Molecular diagnostics and genotyping of MRSA: an update

Staphylococcus aureus

Staphylococcus aureus is a significant human pathogen that can cause a wide variety of diseases owing to its ability to acquire and express various virulence factors and antimicrobial resistance determinants. Colonization is an important step in the pathogenesis of S. aureus infection. Approximately 20–30% of the general human population is persistently colonized with S. aureus, most frequently in the anterior nares, although other body sites, such as the perineum, skin and throat, may also be colonized. Another 30% of the general population is intermittently colonized and the remaining 40–50% appear not to be susceptible to S. aureus carriage [1–2].

Persistent nasal carriers of S. aureus have a three- to six-times higher risk of healthcare-associated infection with S. aureus than noncarriers [3–5]. More than 80% of healthcare-associated S. aureus infections are endogenous [6–8]. Recently, it has been shown that the number of surgical-site S. aureus infections acquired in the hospital can be reduced by rapid screening and decolonization of nasal carriers of S. aureus on admission [9].

Following the introduction of methicillin in 1959, methicillin-resistant S. aureus (MRSA) has quickly emerged as a major nosocomial burden worldwide [10]. Today, MRSA continues to be a major issue in hospitals, but has also emerged as a problem in the community [11]. Methicillin resistance in S. aureus is encoded by the mecA gene, which is embedded within a mobile staphylococcal cassette chromosome (SCC) element known as SCCmec. MRSA can emerge from methicillin-susceptible S. aureus (MSSA) upon site-specific integration of SCCmec into the orfX locus in the chromosome. To date, nine major types of SCCmec have been recognized in S. aureus[12].

Phenotypic detection & identification of MRSA

Rapid, high-throughput diagnostic tools are needed to detect infections caused by MRSA strains and to contain their spread. The traditional screening cultures require at least 48 h until a negative test result for MRSA can be reported. These methods have been superseded by the introduction of selective agar cultivation methods and latex agglutination tests for the specific detection of the product of the mecA gene, penicillin-binding protein 2a. Most of these tests have been evaluated in different studies [13–26]. Sensitivity and specificity for agar cultivation ranged from 40 to 98% and 80 to 100%, respectively. For latex agglutination tests, both sensitivity and specificity were almost 100%.

The first commercial example of a rapid screening test for detection and identification of MRSA, the BacLite Rapid MRSA assay (Acolyte Biomedia; Surrey, UK) is documented as a sensitive (90.4%) and specific (95.7%) test for the detection of MRSA nasal colonization within 5 h [27]. The test measures adenylate kinase activity in a selective broth during a 4-h growth episode.

Recently, Stenholm et al. described the results of a new culture-based method for MRSA screening [28]. This new method employs the two-photon excited fluorescence (TPX) technology to provide a quantitative S. aureus-specific fluorescence signal in a single separation-free process. Different fluorescence signal progressions are recorded for MRSA and MSSA when bacterial growth under conditions of antibiotic pressure is monitored online by continuous or repeated measurements. When monoclonal antibodies were used, the assay was 100% sensitive and 100% specific for screening for MRSA from pure cultured samples and results were available within 8–12 h.

However, most phenotypic techniques still require 4–24 h of cultivation before detection and identification of S. aureus and testing for methicillin resistance can be initiated and may, therefore, not be suitable for fast screening of patients.

Genotypic detection & identification

Nucleic acid-amplification techniques (NAATs) offer clear benefits over traditional culture-based assays, in particular, a reduced time to identification and an improved specificity and sensitivity. Over the past decade, a range of commercial and in-house developed NAATs has been introduced (Table 1) with two main strategies: identification of MRSA via detection of S. aureus-specific genes, such as spa, nuc and fem in combination with the detection of the mecA gene or via detection of the SCCmec region. However, sensitivity and specificity of these assays may be compromised owing to the presence of methicillin-resistant coagulase-negative staphylococci (MRCoNS), variability within the used S. aureus-specific genes or variability within the SCCmec cassette. This may lead to false-negative (e.g., gene mutation or SCCmec variants) or false-positive results (e.g., deletion of the mecA gene from the SCCmec region) [29–32].

The detection of the mecA gene by (real-time) PCR is widely recognized as the gold standard for identification of MRSA. A variety of in-house assays performed on several different instruments has been described in the literature with variable results (Table 2). Most of these are performed on an overnight selective enrichment broth. The major limitations of this PCR strategy for direct testing in clinical samples were the necessity of enrichment and the inability to directly link identification of S. aureus with mecA gene detection because of the confounding effect caused by MRCoNS. In 2004, Huletsky et al. described a novel real-time PCR assay [33]. This assay is able to distinguish between MRSA and mixtures of MSSA and MRCoNS and, therefore is suitable for direct identification of MRSA in clinical specimen. The assay became commercially available as the IDI-MRSA assay and is currently marketed as the BD GeneOhm MRSA assay (BD GeneOhm, CA, USA). When testing nasal swabs, the detection limit was suggested to be 325 colony-forming units (CFU) per swab. In an external quality assessment (EQA) performed by Quality Control for Molecular Diagnostics (QCMD, Glasgow, UK) in 2009, the limit of detection was found to be 10³–10⁴ CFU/ml [34]. Also, when evaluating the performance of this test, Bartels et al. described how a common variant of SCCmec type IVa was not detected by the BD GeneOhm assay [30].

Rossney et al. evaluated a commercial platform based on the BD GeneOhm principal for direct detection of MRSA in clinical samples, the GeneXpert MRSA assay (Cepheid Diagnostics, CA, USA) [35]. In this fully automatic, but low-throughput assay, cells are lysed using ultrasonics and DNA from samples is extracted, amplified and detected in separate chambers of single-use disposable cartridges, which contain freeze-dried beads with all reagents required for the real-time process. Sample preparation time is minimal and the PCR assay time is a maximum of 75 min. The authors reported an average limit of detection of 610 CFU/ml with a sensitivity, specificity, positive predictive value and negative predictive value of 90, 97, 86 and 98% respectively.

In the QCMD EQA study of 2009, 11 samples containing various amounts of inactivated MRSA cells, MSSA, MRCoNS or Escherichia coli were distributed to 82 laboratories [34]. When compared with previous EQA studies on molecular diagnostics of MRSA, a statistically nonsignificant decrease was observed in the overall test sensitivity. However, a minor improvement was observed for the ‘specificity’ and the ‘true-negative’ samples. In this EQA, one normal MSSA strain and two MSSA samples harboring a SCCmec cassette lacking the mecA gene were included. There was a marked difference in the percentage of correct results for the MSSA strain containing the mecA gene compared with the two strains lacking it.

In conclusion, the quality of direct molecular diagnostic tests still needs improvement. Every assay should be evaluated and continuously monitored to determine the assay’s usefulness. Positive results should always be confirmed by a culture method or a second molecular test.

Genotyping

After MRSA detection, genetic typing may be necessary in order to assess whether transmission of MRSA occurred and whether infection-preventive measures are mandatory. In addition, genetic typing is essential for elucidation of (inter)national dissemination of MRSA clones. Currently, many different genotyping methods are in use in the diagnostic laboratory, but pulsed-field gel electrophoresis (PFGE) of SmaI macro restriction analysis of genomic DNA is preferential [36]. However, PFGE is a technically demanding method, with limited portability due to lack of reproducibility.

The development of a commercially available automated rep-PCR assay, the DiversiLab system (bioMérieux, Boxtel, The Netherlands) offers advances in standardization and reproducibility over manual fingerprint-generating systems [37]. However, although two independent studies concluded that the DiversiLab system is a rapid and reproducible technique, it also lacks resolution. DiversiLab analysis does not differentiate genetically and epidemiologically unique MRSA strains; such differentiation is needed for adequate outbreak analysis [38,39].

Multilocus variable number of tandem repeat analysis is a high-throughput genotyping technique that can be used for hospital, national and international genotyping of MRSA, but the discriminatory power depends on the number and types of loci analyzed [40,41].

Sequence-based approaches, such as spa sequence typing and multilocus sequence typing (MLST), have resulted in large sequence databases for MRSA. The determination of sequence polymorphism of the spa gene encoding the staphylococcal surface protein A (spa sequence typing) has become the most popular MRSA typing system, owing to high-throughput capacity and an excellent reproducibility, which allows portability of data and comparison worldwide [42]. MLST defines variation within a very small sample of the genome and often cannot distinguish between closely related isolates.

Full-genome sequencing provides a complete inventory of micro-evolutionary changes, but this approach is impractical for routine diagnostic laboratories. In a recent paper, Harris et al. described a new high-throughput genomics approach based on full-genome sequencing, which provides a high-resolution view of the epidemiology of MRSA with the potential to trace person-to-person transmission within a hospital environment [43].

New techniques

New tools for identification and genotyping of MRSA include different applications of spectroscopy, such as PCR/electrospray ionization-mass spectrometry (MS) [44,45] and MALDI-TOF MS [46]. MALDI-TOF MS is cost effective, analyzes samples in minutes and requires little hands-on time.

In addition, Raman spectroscopy has been described as a promising tool [47,48]. Raman spectroscopy is a fast and reproducible typing technique, which provides strain-specific optical fingerprints in a few minutes instead of several hours to days, as is the case with genotyping methods. Its high throughput and ease of use make it suitable for use in routine diagnostic laboratories. Efforts to develop these technologies for the analysis of single cells are currently in full progress and may in the end compete effectively with the currently preferred nucleic acid-based technologies.

Instead of detecting S. aureus itself, new strategies may look at the host immune response or look at genetic variation in the host.

The rapid detection of antibody levels against S. aureus with Luminex technology demonstrated that antibody levels were associated with the presence of toxin genes in infectious S. aureus isolates [49]. Furthermore, Emonts et al. showed that persistent carriage of S. aureus is influenced by and associated with genetic variation in host inflammatory response genes [50].

Both approaches can be useful in fast screening for (susceptibility) to (methicillin-resistant) S. aureus carriage or infection.

Conclusion

Methicillin-resistant S. aureus is responsible for a large and still growing number of both healthcare and community-associated infections, resulting in increased morbidity and excessive healthcare costs. Screening of individuals, combined with an aggressive infection control program, has become the standard for management of these infections. Rapid screening methods that allow reliable detection of MRSA within hours are now available. The short time-to-result is a clear advantage that has provided a tool for successful infection-control strategies. However, every assay should be evaluated against the local MRSA diversity before being introduced in the diagnostic microbiological laboratory. Continuous evolution of SCCmec, constrains continuously monitoring of the assay performance and positive results of direct MRSA testing should always be confirmed by a culture method or a second molecular test. For laboratories with high false-positivity rates or in regions with low prevalence of MRSA, confirmation is essential.

In conclusion, the quality of molecular diagnostic tests and (geno)typing techniques is still under discussion. Adequate internal and external quality control and international standardization for MRSA diagnostics should be developed over the years to come. To improve the performance and quality of molecular detection, identification and genotyping of MRSA, both laboratories and manufacturers should be encouraged to participate in EQAs.

Genetic and functional studies in large populations are warranted to clarify the contribution of new strategies, such as different applications of spectroscopy, determination of genetic variability in humans or rapid antibody – and/or antigen – detection.

Table 1. Commercial molecular tests for methicillin-resistant Staphylococcus aureus detection and identification.

Testkit (supplier)	Test principle	Sensitivity (%)	Specificity (%)	TAT (h)	Ref.
Culture
Genotype MRSA (Hain)	PCR and reverse line blot detection of mecA and Staphylococcus aureus-specific sequence	91	99	4	[51]
Direct from clinical specimen (swabs)
Hyplex StaphyloResist (Alpha Omega)	PCR-ELISA for detection of mecA, coa and conserved Staphylococcus aureus housekeeping gene	92	90	4	[52]
GenoType MRSA direct (Hain)	PCR and reverse line blot detection of SCCmec/orfX	95	99	4	[53]
GeneOhm IDI-MRSA (BD)	Multiplex real-time PCR for detection of SCCmec/orfX	95	94	2	[33,54,55]
GeneXpert MRSA assay	Multiplex real-time PCR for detection of SCCmec/orfX	90	97	1.3	[35]

MRSA: Methicillin-resistant Staphylococcus aureus; TAT: Turn around time.

Table 2. In-house molecular tests for methicillin-resistant Staphylococcus aureus detection and identification.

Test principle	Sensitivity (%)	Specificity (%)	TAT (h)	Ref.
Enrichment
Multiplex PCR (agarose gel) detecting femB and mecA	100	100	3	[56,57]
Real-time PCR detecting mecA	100	100	2	[58]
High-throughput real-time PCR detecting mecA and nuc	100	100	2.5	[59]
Real-time PCR detecting mecA and SA442	100	100	4	[60]
Isothermal signal amplification (CytAMP) detecting coa and mecA	83	93	3.5	[61]
Real-time PCR detecting nuc and mecA	93	90	2.5	[62]
Direct from clinical specimen (swabs)
Real-time PCR detecting mecA and femA	100	91	6	[63]
Staphylococcus aureus immunocapturing (protein A) and	100	64	6	[64]
Quantitative PCR detecting mecA, femA and femB
Real-time PCR detecting SCCmec/orfX	93 (98)^†	100 (100)^†	3.5	[65]

^†Percentage sensitivity and specificity in brackets are after overnight selective enrichment.

NA: Not applicable; TAT: Turn around time.

Financial & competing interests disclosure

Alex van Belkum is member of the scientific advisory board of Cepheid (Cepheid Diagnostics, CA, USA). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.