antibiotic resistances, and acquisition of exogenous resistance genes by horizontal gene transfers.[226] A large number of in-house qPCR tests have been developed to detect ARGD, whereas a few commercial tests are currently available. None of these tests, however, can replace conventional phenotypic methods such as the antibiogram for antibiotic susceptibility determination because complex genetic alterations may be poorly predictive of the level of antibiotic resistance (MICs); (e.g., PLP gene mutations in S. pneumoniae); the expression of new ARGD may be highly variable in different strains of a same species (e.g., β-lactamase production); variations in the nucleic acid sequence of the ARGD may preclude its detection using available qPCR tests (e.g., the new variant of mecA gene in S. aureus); antibiotic resistance may be due to multiple mechanisms that are difficult to detect simultaneously (e.g., enzyme inactivation and efflux pump mechanisms).

qPCR for Detection of Antibiotic Resistance Gene Determinants

The most widely-used commercial qPCR tests are those allowing detection of the mecA gene responsible for resistance to methicillin in S. aureus,[16,17] and the vanA and vanB genes responsible for resistance to glycopeptides in Enterococcus species.[227–230] In-house and commercial qPCR tests allow rapid detection of methicillin resistance in S. aureus.[21,23,24] Although these tests display high sensitivities and specificities, false-positive results have been reported for methicillin-susceptible S. aureus strains with retained cassette chromosome mec (SCCmec) but missing mecA gene,[231] and false-negative results have occurred for MRSA strains due to lack of detection of some SCCmec variants[232] or the presence of the newly described mecA variant.[233] qPCR tests for detection of vancomycin-resistant Enterococcus sp. in rectal and stool specimens are highly sensitive, but their specificity is limited due to the presence of vanB genes among the nonenterococcal intestinal flora (e.g., Clostridium spp[227–230]). In-house qPCR tests have been developed to detect other ARGDs, including the genes encoding extended-spectrum β-lactamases,[234,235] cephalosporinases,[236] carbapenemases,[237,238] aminoglycoside-inactivating enzymes,[239] methylases responsible for resistance to macrolides[240,241] and efflux systems.[241]

qPCR for Detection of Antibiotic Resistance Mutations

A few commercial tests can detect gene mutations responsible for antibiotic resistances, including resistance to rifampin in M. tuberculosis,[67,68,70,242] and to macrolides in H. pylori.[135,136] A much larger number of in-house tests have been developed for detection of gene mutations responsible for antibiotic resistances, including resistance of S. pneumoniae to penicillins, macrolides and fluoroquinolones,[241,243,244]H. influenzae to fluoroquinolones,[245]N. meningitidis to penicillins and rifampin,[246,247]S. aureus to fluoroquinolones,[248]M. pneumoniae to macrolides,[249]M. tuberculosis to first- and second-line antibiotics,[250–253]C. difficile to the fluoroquinolones,[254] the noncultivable pathogens M. leprae to dapsone, rifampin and fluoroquinolones,[255] and T. pallidum to azithromycin.[256]

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