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Journal of Clinical Microbiology, September 2008, p. 3107-3109, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.02347-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

MVPlex Assay for Direct Detection of Methicillin-Resistant Staphylococcus aureus in Naris and Other Swab Specimens{triangledown}

Raymond P. Podzorski,1* Haijing Li,3 Jian Han,2 and Yi-Wei Tang3,4

Department of Pathology, Waukesha Memorial Hospital, Waukesha, Wisconsin 53188,1 Genaco Biomedical Products, Inc., Huntsville, Alabama 35805,2 Departments of Pathology,3 Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 372324

Received 6 December 2007/ Returned for modification 14 March 2008/ Accepted 3 July 2008


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ABSTRACT
 
We evaluated the MVPlex assay (Geneco Biomedical Products), which uses target-enriched multiplex PCR amplification followed by liquid array identification, for the detection of methicillin-resistant Staphylococcus aureus (MRSA) from 307 dual-swab specimens. By using a combination of culture (Trypticase soy agar-5% sheep blood agar and Columbia CNA agar-5% sheep blood) and an FDA-approved MRSA PCR assay as the "gold standard," the MVPlex MRSA assay and culture were found to have sensitivities of 97.8% and 84.4% (P = 0.002) and specificities of 95.8% and 98.6% (P < 0.05), respectively.


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TEXT
 
Methicillin-resistant Staphylococcus aureus (MRSA) is a well-known cause of hospital-associated nosocomial infections and contributes to significant morbidity and mortality in infected hospitalized patients (17, 20). Because of recent initiatives to provide better and safer care in U.S. hospitals, many health care providers are looking for more rapid and sensitive ways to identify patients who are colonized, or infected with, MRSA (19; see also http://www.ihi.org/IHI/Programs/Campaign). Better and faster detection of MRSA allows the health care provider to more efficiently prevent the spread of MRSA (3).

The aim of this study was to evaluate the performance of the MVPlex MRSA assay (Geneco Biomedical Products, Inc., Huntsville, AL), which incorporates a target-enriched multiplex PCR amplification and a liquid array identification procedure (11), to simultaneously amplify and identify 13 different molecular targets for the detection and differentiation of MRSA, methicillin-susceptible S. aureus, vancomycin-resistant enterococci, and several coagulase-negative Staphylococcus species. The performance of the MVPlex MRSA assay was evaluated by comparing it to a conventional culture method for MRSA detection and the BD GeneOhm MRSA assay (Becton-Dickinson, Inc., Sparks, MD; formerly called the IDI-MRSA assay) (1, 5, 6). This study was approved by the Waukesha Memorial Hospital Institutional Review Board (approval no. 464).

(This study was presented in part at the 107th General Meeting of the American Society for Microbiology, Toronto, Canada, 21 to 25 May 2007.)

Swab specimens. From 6 July to 17 November 2006, dual swabs from the BBL CultureSwab collection and transport system (catalog no. 22019; Becton-Dickinson, Inc., Sparks, MD) were used for the collection and transport of 307 specimens for MRSA screening. Specimens for MRSA screening were obtained from patients at Waukesha Memorial Hospital and individuals residing in extended-care facilities in Waukesha County, WI. The sources of specimens evaluated were the ear (1.3%), nares (76.2%), rectum (2.3%), and skin (20.2%).

Bacterial culture. One swab from a dual-swab specimen was used to inoculate a Trypticase soy agar-5% sheep blood plate and a Columbia CNA agar-5% sheep blood plate (BBL, Becton-Dickinson, Inc., Sparks, MD). The plates were incubated overnight at 35°C and reviewed the next day for colonies suggestive of S. aureus. Possible S. aureus colonies were tested by latex agglutination using Staphaureux (Remel, Inc., Lenexa, KS) to determine if they were S. aureus. Organisms identified as S. aureus were tested for methicillin resistance by using a cefoxitin disk as outlined by the CLSI (2).

DNA extraction and MVPlex assay. The second swab from the dual-swab specimen was eluted into 3 ml of brain heart infusion broth. DNA from a 1-ml aliquot of brain heart infusion broth was extracted using easyMAG (bioMérieux, Durham, NC), an automated nucleic acid extraction instrument, and eluted into 100 µl of water, according to the manufacturer's instructions (16, 21). Amplification of the nucleic acid target regions within the nuc, mecA, staphylococcal cassette chromosome mec (SCCmec)-orfX, vanA, vanB, ddl, and tuf genes was done using the MVPlex assay with 25 µl of extracted DNA added, according to the manufacturer's instructions. The amplified products were detected and characterized using a microsphere suspension array on a Luminex 100 instrument (Luminex, Corp., Austin, TX) and Genaco MDD software according to the MVPlex product insert (11). Results for each target were expressed as median fluorescence intensity (MFI) values.

BD GeneOhm MRSA assay. The easyMAG-extracted DNA (2.8 µl each) from certain patient specimens was used in a second PCR-based MRSA assay, the BD GeneOhm MRSA assay. The testing was done according to manufacturer's instructions as previously published (1, 5, 6). Strict laboratory precautions were used to prevent contamination of patient specimens when the two PCR assays were performed. The DNA from the specimens used in the BD GeneOhm MRSA assay was not extracted according to the assay's package insert; however, it has been well documented that the easyMAG produces concentrated, high-quality DNA from clinical specimens that works well in nucleic acid amplification assays (16, 21).

Result interpretation. DNA extracts that tested positive for the nuc, mecA, and SCCmec-orfX gene targets were considered MRSA. DNA extracts that lacked the mecA gene target were considered negative for MRSA. DNA extracts that tested positive for the nuc gene target and negative for the mecA gene target were considered methicillin-susceptible S. aureus. DNA extracts that tested positive for the mecA and one of the tuf gene targets were considered methicillin-resistant coagulase-negative Staphylococcus spp. DNA extracts that lacked the nuc gene target were considered negative for S. aureus. Amplified products from ddl (encodes a D-alanine:D-alanine ligase used to detect and differentiate Enterococcus faecalis and Enterococcus faecium), vanA and vanB (encode proteins associated with vancomycin resistance in E. faecalis and E. faecium), and tuf (encodes elongation factor Tu used to detect and differentiate many species of coagulase-negative Staphylococcus spp.) were not applicable to the goals of this study and will not be discussed further (4, 12).

During the study period, a total of 307 dual-swab specimens were cultured for MRSA and tested by the MVPlex assay for MRSA. In our study, the hands-on time to run 92 specimens in the MVPlex assay was approximately 65 min and the entire procedure could be completed within 5 h. In contrast, the hands-on time to run 14 specimens and 2 controls using the BD GeneOhm MRSA is approximately 75 min (when specimens are processed according to the manufacturer's instructions) and the entire process can be completed in about 2.5 h. In our laboratory, the Xpert MRSA assay (Cepheid, Sunnyvale, CA) requires approximately 35 min of hands-on time to run 14 specimens and the entire process can be completed in approximately 2 h (10).

The MVPlex assay detected MRSA in 97 of the specimens, while culture detected MRSA in 79 of the specimens (Table 1). Of 234 nares specimens, 26.5% were positive by culture and 33.3% were positive by the MVPlex assay. Of 62 skin specimens, 25.8% were positive by culture and 29.0% were positive by the MVPlex assay. Of four ear specimens, 25.0% were positive by culture and 25.0% were positive by the MVPlex assay. None of the seven rectal specimens were positive for MRSA by either assay. There were 28 discrepant results between the culture and the MVPlex assay. Five specimens were MRSA positive by culture and MRSA negative in the MVPlex assay, and 23 specimens were MRSA negative by culture and MRSA positive in the MVPlex assay. The DNA extracts from these 28 specimens were retested using the BD GeneOhm MRSA assay. When combined results from culture and the BD GeneOhm assay were used as an adjusted "gold standard," the MVPlex MRSA assay had a sensitivity of 97.8% compared to 84.4% for the culture procedure ({chi}2 = 9.89, P = 0.002). There was no significant difference ({chi}2 = 3.08, P > 0.05) between the specificities of the MVPlex MRSA assay (95.8%) and the culture procedure (98.6%) (Table 2).


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  		TABLE 1. Comparison of culture and MVPlex assay target amplification for detection of MRSA


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  		TABLE 2. Comparison of MVPlex assay and adjusted gold standarda for detection of MRSA

Nucleic acid amplification-based procedures approved by the FDA, such as the BD GeneOhm MRSA assay or the Cepheid Xpert MRSA assay, have been reported by many to be sensitive and specific for the detection of MRSA in patient specimens (1, 5, 6, 13, 18). The MVPlex assay has several advantages over these FDA-approved assays. The MVPlex assay is able to detect both MRSA and vancomycin-resistant enterococci, two well-know causes of nosocomial infections. In addition, the MVPlex assay is capable of greater specificity for MRSA than the two FDA-approved MRSA assays because it targets not only the SCCmec-orfX junction, the target region used by the BD GeneOhm MRSA assay and the Cepheid Xpert MRSA assay, but also the mecA gene itself (10, 13). This allows the MVPlex assay to differentiate MRSA from strains of S. aureus that have a truncated SCCmec (and lack the mecA gene) but still contain the SCCmec-orfX target region used in the FDA-approved MRSA assays. Truncation of SCCmec, resulting in the loss of a functional mecA gene, has been shown to lead to false-positive MRSA results in the BD GeneOhm MRSA assay (6, 7, 13, 14, 15). With increasing interest among surgeons to screen patients not only for MRSA but also for methicillin-susceptible S. aureus prior to surgery, the ability of the MVPlex assay to differentiate MRSA from methicillin-susceptible S. aureus makes it a viable option for testing these specimens.

While microsphere suspension arrays such as the Luminex system incorporated in the MVPlex assay can be very powerful tools for infectious disease testing, there are certain challenges to be addressed before they can be routinely used in the clinical laboratory. Background fluorescence was observed to vary substantially for different target-probe combinations in the MVPlex MRSA assay. For this study, the cutoff used for a positive mecA result was set at 1,000 MFI, while the cutoff for a positive SCCmec-orfX was set at 115 MFI. However, the MVPlex MRSA assay detected MRSA in many more specimens than our culture-based screening procedure. This was not surprising, since it has been shown that MRSA culture-based screening using nonselective media has lower sensitivity than culture-based MRSA screening procedures that use selective and/or broth culture enrichment media (8, 9). Using a combination of the BD GeneOhm MRSA assay and the culture procedure as the "gold standard," the MVPlex MRSA assay had a sensitivity of 97.8% and a specificity of 95.8% for MRSA while our culture method had a sensitivity of 84.4% and a specificity of 98.6%.


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ACKNOWLEDGMENTS
 
We thank the Waukesha Memorial Hospital Microbiology Laboratory personnel for their valuable contribution to the study.


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Pathology, Waukesha Memorial Hospital, 725 American Ave., Waukesha, WI 53188. Phone: (262) 928-7635. Fax: (262) 928-7962. E-mail: raymond.podzorski{at}phci.org Back

{triangledown} Published ahead of print on 9 July 2008. Back


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Journal of Clinical Microbiology, September 2008, p. 3107-3109, Vol. 46, No. 9
0095-1137/08/$08.00+0     doi:10.1128/JCM.02347-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Miller, M. B., Tang, Y.-W. (2009). Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology. Clin. Microbiol. Rev. 22: 611-633 [Abstract] [Full Text]  

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