Performance of the Revised Version of an Immunochromatographic Assay for Detection of mecA - and mecC -Mediated Methicillin Resistance in Staphylococci

Abstract

D etection of methicillin resistance in staphylococcal isolates is crucial to quickly optimize treatment in cases of severe staphylococcal infections. Rapid genetic or phenotypic tests have been developed to detect isolates harboring the mecA gene, which is responsible for methicillin resistance related to additional penicillin-binding protein 2a (PBP2a) expression. The discovery in 2011 of a mecA variant, named mecC, encoding PBP2c and showing only 70% nucleotide sequence homology, was a real challenge for laboratories, since most of the tests targeting mecA failed to detect mecC (1). The first version of the immunochromatographic assay developed by Alere (PBP2a culture colony test; Alere, Scarborough, ME), based on blue-colored monoclonal antibodies targeting PBP2a, has been widely evaluated. It demonstrated 100% specificity and sensitivity for mecA-positive Staphylococcus aureus strains but displayed lower sensitivity for Staphylococcus non-aureus (SNA) species such as Staphylococcus epidermidis (2-4). For mecC-positive isolates, this assay revealed a lack of sensitivity unless PBP2c expression was induced by 18-h culture around a cefoxitin disk, which increased the sensitivity from 8.7% to 100% (5). Alere recently updated this test, whose intended use is still the detection of PBP2a from S. aureus isolates, in order to allow storage at room temperature and changed the antibodies immobilized on the nitrocellulose membrane, which are now recombinant monoclonal antibody fragments with a conjugate labeled red (6). Here, we evaluated the revised version of the assay, the PBP2a SA culture colony test (SACCT) (Alere), using a collection of both S. aureus and SNA isolates, in order to determine whether the technical changes, particularly the use of antibody fragments, affected the performance of the immunochromatographic test. A large collection of methicillin-susceptible S. aureus and methicillin-resistant S. aureus (MRSA) isolates (n ϭ 83) belonging to more than 50 different clones and methicillin-susceptible and methicillin-resistant SNA isolates (n ϭ 122) belonging to 14 different species was tested (see Table 1 for details). The presence of mecA and mecC was confirmed using PCR (7, 8). All S. aureus isolates had been previously characterized by spa typing, multilocus sequence typing, and/or microarray analysis (Alere Technologies, Jena, Germany) to ensure diversity of the tested strains, which were representative of the most prevalent clones circulating in Europe. Isolates were grown for 18 to 24 h on Columbia sheep blood agar plates (bioMérieux, La-Balme-les-Grottes, France) and then were tested directly using the PBP2a SACCT, according to the manufacturer's instructions. The assay was also performed after PBP2a/PBP2c induction using colonies harvested around a cefoxitin disk (30 g). The testing was performed by a single

Rapid Detection of PBP2a in Staphylococci from Shortly Incubated Subcultures of Positive Blood Cultures by an Immunochromatographic Assay

“…The improved analytical performance in detecting PBP2a of the SACCT compared to the previous version comes at the cost that it can no longer detect PBP2a in mecC -positive S. aureus isolates ( 14 , 15 ). Since the prevalence of mecC -positive S. aureus isolates is currently very low, we do not consider this a major drawback.…”

“…Previous studies have reported that the SACCT displayed superior sensitivity (100%) and the same specificity (100%) in detecting PBP2a-mediated β-lactam resistance in S. aureus , as well as in CoNS isolates, compared to the old version ( 14 , 15 ). In these studies, the performance of the SACCT has been evaluated with isolates cultivated for 18 to 4 h. This implies that this assay can be employed one workday after the Gram stain identification of staphylococci in native materials at the earliest.…”

“…Phenotypic methods can be alternatively used for detection of the PBP2a protein from primary cultures. The revised version of the immunochromatographic assay developed by Alere (PBP2a SA culture colony test [SACCT]; Abbott Diagnostics Scarborough, Inc., USA) is a rapid, reliable, and easy-to-use test for detection of PBP2a-mediated methicillin resistance in staphylococci ( 14 , 15 ).…”

Recent Developments in Phenotypic and Molecular Diagnostic Methods for Antimicrobial Resistance Detection in Staphylococcus aureus: A Narrative Review

“…Therefore, no improvements were observed in the detection of the PBP2c between the current and the previous version of the PBP2a SA culture colony test (SACCT). Therefore, a negative result with the PBP2a SACCT does not imply methicillin susceptibility, since the isolate can be a mecC -positive MRSA [ 42 ].…”

Rapid Detection of Methicillin-Resistant Staphylococcus aureus Directly from Blood for the Diagnosis of Bloodstream Infections: A Mini-Review

“…For example, the blood culture-dependent three-hour short incubation protocol combines MALDI TOF identification with the qualitative immunochromatographic PBP2a detection test assay (Alere) to detect methicillin resistance, yielding a sensitivity and specificity of 87.5% and 100%, respectively [ 68 , 69 ]. Recently, a revised version of an immunochromatographic assay for detection of mecA - and mecC -positive MRSA has been described [ 70 ].…”