Methods for screening for methicillin-resistant Staphylococcus aureus carriage

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1 REVIEW /j x Methods for screening for methicillin-resistant Staphylococcus aureus carriage G. L. French Department of Infection, King s College London and Guy s and St Thomas Hospital NHS Foundation Trust, St Thomas Hospital, London, UK Abstract Screening patients for methicillin-resistant Staphylococcus aureus (MRSA) carriage at hospital admission is widely accepted as an essential part of MRSA control programmes. It is assumed, although not proven, that rapid reporting of screening results will improve MRSA control, provided that a clear action plan for positive cases is in place and is being followed. An effective culture screening method is direct inoculation of pooled nose, throat and perineal swabs on a well-performing MRSA-selective chromogenic agar; presumptive MRSA colonies can be confirmed rapidly by latex agglutination with antibodies directed against penicillin-binding protein 2a. This method will usually produce a positive result after 24 h of incubation in >95% of true-positive cases, and will be sufficient for most initial treatment and infection control decisions; full antimicrobial susceptibilities will be available on the next day. Inoculation of selective enrichment broth containing a colorimetric growth indicator is an alternative overnight culture method, but there may be problems with overgrowth of other organisms, such as enterococci. PCR methods are now available that can produce same-day results, provided that samples reach the laboratory in time for batch processing, but cultures are required for susceptibility testing. In comparison with culture-based methods, PCR tests are costly, and some have relatively high false-positivity rates; definitive evidence of their clinical costeffectiveness is lacking. New point-of-care PCR tests are being introduced that are potentially even more rapid but are even more expensive; studies on the clinical cost-effectiveness of these very rapid tests are awaited. Keywords: MRSA, screening, laboratory identification, rapid testing, review Clin Microbiol Infect 2009; 15 (Suppl. 7): Corresponding author and reprint requests: G. L. French, Department of Infection, King s College London and Guy s and St Thomas Hospital NHS Foundation Trust, St Thomas Hospital, London SE1 7EH, UK gary.french@kcl.ac.uk Introduction own organism or act as the source of further cross-colonization of others. In order to reduce the risk of endogenous infection and hospital transmission, most guidelines for the prevention and control of MRSA infection recommend the identification of carriers by screening, followed by isolation and/or attempts at decolonization [5 9]. The rationale, evidence and strategies for MRSA screening and surveillance are reviewed in this supplement by Tacconelli [10]; this article reviews the laboratory methods for MRSA screening. True community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infection is now common in the USA [1], but the great majority of MRSA infections in Europe are associated with hospital and healthcare contact [2]. Most of the time, MRSA colonization (which may last for months or years) precedes infection. Patients may acquire colonization during one admission (or healthcare contact) and enter hospital as MRSA carriers on subsequent admissions or transfers. Rates of MRSA carriage among patients on admission varies with geographical location, but in a high-prevalence area such as the UK, the rate is approximately 6.5% of general medical and surgical patients [3,4]. After admission, colonized patients may become endogenously infected with their Rapid Testing, Turn-Around Times, and Patient Management It is widely accepted that rapid reporting of MRSA screening results is important for MRSA control, although the evidence base for this is limited. However, rapid reporting is of no value unless there is a rapid and appropriate response. It is essential that a plan (or care pathway) is in place, detailing who should be informed and what is to be done when an MRSA result is reported, and that these actions are implemented immediately. Similarly, there must be efficient systems for specimen collection and transport, results Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases

2 CMI French Screening for MRSA carriage 11 reporting, and weekend and night-time shifts. Many centres now screen elective surgical patients in pre-admission clinics a week or two before admission. The role of MRSA screening has been recently well reviewed by Dancer [11]. Screening is only part of MRSA control, and all screening methods may give false-negative results. Good hygienic practice and standard infection control procedures should be in place for all patients, whether or not their MRSA status is known. Patients who are assessed at admission as being at high risk for MRSA carriage should be pre-emptively isolated until a negative screen result is reported. In certain high-risk clinical areas, a local risk assessment might conclude that patients who have been previously MRSA-positive should remain in isolation, even if a new screen is negative. Assessment of Screening Methods Screening tests for MRSA can be assessed on the basis of sensitivity, specificity (and positive predictive value (PPV) and negative predictive value (NPV)), turn-around time (time between taking the specimen and reporting the result), ease of performance, and cost. Improving any one of these parameters usually worsens one or more of the others, so the aim should be to optimize cost/performance. As these are biological screening tests, no method is 100% sensitive and specific, and there is therefore no reference standard. This, together with wide variation in detailed methodologies, makes comparisons among test methods difficult. Screening Sites and Pooling MRSA strains colonize various mucosal sites, including the anterior nares, throat, and rectum, and some areas of the skin, especially moist sites such as the axillae, groins and perineum. Wound and skin puncture sites are also often colonized. Rates of colonization vary at different sites but, because the studies are difficult and costly, there is limited information on this, and figures vary. Coello et al. [12] performed the largest reported study on multiple-site screening of 403 MRSA-colonized patients: nasal swabs identified 79% of patients colonized at any site; combined swabs from nose and throat, nose and perineum, and nose, throat and perineum identified 86%, 93% and 98%, respectively (Table 1). Batra et al. [13] more recently reported on 105 intensive-care patients with MRSA, of whom five were positive only at wound sites; among the other 100, approximately two-thirds were detected by pooled nasal, perineal TABLE 1. Methicillin-resistant Staphylococcus aureus (MRSA) screening results from 403 MRSA-positive patients [12] Screening site and axillary swabs, and one-third by rectal and throat swabs. Multiple-site screening (ideally nose, throat and perineum for most patients) is thus necessary to achieve an adequate pickup rate. In order to save time and costs, screening swabs from these sites are usually pooled and processed simultaneously. This further complicates comparisons among screening methods, because different studies often screen different sites and do not give results for individual sites. Agar Culture Tests MRSApositive (no.) MRSAnegative (no.) Nose Perineum Throat Groin Axilla Nose and throat Nose and perineum Nose, throat, and perineum % MRSApositive The standard method for MRSA screening (and one still used for comparisons) is culture of screening swabs on agar. Presumptive S. aureus colonies are identified by standard microbiological tests, and then tested for methicillin resistance by disk diffusion, the whole process taking at least 2 days and usually 3 5 days. More rapid identification of MRSA colonies can be achieved by direct latex agglutination reaction with a monoclonal antibody directed against the variant penicillin-binding protein 2a, responsible for methicillin resistance and encoded by meca. Several studies have shown that the latex agglutination test is accurate, reliable and fast (15 20 min) for differentiating colonies of MRSA from those of methicillinsensitive S. aureus (MSSA) (or confirming them from selective chromogenic agar) [14 16], and this test is widely used [17]. The method requires induction, which provides improved reliability, although cross-reaction with penicillinbinding protein 2 from coagulase-negative staphylococci may sometimes occur. The sensitivity of agar culture screens can be increased by broth pre-enrichment and/or by using non-selective media. However, sensitivity is usually sacrificed for speed, and specificity is reduced by using selective media such as salt or salt mannitol agar (to select S. aureus) and antibiotic (to select for methicillin-resistance). The selective antibiotic is ideally a b-lactam; initially, methicillin or oxacillin were used, but these have been replaced by more stable agents such as

3 12 Clinical Microbiology and Infection, Volume 15, Supplement 7, December 2009 CMI cefoxitin. The quinolone ciprofloxacin was used for a period, because MRSA strains have been, until recently, usually ciprofloxacin-resistant, whereas MSSA strains have generally remained susceptible. However, this differentiation is not reliable especially with the emergence of ciprofloxacin-susceptible community MRSA strains and ciprofloxacin should not now be used for selection [18]. Conventional selective media are now being replaced by chromogenic agars. These allow rapid, direct microbial identification by incorporating chromogenic substrates for specific bacterial enzymes that result in different species producing different-coloured colonies on culture. S. aureus can be identified by a substrate colour change produced by enzymes such as staphylococcal a-glucosidase; adding a selective b-lactam to the agar allows rapid identification of MRSA. These agars are generally more specific and have better PPVs than traditional b-lactam-containing mannitol salt agar [19], and colour identification of MRSA reduces laboratory work time. Results are usually available after h of culture, although sensitivity is often improved at 48 h. A wide range of commercial chromogenic agars is available for MRSA detection by direct inoculation of screening swabs. These have been reviewed recently by Malhotra-Kumar et al. [20], who also provide information on agar composition and a full summary and discussion of performance; readers are referred to their paper for details. Reported sensitivities and specificities vary both among and within studies, and are difficult to summarize, because reference standards, culture sites, pooling, incubation times, and inoculum size vary. Nevertheless, as would be expected, there is a tendency towards high (>95 100%) specificity with direct inoculation at h of incubation, which decreases somewhat after h, and variable sensitivity (c %) at h, which increases to % after h. Reported times from inoculation of agar to confirmation of MRSA range from approximately 1.5 to 3 days, with an average of approximately 2 days. To obtain the best balance between speed and sensitivity, Malhotra-Kumar et al. [20] favour direct inoculation onto chromogenic agar (they use MRSA Select ), (Bio-Rad Laboratories, Nazarethe Eke, Belgium), followed by overnight enrichment culture to improve sensitivity, but at the cost of additional processing time. However, they note that Hansen et al. (46th ICAAC, 2006, Abstract 2719) found Chromogenic MRSA/Denim Blue Agar (Oxoid, Basingstoke, UK) to have a sensitivity of 90% and specificity of 96% with direct plating after 24 h of incubation. We (unpublished data) found a similar sensitivity of 95% after 24 h, and the denim blue colonies of MRSA on this agar are also easy to identify. In our laboratory, therefore, we routinely use Oxoid Chromogenic MRSA agar with direct inoculation and 24 h of incubation. This method is simple and relatively inexpensive, and for the majority of patients provides a reliable culture result on the day after swabbing. Broth Culture Tests Culture of MRSA screening swabs in selective broth can increase the sensitivity of the test, provided that the selective medium is not inhibitory to the MRSA strain involved. A variety of chemical and antibiotic ingredients have been used to favour MRSA while inhibiting other organisms. By adding an indicator of bacterial growth, these broths can produce a definitive negative result overnight, with possible positives being confirmed by further tests and cultures [21]. Kelly et al. [22], demonstrated that swabs can be agitated in the selective broth on the ward and then discarded, the broths being sent to the laboratory, with savings in time and laboratory labour. The original broths contained ciprofloxacin to select for MRSA, but this should now be replaced by cefoxitin because of the increasing incidence of ciprofloxacin-susceptible MRSA. A problem with these broths is that a proportion of tests produce an intermediate colour change after overnight incubation, often due to overgrowth with enterococci that may not be inhibited by the MRSA selective agents. Cultures may therefore have to be prolonged, and suspected positives subcultured on chromogenic or other selective MRSA agar for confirmation. Nucleic Acid Amplification Tests Methicillin resistance is encoded by the meca gene, which is associated with a mobile element designated staphylococcal chromosome cassette mec (SCCmec) [23]. There are several types of SSCmec in different MRSA clonal lineages, and new types are continuously appearing. A range of rapid PCR screening tests for MRSA have been developed, based on simultaneous amplification and detection of meca and genes characteristic of S. aureus. However, methicillin-resistant coagulase-negative staphylococci (MRCNS) also possess meca, and dual colonization with methicillin-resistant coagulase-negative staphylococci and MSSA is not infrequent: for example, this occurred in 3.4% of cardiothoracic patients studied in Germany [24]. PCR systems that use unlinked primers targeting an S. aureus species-specific gene and meca, such as the LightCycler Staphylococcus and MRSA detection kit (LC Assay; Roche Diagnostics, Mannheim, Germany) and

4 CMI French Screening for MRSA carriage 13 the Hyplex StaphyloResist PCR (BAG, Lich, Germany), may give false-positive results with such mixtures [20]. To resolve this problem, the BD GenOhm system (previously known as IDI-MRSA) (BD Diagnostics, San Diego, CA, USA) amplifies MRSA-specific sequences by targeting a single locus that includes the right extremity of SCCmec downstream of meca, and part of the adjacent S. aureus-specific orfx gene. Five primers target SCCmec sequences corresponding to types I, II, III, IVa, IVb, and IVc, and one primer and three molecular beacons are specific for orfx [25]. The test is performed in real time with fluorescence detection. Similar assays are the GeneXpert MRSA (Cepheid, Sunnyvale, CA, USA) [26] and the GenoType MRSA Direct (Hain Lifescience, Nehren, Germany), the latter using hybridization to oligonucleotides on a cellulose strip for detection and identification. In contrast, qmrsa [27] is an in-house triplex quantitative PCR assay that amplifies meca and fema from S. aureus and Staphylococcus epidermidis to identify the origin of the meca signal. Molecular methods can give false-positive results if nonspecific sequences are amplified. Rupp et al. [28] pointed out that single-locus PCR methods for MRSA, such as the IDI/ GenOhm and Hain tests, are potentially especially susceptible to this problem, and described an MSSA isolate containing only small fragments of the right extremity of SCCmec that resulted in false-positive reactions in the Hain test. Desjardins et al. [29] used the IDI-MRSA test to examine nasal and rectal swabs pooled in a selective broth. Of 298 IDI-MRSA assay-positive broths, 103 could not be confirmed by culture; MSSA was recovered from 77 of these 103, and gave positive results with IDI-MRSA. There were 17 different pulsed-field gel electrophoresis genotypes among these MSSA strains, and approximately half of them were similar to common local MRSA genotypes, including the community-acquired MRSA clones USA100 and USA500, which may have been MSSA strains that had not yet acquired meca. Alternatively, MRSA strains may lose meca and become methicillin-susceptible but still retain partial SCCmec elements that react with the primers [20,25]. Furthermore, because these systems target SSCmec elements, they may give false-negative results if as is increasingly the case local strains of MRSA appear to be of unusual or variant SSCmec types [30,31]. Francois et al. [30] found the IDI-MRSA and Hain systems to have poor specificity when tested against 93 MRSA and 89 MSSA strains of diverse genetic backgrounds. Their own multilocus quantitative PCR system was not affected by this problem, and they warn that users of orfx SSCmec-based assays should repeatedly monitor local epidemiology to minimize the risks of failing to detect emerging MRSA clones. A final problem is that topical or systemic antimicrobial therapy may kill MRSA but their DNA may remain, yeilding false-positive results. Further investigations on the accuracy of PCR screening tests after nasal decolonization are required. The IDI/GenOhm system is designed and licensed by the FDA for use with nasal swabs only. Numerous studies of this system in comparison with culture have shown sensitivities, specificities, NPVs and PPVs for nasal swabs of c %, 92 99%, 97 99%, and 83 84%, respectively [3,32 35]. Taking into account the fact that 90 95% of swabs will be true negatives, the high NPV is to be expected, and the low PPV may be a reflection of a relatively high rate of false positivity. However, as discussed previously, interpretation of these figures is complicated by the lack of a valid standard method for comparison. Samples from non-nasal sites and from multiple-site pooled swabs generally give similar results [3,32 34], although a recent study showed that screening with nasal swabs alone would have failed to identify 24% of MRSA-colonized patients [36]. The processing time for these PCR tests is c h, but the total turn-around time from sampling to reporting is much longer. Although many publications have reported extremely rapid turn-arounds, in the order of 4 h, we [3] (unpublished results) found a mean turn-around time for the IDI-MRSA test in routine use in a London hospital located on two sites to be 22 h; because the laboratory processing took place during daytime hours only, much of this delay was due to specimens sent at night and weekends missing batch processing runs, although some was due to slowness of transportation. Similar lengthy turn-around times for laboratory-based PCR tests have been reported by others [37,38]. Some workers have used a broth enrichment step to improve the sensitivity of pooled nasal and rectal swabs at the expense of further turn-around delay [29]. A Scottish systematic review of MRSA screening [39] concluded that in routine clinical practice, where samples require to be batched before testing, PCR does not have a turnaround time advantage over chromogenic agar testing. Many screening swabs can give a same-day result when processed by present PCR systems, but users should be aware that in routine practice a significant proportion will give a result on the day after sampling. Furthermore, good clinical microbiological and infection control practice requires that positive cases will then need culture (or re-swabbing and reculture) to confirm antimicrobial susceptibilities, and other tests for treatment and epidemiological reasons, as well as MRSA confirmation. However, PCR screening systems are becoming available that can be used by clinical staff and allow PCR testing 24 h a day at the bedside or point of care ( GeneXpert ; Cepheid). If these are shown to be practical and effective, turn-around times will become much closer to the processing time of c. 2 h. Independent assessments of point-of-care PCR screening are awaited.

5 14 Clinical Microbiology and Infection, Volume 15, Supplement 7, December 2009 CMI The costs of PCR tests for MRSA screening are significantly higher than for conventional culture. It is difficult to define the exact costs, because reagent and equipment costs differ with rental packages, quantity discounts, and currency rates. However, the Scottish systematic review referred to previously [39] calculated that the cost of a screening test performed with chromogenic agar, latex agglutination and a confirmatory disk test was 4.35 for a negative test and 7.45 for a positive test, and the cost of the IDI-MRSA real-time PCR test was for both positive and negative specimens. We calculated that reagent costs alone were 10 for the PCR test, as compared with less than 2 for conventional tests [3]. The costs for point-of-care PCR screening such as the GeneXpert system are even higher, at about 25 per test. Ritchie et al. [39] concluded that (for the Scottish National Health Service) the resource costs required for PCR screening of swabs are high, meaning that the technology is unlikely to represent a realistic replacement for culture methods. Staff costs may be lower than with conventional tests because hands-on time with automated systems may be shorter; however, costs will increase if rapid testing is to be available 24 h a day. We believe that, for appropriate therapy and epidemiological control, positive screens should also have an MRSA culture and antimicrobial susceptibility test; these costs should be factored into business plans for rapid PCR testing. A strong argument for replacing conventional MRSA culture screening with rapid PCR tests is that the increased costs can be justified by improvements in patient care, resulting in reduced MRSA transmission and infection rates and consequent reductions in mortality, morbidity, and healthcare costs. Indeed, it is argued that the savings in healthcare costs will greatly outweigh the increased costs of rapid testing. Several papers have been published showing that MRSA rates have fallen after the introduction of rapid screening; however, many of these studies have been uncontrolled and/or have had relatively small numbers of patients [3,38,40,41], and it is not known whether MRSA rates would have fallen for other reasons, even if the rapid tests had not been introduced. The literature is reviewed in detail by Tacconelli [10] in this supplement; she concludes that the evidence for the clinical effectiveness of rapid PCR screening is inconclusive. Our own large, randomized, cluster-controlled cross-over study of the impact of the IDI-MRSA rapid screening test at admission to general medical and surgical wards where pre-emptive isolation of high-risk patients was practised showed no reduction in MRSA transmission or infection rates as compared with conventional culture screening [3]. Similar findings have been reported by others [37,38]. It is also striking that national MRSA bacteraemia rates in England have fallen by approximately 30% between 2008 and 2009 as a result of an MRSA improvement programme that did not include rapid screening [42]. Conclusions MRSA screening is only effective if systems are in place to ensure rapid transport of specimens to the laboratory, rapid reporting of results to responsible clinicians, and rapid action to ensure isolation/cohorting and decolonization of colonized patients. For the greatest yield, screening swabs should be taken from multiple sites; pooling of these swabs will reduce laboratory work time and costs. Direct plating on a well-performing selective chromogenic agar of pooled nose and throat swabs (with or without a perineal swab) will usually produce a positive result after 24 h of incubation in >95% of true-positive cases. This will be a sufficient basis for most initial treatment and infection control decisions, and appears to be better than or at least as good as enrichment culture systems. Presumptive MRSA colonies can be confirmed rapidly by a latex agglutination test, and full antimicrobial susceptibilities will be available on the next day. Rapid PCR methods can produce same-day results, provided that samples reach the laboratory in time for batch processing. Commercial systems are validated only for nasal swabs but appear to be effective with most multiple-site pooled swabs. However, some PCR tests may have relatively high false-positive rates (as compared with culture), owing to non-specific amplification, and local quality assurance systems should be in place to ensure the continuing reliability of results. Additional cultures of PCR MRSA-positive cases will be required to obtain antimicrobial susceptibilities and epidemiological characterization of the isolates concerned. There is a lack of evidence so far that these PCR screening tests justify their increased costs. Even more rapid point-of-care PCR screening tests are becoming available, and studies on their cost-effectiveness are awaited. Transparency Declaration The author declares no conflict of interest in conjunction with this review. References 1. Zetola N, Francis JS, Nuermberger EL, Bishai WR. Communityacquired meticillin-resistant Staphylococcus aureus: an emerging threat. Lancet Infect Dis 2005; 5:

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7 16 Clinical Microbiology and Infection, Volume 15, Supplement 7, December 2009 CMI 37. Aldeyab MA, Kearney MP, Hughes CM et al. Can the use of a rapid polymerase chain screening method decrease the incidence of nosocomial meticillin-resistant Staphylococcus aureus? J Hosp Infect 2009; 71: Harbarth S, Fankhauser C, Schrenzel J et al. Universal screening for methicillin-resistant Staphylococcus aureus at hospital admission and nosocomial infection in surgical patients. JAMA 2008; 299: Ritchie K, Bradbury I, Craig J et al. The clinical and cost effectiveness of screening for meticillin-resistant Staphylococcus aureus (MRSA). NHS Quality Improvement Scotland: Health Technology Assessment Report 9, Robicsek A, Beaumont JL, Paule SM et al. Universal surveillance for methicillin-resistant Staphylococcus aureus in 3 affiliated hospitals. Ann Intern Med 2008; 148: Wilcox MH. Screening for MRSA. BMJ 2008; 336: Health Protection Agency Commentary for MRSA bacteraemia. September Available at: HPAwebStandard/HPAweb_C/ ?p= (last accessed 11 November 2009)