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One-year experience with modified BD GeneOhm™ MRSA assay for detection of methicillin-resistant Staphylococcus aureus from pooled nasal, skin, and throat samples
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Diagnostic Microbiology and Infectious Disease 63 (2009) 132 – 139 www.elsevier.com/locate/diagmicrobio
One-year experience with modified BD GeneOhm™ MRSA assay for detection of methicillin-resistant Staphylococcus aureus from pooled nasal, skin, and throat samples Natasa Svent-Kucina⁎, Mateja Pirs, Manica Mueller-Premru, Vesna Cvitkovic-Spik, Romina Kofol, Katja Seme Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia Received 21 July 2008; accepted 10 October 2008
Abstract We report our 1-year experience with modified GeneOhm™ MRSA assay (formerly IDI-MRSA) for pooled surveillance specimens in low methicillin-resistant Staphylococcus aureus (MRSA) prevalence clinical setting. We have successfully modified the GeneOhm™ MRSA assay protocol during the specimen preparation step by adding an extra washing step followed by pooling of up to 3 samples per patient (nose, skin, with or without throat) at the lysis step. The sensitivity of the modified assay compared with conventional cultivation was 94.3%, specificity 99.2%, negative predictive value 99.2%, and positive predictive value 94.3%. The modified test is reliable and performed well compared with conventional culture methods in our clinical setting with low-level prevalence of MRSA colonization. Our findings support the use of pooling of the patients samples as a cost-effective way of screening for MRSA colonization. © 2009 Elsevier Inc. All rights reserved. Keywords: MRSA; Surveillance; Pooling; GeneOhm™ MRSA assay; IDI-MRSA
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens responsible for increased morbidity, mortality, and prolonged hospitalization that results in increased hospital costs taxing the health care system (Cosgrove et al., 2003; Lodise and McKinnon, 2005; Melzer et al., 2003). Methicillin-resistant S. aureus prevalence varies significantly among European countries and among individual hospitals and accounts for less than 1% to more than 50% of bloodstream S. aureus infections. In Slovenia, the proportion of MRSA isolates from blood cultures in hospitalized patients has been steadily decreasing from 21.4% in the year 2000 to 7.1% in the year 2006 (EARSS annual report, 2006). Effective ways to reduce MRSA spread in hospitals are active surveillance cultures, isolation or cohort isolation ⁎ Corresponding author. Tel.: +386-1-54374-48; fax: +386-1-54374-01. E-mail addresses: [email protected], [email protected] (N. Svent-Kucina). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.10.005
of the colonized or infected patients, decolonization procedures performed according to hospital recommendations, as well as continuous education of all of the health care workers (Ministry of Health of the Republic of Slovenia, 2003; Rubinovitch and Pittet, 2001). Current Slovenian recommendations for MRSA screening include culturing of the nose and skin (axilla and groin, or wound) swabs, with or without throat swab from patients who are transferred from another ward (particularly intensive care units) or another hospital or who live in a long-term care facility; patients who were hospitalized at least 3 times during the last year; patients previously infected or colonized with MRSA; patients who have chronic wounds; patients who were hospitalized in the same room as an MRSA carrier for more than 3 days. Screening for MRSA is performed at the time of admission or within the first 48 h (Ministry of Health of the Republic of Slovenia, 2003). The results of the conventional culture-based screening methods with cultivation, identification, and antimicrobial susceptibility testing take 48 to 72 h (Safdar et al., 2003). Although molecular assays promise a quicker identification
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of MRSA colonization, because the results of the test can be available within a few hours, these methods are more expensive. However, quicker MRSA screening tests prevent unnecessary isolation procedures as well as the spread of MRSA in case of colonized patients, thus, reducing overall hospital costs. BD GeneOhm™ MRSA Assay (BD Diagnostics GeneOhm™, Erembodegem, Belgium), formerly IDI-MRSA, is a qualitative molecular diagnostic test that simultaneously detects the staphylococcal cassette chromosome mec (SSCmec) (carrying the mecA gene) and an S. aureusspecific sequence located within the orfX gene. The test is approved by the Food and Drug Administration for direct detection of nasal colonization by MRSA (BD GeneOhm™ Test Product Insert). Optimal screening for MRSA colonization includes testing of multiple sites; however, the use of GeneOhm™ assay for specimens from other sites or pooled surveillance specimens has not been fully validated (Bishop et al., 2006; de San et al., 2007; Sewell et al., 1993). Pooling of the patients' samples significantly reduces the cost of surveillance testing; however, previously described pooling methods prolong the testing procedure, and the results can be delayed (usually for 24 h) (Bishop et al., 2006; Desjardins et al., 2006). Jeyaratnam et al. (2008) published the evaluation of pooling method similar to our method. The aim of our study was to 1) evaluate the diagnostic performance of the GeneOhm™ MRSA assay (BD Diagnostics GeneOhm™) for detection of MRSA from pooled skin, throat, and nasal swabs from patients hospitalized in a tertiary care hospital compared with routine culture methods as a gold standard using enrichment broth and selective agar medium, and 2) evaluate the performance of BD GeneOhm™ MRSA assay modified in such way that enables us to obtain the results on the same day. By pooling the patients' samples, the cost of surveillance testing is significantly reduced while retaining the speed of the original assay. We present the data from our study period with optimization of modified protocol and subsequent routine use of rapid polymerase chain reaction (PCR)-based screening for MRSA colonization.
2. Materials and methods 2.1. Patients 2.1.1. Study period Surveillance specimens were collected from patients attending the 2678-bed Slovenian tertiary and teaching hospital University Medical Centre Ljubljana, Ljubljana, Slovenia (approximately 100 000 admissions, 640 000 patient days in 2006). Samples from nose (both anterior nares), skin (combined groin and axilla), and throat were collected on dry swabs from hospitalized patients and placed into Stuart transport medium (Copan Diagnostics, Corona, CA). During the study period (March to September 2007) for almost two-thirds of the patients (183 pools), the samples
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were taken from 3 surveillance sites (nose, skin, and throat); for the remaining 114 patients, 2 surveillance sites were screened (skin, nose). 2.1.2. Routine rapid PCR screening After successful integration of rapid MRSA screening test into our routine testing, the data from all subsequent patients during the 1st year was also included in our analysis (additional 136 pools, a total of 433 pools). 2.2. Clinical samples 2.2.1. Study period Two sets of swabs (nose, skin with or without throat) were collected from each patient, 1 for the conventional culture detection and the other for PCR assay and cultivation from enrichment broth. In accordance with manufacturer's instructions, patients with documented MRSA infection and patients treated with glycopeptides were not included in the study. The surveillance specimens taken from patients following decolonization procedure to determine the effectiveness of procedure were also not included in the study. We did not include rectal and wound swabs because of the likely presence of interfering substances (van Belkum et al., 2007). 2.2.2. Routine rapid PCR screening One set of swabs (nose, skin with or without throat) was collected from each patient for PCR assay. Because of high negative predictive value (NPV) of the modified GeneOhm™ MRSA assay for pooled samples established during the study period, conventional cultivation was subsequently performed for all unresolved tests to establish true status (see below). Conventional cultivation was also performed for all PCR-positive tests to determine antimicrobial susceptibility and site of colonization. 2.3. Conventional culture detection One set of swabs was plated onto nonselective blood agar (BA) and selective oxacillin resistance screening agar (ORSA; Oxoid Limited, Basingstoke, England) and inoculated into enrichment broth medium (Todd–Hewitt broth) for 24 h at 35 °C and for 48 h at 35 °C and inspected daily. If the enrichment broth became turbid after 24 h, it was subcultured onto BA and ORSA agar plates for 48 h at 35 °C and inspected daily. If turbidity was not observed after 48 h, it was streaked on BA, which was then incubated for 48 h. Yellow or white colonies with or without hemolysis on BA suspected to be S. aureus were further identified by positive tube coagulase test, the production of thermostable deoxyribonuclease (DNase), and mannitol fermentation (Brown et al., 2005). Characteristic colonies that appeared after 24 to 48 h on ORSA medium suggestive of MRSA were identified as S. aureus and tested for methicillin resistance. S. aureus isolates were screened for methicillin resistance by using Mueller–Hinton agar containing 6 mg/L oxacillin and 4% NaCl and by cefoxitin disk diffusion
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method according to Clinical and Laboratory Standards Institute (2007). The other set of swabs used for PCR assay was inoculated into enrichment broth (each swab was cultivated individually) and subcultured on BA and ORSA as described above (Fig. 1). 2.4. Modified BD GeneOhm™ MRSA assay Methicillin-resistant S. aureus-specific primers amplify the genetic target (SSCmec and orfX gene), if present. Internal control is included in the assay to detect PCR inhibitory specimens and, at the same time, to confirm the integrity of the assay reagents (BD GeneOhm™ Test Product Insert). The original protocol was modified during specimen preparation step by 1st adding an extra washing step to reduce the increased concentration of potential inhibitors followed by pooling of up to 3 samples per patient (nose, skin, with or without throat) at the lysis step as advised by the manufacturer (personal communication). Each swab was vortexed separately at high speed in 300 μL of sample buffer (300 μL were used instead of 1 mL according to manufacturer's suggestion for pooling of the samples [personal communication]). An extra washing step was performed according to BD protocol suggestions for preparation of the wound swabs by transferring the entire
suspension into sterile Eppendorf tube and centrifugation for 5 min at 13.000 × g (personal communication). Supernatant was discarded and 300 μL of fresh sample buffer added (additional sample buffer tubes are ordered separately). The pellet was dissolved by vortexing the tube for 60 seconds. The specimens were then pooled by transferring 300 μL of volume of the cell suspension from each swab included into the pool to a lysis tube and further processed as a single sample (Fig. 2). The subsequent procedure was performed according to the original manufacturer's protocol. Smart Cycler® II instrument (Cepheid, Sunnyvale, CA) was used for PCR amplification using Smart Cycler® Dx software version 1.7b. Positive and negative controls were included in each run. Polymerase chain reaction assay was repeated if the internal control was invalid or if the positive or negative control of the run was invalid. The samples with invalid internal control were interpreted as unresolved specimens and the assay as an inhibited test (BD GeneOhm™ Test Product Insert). These specimens were retested using 5-fold dilution of the extracted DNA with nuclease free water. 2.5. Detection of mecA gene The presence of mecA gene was confirmed for isolates with discordant results (culture positive, PCR negative) using a signal-amplified sandwich hybridization kit, MRSA
Fig. 1. Processing of the samples.
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Fig. 2. Sample preparation and DNA extraction.
EVIGENE™ Advan Dx MRSA Culture Identification Kit (AdvanDx, Woburn, MA). 2.6. Data analysis and discordant results During the study period, GeneOhm™ MRSA results were compared with results of conventional cultivation from both sets of samples. In addition, laboratory records of the patients with a negative culture and a positive PCR result were reviewed to collect information on previously documented MRSA colonization or infection or MRSA decolonization procedure as well as concurrent glycopeptide treatment. A true-positive result was defined as PCR assaypositive and culture-positive result (1 or both sets). A truenegative result was defined as PCR assay-negative and culture-negative (both sets) results. False-positive result was defined as PCR assay-positive and culture-negative result (both sets). False-negative result was defined as PCR assaynegative and culture-positive result (1 or both sets). 2.7. Statistical analysis
3. Results 3.1. Evaluation of the GeneOhm™ MRSA assay on clinical samples 3.1.1. Analysis of the performance of the modified BD GeneOhm™ MRSA assay Of the 297 pools included in our study, 229 (77.1%) were PCR negative, 34 (11.4%) were PCR positive, and 34 (11.4%) were unresolved after the 1st run (Tables 1 and 2). Sixteen of 114 (14.0%) nose–skin pools and 18 of 183 (9.8%) nose–skin–throat pools were initially unresolved. As shown in Table 1, all initially unresolved samples were resolved upon retesting using 5-fold dilution (as described above). One was resolved as positive (2.9%), and the others (97.1%) were negative. These results were also
Table 1 Analysis of the inhibited tests (assays with invalid internal control) and the results after resolution of the tests Pool type
Sensitivity, specificity, positive predictive value (PPV), and NPV of the modified GeneOhm™ MRSA assay of the pooled samples were calculated for i) all of the pooled samples, ii) the combination of nasal, throat, and skin (NTS) swabs, and iii) nasal and skin (NS) swabs separately. Additional statistical analysis was performed using SPSS version 15.0 for Windows (SPSS, Chicago, IL).
No. of unresolved tests after 1st run (%) No. of tests resolved upon retesting (%) No. of negative (%) No. of positive (%)
All
NTS
NS
34 (11.4)
18 (9.8)
16 (14.0)
34 (100.0)
18 (100.0)
16 (100.0)
33 (97.1) 1 (2.9)
18 (100.0) 0 (0.0)
15 (93.4) 1 (6.6)
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included in analysis of the performance of the modified BD GeneOhm™ MRSA assay. The analysis of the performance of the modified BD GeneOhm™ MRSA assay was based solely on the study data. To determine the performance of the PCR assay, we included the results from all valid tests (all initially resolved tests and all resolved initially unresolved tests). After the resolution of unresolved tests, 262 (88.2%) pools were PCR negative and 35 (11.8%) were PCR positive as shown in Table 2. The overall sensitivity of the modified PCR assay compared with cultivation was 94.3%, with high specificity 99.2%, NPV 99.2%, and PPV 94.3%. 3.1.2. The results of conventional cultivation No MRSA was detected from 262 (88.2%) pooled samples. Methicillin-resistant S. aureus was isolated from 35 (11.8%) pooled samples; 33 (94.3%) of them were also positive in the GeneOhm™ MRSA assay (true-positive results). The MRSA colonization rate among the study population was 11.0%. 3.1.3. Analysis of unresolved tests As shown above, the rate of inhibition was 11.4%. We have noted that the rate of inhibition depended on the batch of the assay. In 1 assay batch, 5 (4.6%) of 108 tests were initially unresolved; in the other assay batch, 21 (13.5%) of 156 were initially unresolved. To better evaluate the possible effect of different batches of assay, we analyzed the results of tests performed during the 1st year using the modified assay (1-year period; 433 pools tested, 36 initially unresolved) and found similar differences. In the 3rd batch, 9 (14.5%) of 62 pools were initially unresolved; in the 4th batch (79 pools), there were no unresolved tests. There were no statistically significant differences (P = 0.237) in the proportion of unresolved tests based on different pool combination, 20 (7.1%) of 280 NTS pools and 16 (10.0%) of 160 NS pools. We have, however, established that there was a statistically significant difference between Table 2 The overview of results of the study and statistical analysis of performance of the modified BD GeneOhm™ MRSA assay and culture-based methods Pool type
No. of sets of samples PCR assay No. of positive (%) No. of negative (%) No. of false negative (%) No. of false positive (%) Cultivation No. of positive (%) No. of negative (%) Statistical analysis PPV (%) NPV (%) Specificity (%) Sensitivity (%)
All
NTS
NS
297
183
114
35 (11.8) 262 (88.2) 2 (0.8) 2 (5.7)
19 (10.4) 164 (89.6) 2 (1.2) 0 (0.0)
16 (14.0) 98 (86.0) 0 (0.0) 2 (12.5)
35 (11.8) 262 (88.2)
21 (11.5) 162 (88.5)
14 (12.3) 100 (87.7)
100.0 98.8 100.0 90.5
87.5 100.0 98.0 100.0
94.3 99.2 99.2 94.3
the proportion of unresolved tests and different batches of assay (P = 0.019). 3.1.4. Discordant results In 2 cases, PCR assay was false negative; in both cases, nose, skin, and throat swabs were included in the pool. Methicillin-resistant S. aureus was cultured only from swabs used for routine cultivation. No methicillin-susceptible S. aureus was cultured. In both cases, the presence of mecA gene was confirmed. In the 1st case, less than 10 colonyforming units (CFU) were cultured on solid media, which may indicate low bacterial load on the original swab. In the 2nd case, more than 10 CFU of MRSA were cultured on solid media from 1 set of swabs, which does not indicate low-level colonization of the patient. In both cases, the repeated PCR assay performed on isolated DNA with another batch of test kit was negative. We have again repeated the PCR assay using suspension of pure culture (at a concentration of 105 CFU/mL) because we wanted to exclude the possibility of lower sensitivity of the assay for these particular strains. The repeated PCR assay yielded positive result in both cases. In 2 cases, the PCR assay yielded a false-positive result; in both cases, nose and skin swabs were included in the pool. In the 1st case, the repeated PCR assay performed on isolated DNA with another batch of test kit was negative; the review of laboratory records from this patient showed that MRSA was isolated 1 month later from a tracheal aspirate. In the 2nd case, the repeated PCR assay performed on isolated DNA with another batch of test kit was positive. The review of laboratory records from this patient showed that MRSA was isolated only from throat swab during the same period using both PCR assay and conventional cultivation. 3.2. Specimen preparation time An experienced technician prepares the samples from a single patient and both controls in 30 min; every additional patient adds approximately 10 min to the proceedings. Because Smart Cycler® allows for simultaneous testing of 14 samples (and 2 controls), the maximum time for preparation based on our modified procedure is 2 h 40 min. Because the PCR assay itself takes approximately 60 min, the results of the screening test are available in 3 h 40 min at the latest. In case of unresolved test, the dilution of the sample takes another 10 min for sample preparation and another hour for PCR assay. 3.3. Cost-effectiveness of pooling method Material costs of our pooling method were compared with material costs of unmodified PCR assay. When using the unmodified method, 2 or 3 PCR assays are performed per patient (depending on the number of surveillance sites). One PCR assay costs approximately €25 (Rossney et al., 2007). By pooling of the samples at the lysis step, only 1 PCR assay per patient is performed; additional sample buffer (1 tube per swab) in extra washing step costs
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approximately €3.74. Even when increased rate of inhibition is taken into account, the material costs of our pooling method are still reduced. Another factor to consider is the fact that positive and negative controls are included in each run regardless of the number of tested samples, which influences the cost-effectiveness of the test. When 2 samples per patient are pooled, the material costs are thus reduced by 17% (1 patient) to 46% (14 simultaneously tested patients). When 3 samples per patient are pooled, the material costs are reduced by 30% (1 patient) to 62% (14 simultaneously tested patients).
4. Discussion Reliable rapid detection of MRSA colonized patients is essential for successful control and prevention of MRSA spread in hospital, which improves patient care and hospital infection control. BD GeneOhm™ MRSA assay has the advantage of speed because the results can be available within a few hours. As shown in previous studies, BD GeneOhm™ MRSA assay is both specific and sensitive (Bishop et al., 2006; Desjardins et al., 2006; Rossney et al., 2007; van Hal et al., 2007). The main disadvantages of PCR assays are the increase in expenses especially for screening of the multiple surveillance specimens and the possibility of detection of residual DNA from nonviable bacteria (Rossney et al., 2007). To reduce the cost of patient screening, an alternative approach that allows pooling of multiple specimens and testing of the pooled specimens in a single PCR reaction is a cost-effective alternative. Several approaches are possible. One is brothPCR method with pooling of the surveillance specimens from the same patient in enrichment broth followed by PCR assay from the broth as described previously (Bishop et al., 2006; Desjardins et al., 2006). This screening process still requires several hours for broth incubation, which is an important drawback because the results are delayed for up to 24 h. We have opted for a different approach and modified the original protocol by pooling the samples at the lysis step. The procedure is faster because the results are available within a few hours even in the case of unresolved test. Pooling of the samples adds only 10 min per patient during the sample preparation step. Moderately increased duration of sample preparation was previously described for other pooling methods as well (Bishop et al., 2006; Rossney et al., 2007). Similar method for pooling of the patients samples was described by Jeyaratnam et al. (2008). They have evaluated the performance of the modified assay on selected patient population with positivity rate of MRSA of 50%. As shown in Table 2, the performance of the modified GeneOhm™ MRSA assay depended on the type of pooled samples. Sensitivity of the assay was very good for pools that contained only nose and skin samples (sensitivity, 100.0%; specificity, 98.0%); we have however noted lower sensitivity of the test when throat swabs were included in the pool
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(sensitivity, 90.5%; specificity, 100.0%). Jeyaratnam et al. (2008) described the sensitivity and specificity for nose and skin pools of 85% and 95%, respectively. Similar results regarding the sensitivity of the GeneOhm™ MRSA for single throat swabs were already described by Rossney et al. (2007) (89.0% sensitivity and 99.0% specificity). van Hal et al. (2007) described combined sensitivity and specificity of 90.0% and 96.0%, respectively, for separate nose, groin, and axilla samples. In 2 cases, GeneOhm™ MRSA assay gave false-negative result; MRSA was however detected by repeated PCR assay performed on pure culture. The discordant results where PCR assay gave negative results while the cultures from 1 set of samples were positive could be related to low bacterial load on the original swab or the problems the kit may have in extracting DNA from some isolates (Rossney et al., 2007). There is however also a possibility of inadequate quality of the other set of swabs because both PCR assay and the results of subcultivation of the enrichment broth were negative (Bishop et al., 2006). In 2 cases, the GeneOhm™ MRSA assay gave false-positive result. The discordant results where PCR assay gave positive result, although ORSA failed to grow MRSA, could be related to a molecular test having a higher sensitivity than the standard cultivation method used at our institution, so it may be possible that these discordant results were actually true-positive results (de San et al., 2007; Drews et al., 2006). Some might argue that the problem with our study design is the fact that we used 2 sets of samples from the same body site. We did randomly assign one of the sets of samples for the cultivation and the other for PCR assay, thus, reducing the possible effect of sampling variation. The most important drawback of our pooling method is the increased rate of inhibition. We have noted that the rate of inhibition depended on the batch of the assay. Similar observations regarding variations in rate of inhibition in different batches of assay were also reported by Oberdorfer et al. (2006). Desjardins et al. (2006) described significantly lower rate of inhibition (b1.0%) when PCR assay was performed from samples pooled in a selective broth. Their pooling method reduced the concentration of potential inhibitors, which was especially important because they included rectal swabs. Bishop et al. (2006) found 4.0% inhibition rate for pools containing nose and skin (groin) samples and 7.5% for nose samples only. Jeyaratnam et al. (2008) who used a similar pooling method described a significantly lower level of inhibition of 1.5%. Their low inhibition rate could be the result of extra heating step they have used to liquefy the Amies transport gel, which could have reduced the concentration of potential inhibitors. For resolving the unresolved tests, we have decided to use the dilution method similar to that previously described by Drews et al. (2006). In our case, 5-fold dilution of the sample proved successful. The main disadvantage of freezing, the most frequently used method, which is also recommended by the manufacturer, is the prolonged testing procedure
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(Bishop et al., 2006; Desjardins et al., 2006; Rossney et al., 2007). The disadvantage of the dilution method is however the possibility of diluting the DNA beyond limit of detection. Dilution method affected the performance of the assay when the initial inoculum was low, close to the limit of detection (data not shown). We therefore recommend that concurrent enrichment broth cultivation be performed for all unresolved tests. According to our national guidelines, it is recommended that a throat swab is included in screening for MRSA colonization. During our study, 297 pools were tested using PCR assay. Among 35 PCR-positive assays, 33 were true positive. In 5 cases (14.3%), throat swab was the only site from which MRSA was cultured. Had the throat swab not been included, these 5 patients would have been missed. We have been using the modified BD GeneOhm™ MRSA assay as a rapid PCR screening of pooled samples for more than 1 year. During the 1st year, 433 pools were tested. This may seem a low number of tests; however, the test is mostly used for rapid screening of patients in the intensive care units and for patients awaiting major surgical procedure such as major cardiovascular surgery. 5. Conclusion We have successfully modified the BD GeneOhm™ MRSA assay to allow pooling of the surveillance specimens with the results available the same day even in case of unresolved test with immediate retesting using the dilution method. Our modified protocol for pooling of the surveillance specimens did not interfere with the performance of the GeneOhm™ MRSA assay. The test has been subsequently established as a part of routine screening test and has now been in use for more than 1 year. The modified test is reliable and performs well compared with routine cultivation methods in our clinical setting with low MRSA prevalence. Our findings support the use of pooling of patient samples as a cost-effective way of rapid screening for MRSA colonization in low-level prevalence clinical settings. Acknowledgments We are grateful to BD for providing us with the Smart Cycler® platform and GeneOhm™ MRSA kit at evaluation rate. We are grateful to the Department of Infectious Diseases, University Medical Centre Ljubljana for providing us with majority of the samples and the Department of Infection Control, University Medical Centre Ljubljana, for their help in promoting the test to clinicians. References BD GeneOhm™ Test Product Insert. Bishop EJ, Grabsch EA, Ballard SA, Mayall B, Xie S, Martin R, Grayson ML (2006) Concurrent analysis of nose and groin swab specimens by the IDIMRSA PCR assay is comparable to analysis by individual-specimen
PCR and routine culture assays for detection of colonization by methicillin-resistant Staphylococcus aureus. J Clin Microbiol 44: 2904–2908. Brown DF, Edwards DI, Hawkey PM, Morrison D, Ridgway GL, Towner KJ, Wren MW (2005) Guidelines for the laboratory diagnosis and susceptibility testing of methicillin-resistant Staphylococcus aureus (MRSA). J Antimicrob Chemother 56:1000–1018. Clinical and Laboratory Standards Institute (2007) Performance standards for antimicrobial susceptibility testing. Seventeenth informational supplement. CLSI document M100-S17. Wayne, PA: Clinical and Laboratory Standards Institute. Cosgrove SE, Sakoulas G, Perencevich EN, Schwaber MJ, Karchmer AW, Carmeli Y (2003) Comparison of mortality associated with methicillinresistant and methicillin-susceptible Staphylococcus aureus bacteremia: a meta-analysis. Clin Infect Dis 36:53–59. de San N, Denis O, Gasasira MF, De Mendonca R, Nonhoff C, Struelens MJ (2007) Controlled evaluation of the IDI-MRSA assay for detection of colonization by methicillin-resistant Staphylococcus aureus in diverse mucocutaneous specimens. J Clin Microbiol 45: 1098–1101. Desjardins M, Guibord C, Lalonde B, Toye B, Ramotar K (2006) Evaluation of the IDI-MRSA assay for detection of methicillin-resistant Staphylococcus aureus from nasal and rectal specimens pooled in a selective broth. J Clin Microbiol 44:1219–1223. Drews SJ, Willey BM, Kreiswirth N, Wang M, Ianes T, Mitchell J, Latchford M, McGeer AJ, Katz KC (2006) Verification of the IDI-MRSA assay for detecting methicillin-resistant Staphylococcus aureus in diverse specimen types in a core clinical laboratory setting. J Clin Microbiol 44: 3794–3796. EARSS Management Team (2006) EARSS annual report 2006. Bilthoven, The Netherlands: EARSS Management Team. [Online.] http://www. rivm.nl/earss/database/. Cited June 21, 2008. Jeyaratnam D, Gottlieb A, Ajoku U, French GL (2008) Validation of the IDIMRSA™ system for use on pooled nose, axilla, and groin swabs and single swabs from other screening sites. Diagn Microbiol Infect Dis 61: 1–5. Lodise TP, McKinnon PS (2005) Clinical and economic impact of methicillin resistance in patients with Staphylococcus aureus bacteremia. Diagn Microbiol Infect Dis 52:113–122. Melzer M, Eykyn SJ, Gransden WR, Chinn S (2003) Is methicillin-resistant Staphylococcus aureus more virulent than methicillin-susceptible S. aureus? A comparative cohort study of British patients with nosocomial infection and bacteremia. Clin Infect Dis 37:1453–1460. Ministry of Health of the Republic of Slovenia Multidisciplinary working group (2003) First national guidelines 2000–2003 for preventing HAIs. Ljubljana, Slovenia: Hospital Infection Control Committee. Oberdorfer K, Pohl S, Frey M, Heeg K, Wendt C (2006) Evaluation of a single-locus real-time polymerase chain reaction as a screening test for specific detection of methicillin-resistant Staphylococcus aureus in ICU patients. Eur J Clin Microbiol Infect Dis 10:657–663. Rossney AS, Herra CM, Fitzgibbon MM, Morgan PM, Lawrence MJ, O'Connell B (2007) Evaluation of the IDI-MRSA assay on the Smart Cycler® real-time PCR platform for rapid detection of MRSA from screening specimens. Eur J Clin Microbiol Infect Dis 26: 459–466. Rubinovitch B, Pittet D (2001) Screening for methicillin-resistant Staphylococcus aureus in the endemic hospital: what have we learned? J Hosp Infect 47:9–18. Safdar N, Narans L, Gordon B, Maki DG (2003) Comparison of culture screening methods for detection of nasal carriage of methicillin-resistant Staphylococcus aureus: a prospective study comparing 32 methods. J Clin Microbiol 41:3163–3166. Sewell DL, Potter SA, Jacobson CM, Strausbaugh LJ, Ward TT (1993) Sensitivity of surveillance cultures for the detection of methicillinresistant Staphylococcus aureus in a nursing-home-care unit. Diagn Microbiol Infect Dis 17:53–56.
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Diagnostic Microbiology and Infectious Disease 63 (2009) 132 – 139 www.elsevier.com/locate/diagmicrobio
One-year experience with modified BD GeneOhm™ MRSA assay for detection of methicillin-resistant Staphylococcus aureus from pooled nasal, skin, and throat samples Natasa Svent-Kucina⁎, Mateja Pirs, Manica Mueller-Premru, Vesna Cvitkovic-Spik, Romina Kofol, Katja Seme Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 1000 Ljubljana, Slovenia Received 21 July 2008; accepted 10 October 2008
Abstract We report our 1-year experience with modified GeneOhm™ MRSA assay (formerly IDI-MRSA) for pooled surveillance specimens in low methicillin-resistant Staphylococcus aureus (MRSA) prevalence clinical setting. We have successfully modified the GeneOhm™ MRSA assay protocol during the specimen preparation step by adding an extra washing step followed by pooling of up to 3 samples per patient (nose, skin, with or without throat) at the lysis step. The sensitivity of the modified assay compared with conventional cultivation was 94.3%, specificity 99.2%, negative predictive value 99.2%, and positive predictive value 94.3%. The modified test is reliable and performed well compared with conventional culture methods in our clinical setting with low-level prevalence of MRSA colonization. Our findings support the use of pooling of the patients samples as a cost-effective way of screening for MRSA colonization. © 2009 Elsevier Inc. All rights reserved. Keywords: MRSA; Surveillance; Pooling; GeneOhm™ MRSA assay; IDI-MRSA
1. Introduction Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major nosocomial pathogens responsible for increased morbidity, mortality, and prolonged hospitalization that results in increased hospital costs taxing the health care system (Cosgrove et al., 2003; Lodise and McKinnon, 2005; Melzer et al., 2003). Methicillin-resistant S. aureus prevalence varies significantly among European countries and among individual hospitals and accounts for less than 1% to more than 50% of bloodstream S. aureus infections. In Slovenia, the proportion of MRSA isolates from blood cultures in hospitalized patients has been steadily decreasing from 21.4% in the year 2000 to 7.1% in the year 2006 (EARSS annual report, 2006). Effective ways to reduce MRSA spread in hospitals are active surveillance cultures, isolation or cohort isolation ⁎ Corresponding author. Tel.: +386-1-54374-48; fax: +386-1-54374-01. E-mail addresses: [email protected], [email protected] (N. Svent-Kucina). 0732-8893/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2008.10.005
of the colonized or infected patients, decolonization procedures performed according to hospital recommendations, as well as continuous education of all of the health care workers (Ministry of Health of the Republic of Slovenia, 2003; Rubinovitch and Pittet, 2001). Current Slovenian recommendations for MRSA screening include culturing of the nose and skin (axilla and groin, or wound) swabs, with or without throat swab from patients who are transferred from another ward (particularly intensive care units) or another hospital or who live in a long-term care facility; patients who were hospitalized at least 3 times during the last year; patients previously infected or colonized with MRSA; patients who have chronic wounds; patients who were hospitalized in the same room as an MRSA carrier for more than 3 days. Screening for MRSA is performed at the time of admission or within the first 48 h (Ministry of Health of the Republic of Slovenia, 2003). The results of the conventional culture-based screening methods with cultivation, identification, and antimicrobial susceptibility testing take 48 to 72 h (Safdar et al., 2003). Although molecular assays promise a quicker identification
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of MRSA colonization, because the results of the test can be available within a few hours, these methods are more expensive. However, quicker MRSA screening tests prevent unnecessary isolation procedures as well as the spread of MRSA in case of colonized patients, thus, reducing overall hospital costs. BD GeneOhm™ MRSA Assay (BD Diagnostics GeneOhm™, Erembodegem, Belgium), formerly IDI-MRSA, is a qualitative molecular diagnostic test that simultaneously detects the staphylococcal cassette chromosome mec (SSCmec) (carrying the mecA gene) and an S. aureusspecific sequence located within the orfX gene. The test is approved by the Food and Drug Administration for direct detection of nasal colonization by MRSA (BD GeneOhm™ Test Product Insert). Optimal screening for MRSA colonization includes testing of multiple sites; however, the use of GeneOhm™ assay for specimens from other sites or pooled surveillance specimens has not been fully validated (Bishop et al., 2006; de San et al., 2007; Sewell et al., 1993). Pooling of the patients' samples significantly reduces the cost of surveillance testing; however, previously described pooling methods prolong the testing procedure, and the results can be delayed (usually for 24 h) (Bishop et al., 2006; Desjardins et al., 2006). Jeyaratnam et al. (2008) published the evaluation of pooling method similar to our method. The aim of our study was to 1) evaluate the diagnostic performance of the GeneOhm™ MRSA assay (BD Diagnostics GeneOhm™) for detection of MRSA from pooled skin, throat, and nasal swabs from patients hospitalized in a tertiary care hospital compared with routine culture methods as a gold standard using enrichment broth and selective agar medium, and 2) evaluate the performance of BD GeneOhm™ MRSA assay modified in such way that enables us to obtain the results on the same day. By pooling the patients' samples, the cost of surveillance testing is significantly reduced while retaining the speed of the original assay. We present the data from our study period with optimization of modified protocol and subsequent routine use of rapid polymerase chain reaction (PCR)-based screening for MRSA colonization.
2. Materials and methods 2.1. Patients 2.1.1. Study period Surveillance specimens were collected from patients attending the 2678-bed Slovenian tertiary and teaching hospital University Medical Centre Ljubljana, Ljubljana, Slovenia (approximately 100 000 admissions, 640 000 patient days in 2006). Samples from nose (both anterior nares), skin (combined groin and axilla), and throat were collected on dry swabs from hospitalized patients and placed into Stuart transport medium (Copan Diagnostics, Corona, CA). During the study period (March to September 2007) for almost two-thirds of the patients (183 pools), the samples
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were taken from 3 surveillance sites (nose, skin, and throat); for the remaining 114 patients, 2 surveillance sites were screened (skin, nose). 2.1.2. Routine rapid PCR screening After successful integration of rapid MRSA screening test into our routine testing, the data from all subsequent patients during the 1st year was also included in our analysis (additional 136 pools, a total of 433 pools). 2.2. Clinical samples 2.2.1. Study period Two sets of swabs (nose, skin with or without throat) were collected from each patient, 1 for the conventional culture detection and the other for PCR assay and cultivation from enrichment broth. In accordance with manufacturer's instructions, patients with documented MRSA infection and patients treated with glycopeptides were not included in the study. The surveillance specimens taken from patients following decolonization procedure to determine the effectiveness of procedure were also not included in the study. We did not include rectal and wound swabs because of the likely presence of interfering substances (van Belkum et al., 2007). 2.2.2. Routine rapid PCR screening One set of swabs (nose, skin with or without throat) was collected from each patient for PCR assay. Because of high negative predictive value (NPV) of the modified GeneOhm™ MRSA assay for pooled samples established during the study period, conventional cultivation was subsequently performed for all unresolved tests to establish true status (see below). Conventional cultivation was also performed for all PCR-positive tests to determine antimicrobial susceptibility and site of colonization. 2.3. Conventional culture detection One set of swabs was plated onto nonselective blood agar (BA) and selective oxacillin resistance screening agar (ORSA; Oxoid Limited, Basingstoke, England) and inoculated into enrichment broth medium (Todd–Hewitt broth) for 24 h at 35 °C and for 48 h at 35 °C and inspected daily. If the enrichment broth became turbid after 24 h, it was subcultured onto BA and ORSA agar plates for 48 h at 35 °C and inspected daily. If turbidity was not observed after 48 h, it was streaked on BA, which was then incubated for 48 h. Yellow or white colonies with or without hemolysis on BA suspected to be S. aureus were further identified by positive tube coagulase test, the production of thermostable deoxyribonuclease (DNase), and mannitol fermentation (Brown et al., 2005). Characteristic colonies that appeared after 24 to 48 h on ORSA medium suggestive of MRSA were identified as S. aureus and tested for methicillin resistance. S. aureus isolates were screened for methicillin resistance by using Mueller–Hinton agar containing 6 mg/L oxacillin and 4% NaCl and by cefoxitin disk diffusion
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method according to Clinical and Laboratory Standards Institute (2007). The other set of swabs used for PCR assay was inoculated into enrichment broth (each swab was cultivated individually) and subcultured on BA and ORSA as described above (Fig. 1). 2.4. Modified BD GeneOhm™ MRSA assay Methicillin-resistant S. aureus-specific primers amplify the genetic target (SSCmec and orfX gene), if present. Internal control is included in the assay to detect PCR inhibitory specimens and, at the same time, to confirm the integrity of the assay reagents (BD GeneOhm™ Test Product Insert). The original protocol was modified during specimen preparation step by 1st adding an extra washing step to reduce the increased concentration of potential inhibitors followed by pooling of up to 3 samples per patient (nose, skin, with or without throat) at the lysis step as advised by the manufacturer (personal communication). Each swab was vortexed separately at high speed in 300 μL of sample buffer (300 μL were used instead of 1 mL according to manufacturer's suggestion for pooling of the samples [personal communication]). An extra washing step was performed according to BD protocol suggestions for preparation of the wound swabs by transferring the entire
suspension into sterile Eppendorf tube and centrifugation for 5 min at 13.000 × g (personal communication). Supernatant was discarded and 300 μL of fresh sample buffer added (additional sample buffer tubes are ordered separately). The pellet was dissolved by vortexing the tube for 60 seconds. The specimens were then pooled by transferring 300 μL of volume of the cell suspension from each swab included into the pool to a lysis tube and further processed as a single sample (Fig. 2). The subsequent procedure was performed according to the original manufacturer's protocol. Smart Cycler® II instrument (Cepheid, Sunnyvale, CA) was used for PCR amplification using Smart Cycler® Dx software version 1.7b. Positive and negative controls were included in each run. Polymerase chain reaction assay was repeated if the internal control was invalid or if the positive or negative control of the run was invalid. The samples with invalid internal control were interpreted as unresolved specimens and the assay as an inhibited test (BD GeneOhm™ Test Product Insert). These specimens were retested using 5-fold dilution of the extracted DNA with nuclease free water. 2.5. Detection of mecA gene The presence of mecA gene was confirmed for isolates with discordant results (culture positive, PCR negative) using a signal-amplified sandwich hybridization kit, MRSA
Fig. 1. Processing of the samples.
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Fig. 2. Sample preparation and DNA extraction.
EVIGENE™ Advan Dx MRSA Culture Identification Kit (AdvanDx, Woburn, MA). 2.6. Data analysis and discordant results During the study period, GeneOhm™ MRSA results were compared with results of conventional cultivation from both sets of samples. In addition, laboratory records of the patients with a negative culture and a positive PCR result were reviewed to collect information on previously documented MRSA colonization or infection or MRSA decolonization procedure as well as concurrent glycopeptide treatment. A true-positive result was defined as PCR assaypositive and culture-positive result (1 or both sets). A truenegative result was defined as PCR assay-negative and culture-negative (both sets) results. False-positive result was defined as PCR assay-positive and culture-negative result (both sets). False-negative result was defined as PCR assaynegative and culture-positive result (1 or both sets). 2.7. Statistical analysis
3. Results 3.1. Evaluation of the GeneOhm™ MRSA assay on clinical samples 3.1.1. Analysis of the performance of the modified BD GeneOhm™ MRSA assay Of the 297 pools included in our study, 229 (77.1%) were PCR negative, 34 (11.4%) were PCR positive, and 34 (11.4%) were unresolved after the 1st run (Tables 1 and 2). Sixteen of 114 (14.0%) nose–skin pools and 18 of 183 (9.8%) nose–skin–throat pools were initially unresolved. As shown in Table 1, all initially unresolved samples were resolved upon retesting using 5-fold dilution (as described above). One was resolved as positive (2.9%), and the others (97.1%) were negative. These results were also
Table 1 Analysis of the inhibited tests (assays with invalid internal control) and the results after resolution of the tests Pool type
Sensitivity, specificity, positive predictive value (PPV), and NPV of the modified GeneOhm™ MRSA assay of the pooled samples were calculated for i) all of the pooled samples, ii) the combination of nasal, throat, and skin (NTS) swabs, and iii) nasal and skin (NS) swabs separately. Additional statistical analysis was performed using SPSS version 15.0 for Windows (SPSS, Chicago, IL).
No. of unresolved tests after 1st run (%) No. of tests resolved upon retesting (%) No. of negative (%) No. of positive (%)
All
NTS
NS
34 (11.4)
18 (9.8)
16 (14.0)
34 (100.0)
18 (100.0)
16 (100.0)
33 (97.1) 1 (2.9)
18 (100.0) 0 (0.0)
15 (93.4) 1 (6.6)
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included in analysis of the performance of the modified BD GeneOhm™ MRSA assay. The analysis of the performance of the modified BD GeneOhm™ MRSA assay was based solely on the study data. To determine the performance of the PCR assay, we included the results from all valid tests (all initially resolved tests and all resolved initially unresolved tests). After the resolution of unresolved tests, 262 (88.2%) pools were PCR negative and 35 (11.8%) were PCR positive as shown in Table 2. The overall sensitivity of the modified PCR assay compared with cultivation was 94.3%, with high specificity 99.2%, NPV 99.2%, and PPV 94.3%. 3.1.2. The results of conventional cultivation No MRSA was detected from 262 (88.2%) pooled samples. Methicillin-resistant S. aureus was isolated from 35 (11.8%) pooled samples; 33 (94.3%) of them were also positive in the GeneOhm™ MRSA assay (true-positive results). The MRSA colonization rate among the study population was 11.0%. 3.1.3. Analysis of unresolved tests As shown above, the rate of inhibition was 11.4%. We have noted that the rate of inhibition depended on the batch of the assay. In 1 assay batch, 5 (4.6%) of 108 tests were initially unresolved; in the other assay batch, 21 (13.5%) of 156 were initially unresolved. To better evaluate the possible effect of different batches of assay, we analyzed the results of tests performed during the 1st year using the modified assay (1-year period; 433 pools tested, 36 initially unresolved) and found similar differences. In the 3rd batch, 9 (14.5%) of 62 pools were initially unresolved; in the 4th batch (79 pools), there were no unresolved tests. There were no statistically significant differences (P = 0.237) in the proportion of unresolved tests based on different pool combination, 20 (7.1%) of 280 NTS pools and 16 (10.0%) of 160 NS pools. We have, however, established that there was a statistically significant difference between Table 2 The overview of results of the study and statistical analysis of performance of the modified BD GeneOhm™ MRSA assay and culture-based methods Pool type
No. of sets of samples PCR assay No. of positive (%) No. of negative (%) No. of false negative (%) No. of false positive (%) Cultivation No. of positive (%) No. of negative (%) Statistical analysis PPV (%) NPV (%) Specificity (%) Sensitivity (%)
All
NTS
NS
297
183
114
35 (11.8) 262 (88.2) 2 (0.8) 2 (5.7)
19 (10.4) 164 (89.6) 2 (1.2) 0 (0.0)
16 (14.0) 98 (86.0) 0 (0.0) 2 (12.5)
35 (11.8) 262 (88.2)
21 (11.5) 162 (88.5)
14 (12.3) 100 (87.7)
100.0 98.8 100.0 90.5
87.5 100.0 98.0 100.0
94.3 99.2 99.2 94.3
the proportion of unresolved tests and different batches of assay (P = 0.019). 3.1.4. Discordant results In 2 cases, PCR assay was false negative; in both cases, nose, skin, and throat swabs were included in the pool. Methicillin-resistant S. aureus was cultured only from swabs used for routine cultivation. No methicillin-susceptible S. aureus was cultured. In both cases, the presence of mecA gene was confirmed. In the 1st case, less than 10 colonyforming units (CFU) were cultured on solid media, which may indicate low bacterial load on the original swab. In the 2nd case, more than 10 CFU of MRSA were cultured on solid media from 1 set of swabs, which does not indicate low-level colonization of the patient. In both cases, the repeated PCR assay performed on isolated DNA with another batch of test kit was negative. We have again repeated the PCR assay using suspension of pure culture (at a concentration of 105 CFU/mL) because we wanted to exclude the possibility of lower sensitivity of the assay for these particular strains. The repeated PCR assay yielded positive result in both cases. In 2 cases, the PCR assay yielded a false-positive result; in both cases, nose and skin swabs were included in the pool. In the 1st case, the repeated PCR assay performed on isolated DNA with another batch of test kit was negative; the review of laboratory records from this patient showed that MRSA was isolated 1 month later from a tracheal aspirate. In the 2nd case, the repeated PCR assay performed on isolated DNA with another batch of test kit was positive. The review of laboratory records from this patient showed that MRSA was isolated only from throat swab during the same period using both PCR assay and conventional cultivation. 3.2. Specimen preparation time An experienced technician prepares the samples from a single patient and both controls in 30 min; every additional patient adds approximately 10 min to the proceedings. Because Smart Cycler® allows for simultaneous testing of 14 samples (and 2 controls), the maximum time for preparation based on our modified procedure is 2 h 40 min. Because the PCR assay itself takes approximately 60 min, the results of the screening test are available in 3 h 40 min at the latest. In case of unresolved test, the dilution of the sample takes another 10 min for sample preparation and another hour for PCR assay. 3.3. Cost-effectiveness of pooling method Material costs of our pooling method were compared with material costs of unmodified PCR assay. When using the unmodified method, 2 or 3 PCR assays are performed per patient (depending on the number of surveillance sites). One PCR assay costs approximately €25 (Rossney et al., 2007). By pooling of the samples at the lysis step, only 1 PCR assay per patient is performed; additional sample buffer (1 tube per swab) in extra washing step costs
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approximately €3.74. Even when increased rate of inhibition is taken into account, the material costs of our pooling method are still reduced. Another factor to consider is the fact that positive and negative controls are included in each run regardless of the number of tested samples, which influences the cost-effectiveness of the test. When 2 samples per patient are pooled, the material costs are thus reduced by 17% (1 patient) to 46% (14 simultaneously tested patients). When 3 samples per patient are pooled, the material costs are reduced by 30% (1 patient) to 62% (14 simultaneously tested patients).
4. Discussion Reliable rapid detection of MRSA colonized patients is essential for successful control and prevention of MRSA spread in hospital, which improves patient care and hospital infection control. BD GeneOhm™ MRSA assay has the advantage of speed because the results can be available within a few hours. As shown in previous studies, BD GeneOhm™ MRSA assay is both specific and sensitive (Bishop et al., 2006; Desjardins et al., 2006; Rossney et al., 2007; van Hal et al., 2007). The main disadvantages of PCR assays are the increase in expenses especially for screening of the multiple surveillance specimens and the possibility of detection of residual DNA from nonviable bacteria (Rossney et al., 2007). To reduce the cost of patient screening, an alternative approach that allows pooling of multiple specimens and testing of the pooled specimens in a single PCR reaction is a cost-effective alternative. Several approaches are possible. One is brothPCR method with pooling of the surveillance specimens from the same patient in enrichment broth followed by PCR assay from the broth as described previously (Bishop et al., 2006; Desjardins et al., 2006). This screening process still requires several hours for broth incubation, which is an important drawback because the results are delayed for up to 24 h. We have opted for a different approach and modified the original protocol by pooling the samples at the lysis step. The procedure is faster because the results are available within a few hours even in the case of unresolved test. Pooling of the samples adds only 10 min per patient during the sample preparation step. Moderately increased duration of sample preparation was previously described for other pooling methods as well (Bishop et al., 2006; Rossney et al., 2007). Similar method for pooling of the patients samples was described by Jeyaratnam et al. (2008). They have evaluated the performance of the modified assay on selected patient population with positivity rate of MRSA of 50%. As shown in Table 2, the performance of the modified GeneOhm™ MRSA assay depended on the type of pooled samples. Sensitivity of the assay was very good for pools that contained only nose and skin samples (sensitivity, 100.0%; specificity, 98.0%); we have however noted lower sensitivity of the test when throat swabs were included in the pool
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(sensitivity, 90.5%; specificity, 100.0%). Jeyaratnam et al. (2008) described the sensitivity and specificity for nose and skin pools of 85% and 95%, respectively. Similar results regarding the sensitivity of the GeneOhm™ MRSA for single throat swabs were already described by Rossney et al. (2007) (89.0% sensitivity and 99.0% specificity). van Hal et al. (2007) described combined sensitivity and specificity of 90.0% and 96.0%, respectively, for separate nose, groin, and axilla samples. In 2 cases, GeneOhm™ MRSA assay gave false-negative result; MRSA was however detected by repeated PCR assay performed on pure culture. The discordant results where PCR assay gave negative results while the cultures from 1 set of samples were positive could be related to low bacterial load on the original swab or the problems the kit may have in extracting DNA from some isolates (Rossney et al., 2007). There is however also a possibility of inadequate quality of the other set of swabs because both PCR assay and the results of subcultivation of the enrichment broth were negative (Bishop et al., 2006). In 2 cases, the GeneOhm™ MRSA assay gave false-positive result. The discordant results where PCR assay gave positive result, although ORSA failed to grow MRSA, could be related to a molecular test having a higher sensitivity than the standard cultivation method used at our institution, so it may be possible that these discordant results were actually true-positive results (de San et al., 2007; Drews et al., 2006). Some might argue that the problem with our study design is the fact that we used 2 sets of samples from the same body site. We did randomly assign one of the sets of samples for the cultivation and the other for PCR assay, thus, reducing the possible effect of sampling variation. The most important drawback of our pooling method is the increased rate of inhibition. We have noted that the rate of inhibition depended on the batch of the assay. Similar observations regarding variations in rate of inhibition in different batches of assay were also reported by Oberdorfer et al. (2006). Desjardins et al. (2006) described significantly lower rate of inhibition (b1.0%) when PCR assay was performed from samples pooled in a selective broth. Their pooling method reduced the concentration of potential inhibitors, which was especially important because they included rectal swabs. Bishop et al. (2006) found 4.0% inhibition rate for pools containing nose and skin (groin) samples and 7.5% for nose samples only. Jeyaratnam et al. (2008) who used a similar pooling method described a significantly lower level of inhibition of 1.5%. Their low inhibition rate could be the result of extra heating step they have used to liquefy the Amies transport gel, which could have reduced the concentration of potential inhibitors. For resolving the unresolved tests, we have decided to use the dilution method similar to that previously described by Drews et al. (2006). In our case, 5-fold dilution of the sample proved successful. The main disadvantage of freezing, the most frequently used method, which is also recommended by the manufacturer, is the prolonged testing procedure
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(Bishop et al., 2006; Desjardins et al., 2006; Rossney et al., 2007). The disadvantage of the dilution method is however the possibility of diluting the DNA beyond limit of detection. Dilution method affected the performance of the assay when the initial inoculum was low, close to the limit of detection (data not shown). We therefore recommend that concurrent enrichment broth cultivation be performed for all unresolved tests. According to our national guidelines, it is recommended that a throat swab is included in screening for MRSA colonization. During our study, 297 pools were tested using PCR assay. Among 35 PCR-positive assays, 33 were true positive. In 5 cases (14.3%), throat swab was the only site from which MRSA was cultured. Had the throat swab not been included, these 5 patients would have been missed. We have been using the modified BD GeneOhm™ MRSA assay as a rapid PCR screening of pooled samples for more than 1 year. During the 1st year, 433 pools were tested. This may seem a low number of tests; however, the test is mostly used for rapid screening of patients in the intensive care units and for patients awaiting major surgical procedure such as major cardiovascular surgery. 5. Conclusion We have successfully modified the BD GeneOhm™ MRSA assay to allow pooling of the surveillance specimens with the results available the same day even in case of unresolved test with immediate retesting using the dilution method. Our modified protocol for pooling of the surveillance specimens did not interfere with the performance of the GeneOhm™ MRSA assay. The test has been subsequently established as a part of routine screening test and has now been in use for more than 1 year. The modified test is reliable and performs well compared with routine cultivation methods in our clinical setting with low MRSA prevalence. Our findings support the use of pooling of patient samples as a cost-effective way of rapid screening for MRSA colonization in low-level prevalence clinical settings. Acknowledgments We are grateful to BD for providing us with the Smart Cycler® platform and GeneOhm™ MRSA kit at evaluation rate. We are grateful to the Department of Infectious Diseases, University Medical Centre Ljubljana for providing us with majority of the samples and the Department of Infection Control, University Medical Centre Ljubljana, for their help in promoting the test to clinicians. References BD GeneOhm™ Test Product Insert. Bishop EJ, Grabsch EA, Ballard SA, Mayall B, Xie S, Martin R, Grayson ML (2006) Concurrent analysis of nose and groin swab specimens by the IDIMRSA PCR assay is comparable to analysis by individual-specimen
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