- Email: [email protected]
Comparison of the BD MAX MRSA XT to the Cepheid™ Xpert® MRSA assay for the molecular detection of methicillin-resistant Staphylococcus aureus from nasal swabs
Comparison of the BD MAX MRSA XT to the Cepheid Xpert MRSA Assay for the Molecular Detection of methicillin-resistant Staphylococcus aureus (MRSA) from Nasal Swabs Sanjay R. Mehta, Jasmine Estrada, Juan Ybarra, Joshua Fierer PII: DOI: Reference:
S0732-8893(16)30429-1 doi: 10.1016/j.diagmicrobio.2016.12.011 DMB 14263
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
4 October 2016 12 December 2016 13 December 2016
Please cite this article as: Mehta Sanjay R., Estrada Jasmine, Ybarra Juan, Fierer Joshua, Comparison of the BD MAX MRSA XT to the Cepheid Xpert MRSA Assay for the Molecular Detection of methicillin-resistant Staphylococcus aureus (MRSA) from Nasal Swabs, Diagnostic Microbiology and Infectious Disease (2016), doi: 10.1016/j.diagmicrobio.2016.12.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Original Article Title: Comparison of the BD MAX MRSA XT to the Cepheid Xpert MRSA Assay for the
RI P
T
Molecular Detection of methicillin-resistant Staphylococcus aureus (MRSA) from Nasal Swabs
Authors:
SC
Sanjay R. Mehta,a,b# Jasmine Estrada,a Juan Ybarra,a and Joshua Fierer, a,b
NU
Department of Pathology, San Diego Veterans Affairs Medical Center, San Diego, California,
MA
USAa; Division of Infectious Diseases, University of California, San Diego, California, USAb
CE
Abstract Word Count: 276
PT
Manuscript Word Count: 1540
ED
Running Head: BD MAX vs Cepheid Xpert for detection of MRSA
#Address correspondence to:
AC
Sanjay R. Mehta
Address: 9500 Gilman Drive MC 8208, San Diego, CA 92130 Phone: 1-619-543-3150 Fax: 1-619-543-5094 Email: [email protected].
1
ACCEPTED MANUSCRIPT Abstract Introduction: Variation in MRSA genotypes may affect the sensitivity of molecular assays to
T
detect this organism. Methods: We compared two commonly used screening assays, the
RI P
CepheidTM Xpert MRSA and the BD MAXTM MRSA XT on consecutively obtained nasal swabs from 479 subjects. Specimens giving discordant results were subjected to additional
SC
microbiologic and molecular testing. Results: 642/658 (97.6%) of the test results were concordant. Of the 16 discordant results from 12 subjects, additional results suggested that for
NU
9/15 (60%) the MRSA XT assay was likely correct, and for 6/15 (40%) the Xpert assay was
MA
likely correct. One discordant result could not be resolved. A mecA dropout and novel MREJ sites led to false positive and negative results by Xpert. Conclusion: While both assays performed well, continued vigilance is needed to monitor for Staphylococcus aureus with novel
ED
MREJ sites, mecA dropouts, and mecC, leading to inaccurate results in screening assays.
AC
CE
PT
Keywords: MRSA, screening, molecular, mecC, MREJ, mecA dropout
2
ACCEPTED MANUSCRIPT Introduction Staphylococcus aureus remains an important human pathogen, causing a substantial proportion
T
of hospital acquired infections(1–3). Screening patients admitted to the hospital for methicillin
RI P
resistant Staphylococcus aureus (MRSA) colonization and placing them in contact isolation has been proposed as a tool to prevent the nosocomial spread of MRSA between patients. This
SC
screening approach is mandated by the State of California and was championed by the Department of Veterans Affairs as part of a multipronged strategy endorsed by the Department
NU
of Veterans Affairs to reduce the incidence of nosocomial MRSA infections (4,5,6). Many
MA
hospitals in the US have now adopted a policy of screening patients on admission for MRSA nasal colonization. Molecular detection of MRSA in nasal swabs is a rapid method to identify colonized individuals. However, due to genetic variation in the SCCmec elements responsible
ED
for the MRSA phenotype, there is not a consensus what are the best molecular targets to use to
PT
identify MRSA strains by gene amplification. Here we compared two commercial molecular
CE
assays used to screen for MRSA colonization, using nasal swabs collected during routine care.
Materials and Methods
AC
This study was approved by the institutional review board of the San Diego Veterans Affairs Medical Center located in the United States. During the study period between 11/2/2015 and 11/30/2015, there were 479 patients admitted to the San Diego Veterans Affairs Medical Center. For the study, we continued our policy of obtaining nasal swabs for PCR identification of MRSA carriers on admission to the hospital and whenever there was a transfer from one service to another. Nursing staff swabbed both anterior nares using the paired swabs found in the sterile swab transport kit (Copan Diagnostics, Murrieta, CA). Swabs were then transported to the microbiology laboratory. Before processing the swabs were rubbed together to ensure comparability and then one was used in the CepheidTM Xpert MRSA assay according to the manufacturer’s instructions, and the other was used in the BD MAXTM MRSA XT assay. The BD
3
ACCEPTED MANUSCRIPT assay involves vortexing the swab in a buffer. The buffer was used for detection of MRSA
T
according to manufacturer’s instructions and residual fluid was used for culturing for MRSA.
RI P
Results from each assay were recorded in a de-identified manner. No further analysis was performed on concordant results. For discordant results, 100 µl of residual BD MAX™ MSRA
SC
XT buffer was used to inoculate an enrichment broth that was incubated overnight at 35°C in a 5% CO2 atmosphere. Growth from the broth was then sub-cultured onto chromIDTM MRSA
NU
plates (bioMerieux, Durham, NC) and Columbia blood agar plates (Hardy Diagnostics, Santa
MA
Maria, CA) and incubated for up to 24h. All colonies chromogenically consistent with MRSA from the chromID plates were confirmed as MRSA by testing using isolates from the blood agar plates on the Vitek2 (bioMerieux). False positive results were defined as a positive PCR result
ED
for MRSA but a negative culture result on the same specimen after overnight incubation at 35°
PT
in tryptic soy enrichment broth. A false negative result was defined as a negative PCR result with growth of MRSA on culture. For all specimens that showed discordant results by the two
CE
molecular assays, 100 µl of residual BD MAX™ MSRA XT buffer (frozen) and the isolate, if available, were sent to the BD Molecular Research Lab (Montreal, Quebec) for reanalysis using
AC
the BD MAX™ MRSA XT assay, and a proprietary PCR and SCCmec sequencing based approach for detection of mecA and mecC (7) and molecular characterization of the MREJ junction site for that isolate. Further details of the analysis plan for the discordant results are shown in Table 1.
If only one of the molecular assays was positive for MRSA, and we had an isolate to analyze, we also determined the SCCmec right extremity junction (MREJ) genotypes . To determine which MREJ were detected by the two assays, we used a panel of 14 well characterized S. aureus strains (Microbiologics Ind., St. Cloud, MN) to compare the two assays. The panel consisted of 10 MRSA strains with different MREJ, one MRSA strain carrying a mecC gene, one
4
ACCEPTED MANUSCRIPT methicillin-susceptible strain, one methicillin-susceptible strain with a SCC but lacking the mecA gene (mecA dropout), and one methicillin-susceptible coagulase negative Staphylococcus
RI P
T
epidermidis.
SC
Results
We analyzed 658 specimens from 479 patients and the results of the two assays for MRSA
NU
were concordant in 97.6% of the samples. 38 specimens were positive on both assays, while
MA
604 were concordantly negative. Sixteen specimens from 12 patients (2.4% of all specimens) demonstrated discordant results. Three specimens were positive using the Xpert assay but negative with the MRSA XT, and 13 specimens were negative with the Xpert but positive with
ED
the MRSA XT. Overall there was very good concordance between the two assays (kappa =
PT
0.813, 95% CI [0.724 to 0.902]. Staphylococci were not isolated by subculture from two of the discordant specimens, one identified as MRSA positive by the Xpert and the other by the MRSA
CE
XT assay, so no further analysis of those discrepant results was possible.
AC
Using the challenge panel, we determined that the Xpert and the MRSA XT correctly detected MRSA strains with MREJ junction types i-v, vii, ix, xiv. In addition, the MRSA XT assay also detected the MRSA strains with MREJ junction types vi and xiii, and MRSA XT also correctly identified an MRSA strain carrying the mecC gene and an MSSA strain with a SCC cassette but dropout of the mecA gene, while the Xpert misidentified these isolates. We then applied that information to our samples. We sequenced the SCCmec insertion site for all discordant samples that had a positive culture and found that differences in MREJ type detection explained four of the13 Xpert negative but MRSA XT positive discordant test results from three individuals. One of those individuals (participant C) had two additional specimens sent because he was transferred to different services during the time span of the study; both of them were positive
5
ACCEPTED MANUSCRIPT only in the BD MAX assay, even though neither of them grew MRSA. This patient was on antibiotic treatment for an MRSA infection when the follow-up cultures were obtained so it is
T
likely these apparent false positive results were due to detection of residual MRSA DNA or to
RI P
false negative cultures do to residual antibiotics on the swabs. Three other MRSA XT positive, Xpert negative specimens were confirmed to be MRSA positive by Chromagar, but for one of
SC
these the Vitek2 result was MSSA. MREJ sequencing was not successful for that isolate so we do not have an explanation for the discrepancy. For the remaining four MRSA XT but Xpert
NU
negative specimens, based on the results of culture, the MRSA XT provided false positive
MA
results. Three of these four samples, as well as the two follow-up specimens on partient C all had high Ct values, indicating very little target DNA was present in the sample. None of our
ED
isolates were positive for mecC.
PT
Of the three specimens positive by Xpert, but negative by MRSA XT, SCCmec cassette sequencing revealed a dropout of the mecA gene in one specimen. Another two specimens
CE
were found to be a false positive for the Xpert, as MRSA was not recovered on culture. Results
Discussion
AC
from the analysis of the discordant specimens are shown in Table 2.
The Xpert and MRSA XT assays for nasal carriage of MRSA were concordant for >95% of patients tested, similar to a previous comparison of the Xpert and previous BD MAX MRSA assay (8). The MRSA XT assay did detect four MRSA carriers that were missed by the Xpert assay, highlighting its ability to identify MREJ junctions not detected by the current Xpert assay. Cepheid does have a research use only MRSA assay that detects mecC and additional MREJ junction sites, but at the time of writing of this manuscript, it was not available for clinical use(9).
6
ACCEPTED MANUSCRIPT In this study the MRSA XT assay falsely identified six samples from five subjects as carrying MRSA, however, five of these results had high cycle threshold (Ct) values suggesting either
T
cross-reaction or a low inoculum of bacteria resulting in a negative culture. The Cepheid assay
RI P
falsely identified three subjects as being carriers of MRSA, including one whose organism had drop out of the mecA gene from the SCCmec. Since isolation of patients is expensive, and there
SC
are potential negative consequences for patients in contact isolation (10), these false positive results can have significant consequences. However, in our population, mecA dropouts do not
MA
NU
seem to be a major issue.
A strength of this study was that it was performed under clinical conditions using samples collected directly from patients during routine clinical care, and the same set of swabs was used
ED
for both molecular assays. Both swabs were rubbed into both nares when the sample is
PT
collected so they should have been very similar, but they were collected by nurses in the course of their clinical care not by study personnel. Limitations of this study include a small number of
CE
MRSA positive samples due to a lower than anticipated rate of nasal carriage. Although we made every effort to be sure that the two swabs were comparable by rolling the two swabs
AC
rogether before testing, that may not have adequately mixed the swab contents together. If transfer was incomplete, this could have affected the led to false negative results on one of the assays.
In conclusion, both assays performed very well. There was a low prevalence of MRSA strains in our patients that were not currently identifiable by the Cepheid Xpert assay, and a low prevalence of isolates carrying the SCCmec cassette with mecA gene dropout. Prior published comparisons between the Cepheid and BD MRSA screening assays have used earlier versions of the BD assay with a more limited repertoire of detection. As screening assays improve, clinicians will be able to be more confident in the results (7,10). Continued vigilance will be
7
ACCEPTED MANUSCRIPT important to monitor other patient populations to determine if there is a change on the prevalence of Staphylococcus aureus with SCCmec cassettes with atypical or novel MREJ
T
sites, mecA dropouts, and mecC, all of which can result in false positive or false negative results
RI P
in screening assays.
SC
Acknowledgements. We thank the staff of SDVAMC microbiology laboratory for their excellent technical assistance, Dustin Lisama, Diane Kawa, and Brian Manning from BD Life Sciences,
NU
and the BD Quebec Molecular support team. This research did not receive any specific grant
AC
CE
PT
ED
MA
from funding agencies in the public, commercial, or not-for-profit sector.
8
ACCEPTED MANUSCRIPT
T
References 1. Seybold U, Kourbatova EV, Johnson JG, Halvosa SJ, Wang YF, King MD, et al. Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections. Clin Infect Dis Off Publ Infect Dis Soc Am. 2006 Mar 1;42(5):647–56.
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2. Pasquale TR, Jabrocki B, Salstrom S-J, Wiemken TL, Peyrani P, Haque NZ, et al. Emergence of methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of late-onset nosocomial pneumonia in intensive care patients in the USA. Int J Infect Dis IJID Off Publ Int Soc Infect Dis. 2013 Jun;17(6):e398–403.
NU
SC
3. Quartin AA, Scerpella EG, Puttagunta S, Kett DH. A comparison of microbiology and demographics among patients with healthcare-associated, hospital-acquired, and ventilator-associated pneumonia: a retrospective analysis of 1184 patients from a large, international study. BMC Infect Dis. 2013;13:561.
MA
4. Jain R, Kralovic SM, Evans ME, Ambrose M, Simbartl LA, Obrosky DS, et al. Veterans Affairs Initiative to Prevent Methicillin-Resistant Staphylococcus aureus Infections. N Engl J Med. 2011 Apr 14;364(15):1419–30.
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5. California Law SB1058.pdf [Internet]. [cited 2016 May 13]. Available from: https://www.cdph.ca.gov/services/boards/Documents/SB1058chaptered09_25_08.pdf
PT
6. Paterson GK, Harrison EM, Holmes MA. The emergence of mecC methicillin-resistant Staphylococcus aureus. Trends Microbiol. 2014 Jan;22(1):42–7. 7. Widen R, Healer V, Silbert S. Laboratory evaluation of the BD MAX MRSA assay. J Clin Microbiol. 2014 Jul;52(7):2686–8.
AC
CE
8. Lepainteur M, Delattre S, Cozza S, Lawrence C, Roux A-L, Rottman M. Comparative Evaluation of Two PCR-Based Methods for Detection of Methicillin-Resistant Staphylococcus aureus (MRSA): Xpert MRSA Gen 3 and BD-Max MRSA XT. J Clin Microbiol. 2015 Jun;53(6):1955–8. 9. Kullar R, Vassallo A, Turkel S, Chopra T, Kaye KS, Dhar S. Degowning the controversies of contact precautions for methicillin-resistant Staphylococcus aureus: A review. Am J Infect Control. 2016 Jan 1;44(1):97–103. 10. Patel PA, Robicsek A, Grayes A, Schora DM, Peterson KE, Wright MO, et al. Evaluation of multiple real-time PCR tests on nasal samples in a large MRSA surveillance program. Am J Clin Pathol. 2015 May;143(5):652–8.
9
ACCEPTED MANUSCRIPT
Cepheid result
BD MAX result
ChromID result
Positive
Negative
MSSA
Action Performed
AC
CE
PT
ED
MA
NU
SC
RI P
T
Molecular evaluation for presence of mecA gene Positive Negative MRSA Molecular evaluation of MREJ junction type and presence of mecC Negative Positive MSSA Molecular evaluation for presence of mecA gene Negative Positive MRSA Molecular evaluation of MREJ junction type and presence of mecC Table 1. Discordant Sample Analysis plan. Plan to analyze discordant results between BD MAX and Cepheid assays. Molecular evaluation performed by a PCR assay specific to the mecA or mecC genes, and by an in-house PCR assay and sequencing of the right SCCmec integration site within the orfX gene.
10
ACCEPTED MANUSCRIPT Subject
MRSA XT
Xpert
TSB
CHROM
VITEK2
MREJ Type
1 2 3 4 5 6 7 8
A B C C C C D E
POS POS POS POS POS POS POS NEG
NEG NEG NEG NEG NEG NEG NEG POS
growth growth growth growth growth growth growth growth
MRSA NEG MRSA NEG MRSA NEG NEG NEG
MRSA SE MRSA SE MRSA SE SE MSSA
9 10 11 12 13
F G G H I
NEG POS POS POS POS
POS NEG NEG NEG NEG
NEG MRSA MRSA MRSA -
MSSA MRSA MRSA MSSA -
14 15
J K
POS NEG
NEG POS
MRSA -
MRSA -
16
L
POS
NEG
growth growth growth growth no growth growth no growth growth
6 13 13 mecA DO 2 2 -
NEG
SE
Xpert Correct
Final ID
X X X X -
MRSA SE MRSA SE* MRSA SE* SE MSSA
X X X -
X
MSSA MRSA MRSA
13 -
X X
-
-
-
X
T
MRSA XT Correct X X X X
RI P
SC
NU
MA
ED
Discordant Specimen
1
No MRSA MRSA No MRSA SE
AC
CE
PT
Table 2. Discordant Specimen Analysis. Results from analysis of specimens discordant between Xpert and MRSA XT assays. (CHROM= Chromagar, TSB= subculture to tryptic soy broth, SBAP= subculture to blood agar, VITEK2 = identification and sensitivity testing performed on subculture using The VITEK2 (bioMerieux) MRSA = methicillin resistant Staphylococcus aureus, MSSA = methicillin resistant Staphylococcus aureus, SE= Staphylococcus epidermidis, mecA DO= mecA gene dropout); Shaded columns = work performed at BD Research Lab in Québec; *other specimens from same subject did grow MRSA; 1 unable to interpret given discordance between Vitek2 and Chromagar.
11
S0732-8893(16)30429-1 doi: 10.1016/j.diagmicrobio.2016.12.011 DMB 14263
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
4 October 2016 12 December 2016 13 December 2016
Please cite this article as: Mehta Sanjay R., Estrada Jasmine, Ybarra Juan, Fierer Joshua, Comparison of the BD MAX MRSA XT to the Cepheid Xpert MRSA Assay for the Molecular Detection of methicillin-resistant Staphylococcus aureus (MRSA) from Nasal Swabs, Diagnostic Microbiology and Infectious Disease (2016), doi: 10.1016/j.diagmicrobio.2016.12.011
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT Original Article Title: Comparison of the BD MAX MRSA XT to the Cepheid Xpert MRSA Assay for the
RI P
T
Molecular Detection of methicillin-resistant Staphylococcus aureus (MRSA) from Nasal Swabs
Authors:
SC
Sanjay R. Mehta,a,b# Jasmine Estrada,a Juan Ybarra,a and Joshua Fierer, a,b
NU
Department of Pathology, San Diego Veterans Affairs Medical Center, San Diego, California,
MA
USAa; Division of Infectious Diseases, University of California, San Diego, California, USAb
CE
Abstract Word Count: 276
PT
Manuscript Word Count: 1540
ED
Running Head: BD MAX vs Cepheid Xpert for detection of MRSA
#Address correspondence to:
AC
Sanjay R. Mehta
Address: 9500 Gilman Drive MC 8208, San Diego, CA 92130 Phone: 1-619-543-3150 Fax: 1-619-543-5094 Email: [email protected].
1
ACCEPTED MANUSCRIPT Abstract Introduction: Variation in MRSA genotypes may affect the sensitivity of molecular assays to
T
detect this organism. Methods: We compared two commonly used screening assays, the
RI P
CepheidTM Xpert MRSA and the BD MAXTM MRSA XT on consecutively obtained nasal swabs from 479 subjects. Specimens giving discordant results were subjected to additional
SC
microbiologic and molecular testing. Results: 642/658 (97.6%) of the test results were concordant. Of the 16 discordant results from 12 subjects, additional results suggested that for
NU
9/15 (60%) the MRSA XT assay was likely correct, and for 6/15 (40%) the Xpert assay was
MA
likely correct. One discordant result could not be resolved. A mecA dropout and novel MREJ sites led to false positive and negative results by Xpert. Conclusion: While both assays performed well, continued vigilance is needed to monitor for Staphylococcus aureus with novel
ED
MREJ sites, mecA dropouts, and mecC, leading to inaccurate results in screening assays.
AC
CE
PT
Keywords: MRSA, screening, molecular, mecC, MREJ, mecA dropout
2
ACCEPTED MANUSCRIPT Introduction Staphylococcus aureus remains an important human pathogen, causing a substantial proportion
T
of hospital acquired infections(1–3). Screening patients admitted to the hospital for methicillin
RI P
resistant Staphylococcus aureus (MRSA) colonization and placing them in contact isolation has been proposed as a tool to prevent the nosocomial spread of MRSA between patients. This
SC
screening approach is mandated by the State of California and was championed by the Department of Veterans Affairs as part of a multipronged strategy endorsed by the Department
NU
of Veterans Affairs to reduce the incidence of nosocomial MRSA infections (4,5,6). Many
MA
hospitals in the US have now adopted a policy of screening patients on admission for MRSA nasal colonization. Molecular detection of MRSA in nasal swabs is a rapid method to identify colonized individuals. However, due to genetic variation in the SCCmec elements responsible
ED
for the MRSA phenotype, there is not a consensus what are the best molecular targets to use to
PT
identify MRSA strains by gene amplification. Here we compared two commercial molecular
CE
assays used to screen for MRSA colonization, using nasal swabs collected during routine care.
Materials and Methods
AC
This study was approved by the institutional review board of the San Diego Veterans Affairs Medical Center located in the United States. During the study period between 11/2/2015 and 11/30/2015, there were 479 patients admitted to the San Diego Veterans Affairs Medical Center. For the study, we continued our policy of obtaining nasal swabs for PCR identification of MRSA carriers on admission to the hospital and whenever there was a transfer from one service to another. Nursing staff swabbed both anterior nares using the paired swabs found in the sterile swab transport kit (Copan Diagnostics, Murrieta, CA). Swabs were then transported to the microbiology laboratory. Before processing the swabs were rubbed together to ensure comparability and then one was used in the CepheidTM Xpert MRSA assay according to the manufacturer’s instructions, and the other was used in the BD MAXTM MRSA XT assay. The BD
3
ACCEPTED MANUSCRIPT assay involves vortexing the swab in a buffer. The buffer was used for detection of MRSA
T
according to manufacturer’s instructions and residual fluid was used for culturing for MRSA.
RI P
Results from each assay were recorded in a de-identified manner. No further analysis was performed on concordant results. For discordant results, 100 µl of residual BD MAX™ MSRA
SC
XT buffer was used to inoculate an enrichment broth that was incubated overnight at 35°C in a 5% CO2 atmosphere. Growth from the broth was then sub-cultured onto chromIDTM MRSA
NU
plates (bioMerieux, Durham, NC) and Columbia blood agar plates (Hardy Diagnostics, Santa
MA
Maria, CA) and incubated for up to 24h. All colonies chromogenically consistent with MRSA from the chromID plates were confirmed as MRSA by testing using isolates from the blood agar plates on the Vitek2 (bioMerieux). False positive results were defined as a positive PCR result
ED
for MRSA but a negative culture result on the same specimen after overnight incubation at 35°
PT
in tryptic soy enrichment broth. A false negative result was defined as a negative PCR result with growth of MRSA on culture. For all specimens that showed discordant results by the two
CE
molecular assays, 100 µl of residual BD MAX™ MSRA XT buffer (frozen) and the isolate, if available, were sent to the BD Molecular Research Lab (Montreal, Quebec) for reanalysis using
AC
the BD MAX™ MRSA XT assay, and a proprietary PCR and SCCmec sequencing based approach for detection of mecA and mecC (7) and molecular characterization of the MREJ junction site for that isolate. Further details of the analysis plan for the discordant results are shown in Table 1.
If only one of the molecular assays was positive for MRSA, and we had an isolate to analyze, we also determined the SCCmec right extremity junction (MREJ) genotypes . To determine which MREJ were detected by the two assays, we used a panel of 14 well characterized S. aureus strains (Microbiologics Ind., St. Cloud, MN) to compare the two assays. The panel consisted of 10 MRSA strains with different MREJ, one MRSA strain carrying a mecC gene, one
4
ACCEPTED MANUSCRIPT methicillin-susceptible strain, one methicillin-susceptible strain with a SCC but lacking the mecA gene (mecA dropout), and one methicillin-susceptible coagulase negative Staphylococcus
RI P
T
epidermidis.
SC
Results
We analyzed 658 specimens from 479 patients and the results of the two assays for MRSA
NU
were concordant in 97.6% of the samples. 38 specimens were positive on both assays, while
MA
604 were concordantly negative. Sixteen specimens from 12 patients (2.4% of all specimens) demonstrated discordant results. Three specimens were positive using the Xpert assay but negative with the MRSA XT, and 13 specimens were negative with the Xpert but positive with
ED
the MRSA XT. Overall there was very good concordance between the two assays (kappa =
PT
0.813, 95% CI [0.724 to 0.902]. Staphylococci were not isolated by subculture from two of the discordant specimens, one identified as MRSA positive by the Xpert and the other by the MRSA
CE
XT assay, so no further analysis of those discrepant results was possible.
AC
Using the challenge panel, we determined that the Xpert and the MRSA XT correctly detected MRSA strains with MREJ junction types i-v, vii, ix, xiv. In addition, the MRSA XT assay also detected the MRSA strains with MREJ junction types vi and xiii, and MRSA XT also correctly identified an MRSA strain carrying the mecC gene and an MSSA strain with a SCC cassette but dropout of the mecA gene, while the Xpert misidentified these isolates. We then applied that information to our samples. We sequenced the SCCmec insertion site for all discordant samples that had a positive culture and found that differences in MREJ type detection explained four of the13 Xpert negative but MRSA XT positive discordant test results from three individuals. One of those individuals (participant C) had two additional specimens sent because he was transferred to different services during the time span of the study; both of them were positive
5
ACCEPTED MANUSCRIPT only in the BD MAX assay, even though neither of them grew MRSA. This patient was on antibiotic treatment for an MRSA infection when the follow-up cultures were obtained so it is
T
likely these apparent false positive results were due to detection of residual MRSA DNA or to
RI P
false negative cultures do to residual antibiotics on the swabs. Three other MRSA XT positive, Xpert negative specimens were confirmed to be MRSA positive by Chromagar, but for one of
SC
these the Vitek2 result was MSSA. MREJ sequencing was not successful for that isolate so we do not have an explanation for the discrepancy. For the remaining four MRSA XT but Xpert
NU
negative specimens, based on the results of culture, the MRSA XT provided false positive
MA
results. Three of these four samples, as well as the two follow-up specimens on partient C all had high Ct values, indicating very little target DNA was present in the sample. None of our
ED
isolates were positive for mecC.
PT
Of the three specimens positive by Xpert, but negative by MRSA XT, SCCmec cassette sequencing revealed a dropout of the mecA gene in one specimen. Another two specimens
CE
were found to be a false positive for the Xpert, as MRSA was not recovered on culture. Results
Discussion
AC
from the analysis of the discordant specimens are shown in Table 2.
The Xpert and MRSA XT assays for nasal carriage of MRSA were concordant for >95% of patients tested, similar to a previous comparison of the Xpert and previous BD MAX MRSA assay (8). The MRSA XT assay did detect four MRSA carriers that were missed by the Xpert assay, highlighting its ability to identify MREJ junctions not detected by the current Xpert assay. Cepheid does have a research use only MRSA assay that detects mecC and additional MREJ junction sites, but at the time of writing of this manuscript, it was not available for clinical use(9).
6
ACCEPTED MANUSCRIPT In this study the MRSA XT assay falsely identified six samples from five subjects as carrying MRSA, however, five of these results had high cycle threshold (Ct) values suggesting either
T
cross-reaction or a low inoculum of bacteria resulting in a negative culture. The Cepheid assay
RI P
falsely identified three subjects as being carriers of MRSA, including one whose organism had drop out of the mecA gene from the SCCmec. Since isolation of patients is expensive, and there
SC
are potential negative consequences for patients in contact isolation (10), these false positive results can have significant consequences. However, in our population, mecA dropouts do not
MA
NU
seem to be a major issue.
A strength of this study was that it was performed under clinical conditions using samples collected directly from patients during routine clinical care, and the same set of swabs was used
ED
for both molecular assays. Both swabs were rubbed into both nares when the sample is
PT
collected so they should have been very similar, but they were collected by nurses in the course of their clinical care not by study personnel. Limitations of this study include a small number of
CE
MRSA positive samples due to a lower than anticipated rate of nasal carriage. Although we made every effort to be sure that the two swabs were comparable by rolling the two swabs
AC
rogether before testing, that may not have adequately mixed the swab contents together. If transfer was incomplete, this could have affected the led to false negative results on one of the assays.
In conclusion, both assays performed very well. There was a low prevalence of MRSA strains in our patients that were not currently identifiable by the Cepheid Xpert assay, and a low prevalence of isolates carrying the SCCmec cassette with mecA gene dropout. Prior published comparisons between the Cepheid and BD MRSA screening assays have used earlier versions of the BD assay with a more limited repertoire of detection. As screening assays improve, clinicians will be able to be more confident in the results (7,10). Continued vigilance will be
7
ACCEPTED MANUSCRIPT important to monitor other patient populations to determine if there is a change on the prevalence of Staphylococcus aureus with SCCmec cassettes with atypical or novel MREJ
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sites, mecA dropouts, and mecC, all of which can result in false positive or false negative results
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in screening assays.
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Acknowledgements. We thank the staff of SDVAMC microbiology laboratory for their excellent technical assistance, Dustin Lisama, Diane Kawa, and Brian Manning from BD Life Sciences,
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and the BD Quebec Molecular support team. This research did not receive any specific grant
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from funding agencies in the public, commercial, or not-for-profit sector.
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References 1. Seybold U, Kourbatova EV, Johnson JG, Halvosa SJ, Wang YF, King MD, et al. Emergence of community-associated methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of health care-associated blood stream infections. Clin Infect Dis Off Publ Infect Dis Soc Am. 2006 Mar 1;42(5):647–56.
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2. Pasquale TR, Jabrocki B, Salstrom S-J, Wiemken TL, Peyrani P, Haque NZ, et al. Emergence of methicillin-resistant Staphylococcus aureus USA300 genotype as a major cause of late-onset nosocomial pneumonia in intensive care patients in the USA. Int J Infect Dis IJID Off Publ Int Soc Infect Dis. 2013 Jun;17(6):e398–403.
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3. Quartin AA, Scerpella EG, Puttagunta S, Kett DH. A comparison of microbiology and demographics among patients with healthcare-associated, hospital-acquired, and ventilator-associated pneumonia: a retrospective analysis of 1184 patients from a large, international study. BMC Infect Dis. 2013;13:561.
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4. Jain R, Kralovic SM, Evans ME, Ambrose M, Simbartl LA, Obrosky DS, et al. Veterans Affairs Initiative to Prevent Methicillin-Resistant Staphylococcus aureus Infections. N Engl J Med. 2011 Apr 14;364(15):1419–30.
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5. California Law SB1058.pdf [Internet]. [cited 2016 May 13]. Available from: https://www.cdph.ca.gov/services/boards/Documents/SB1058chaptered09_25_08.pdf
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6. Paterson GK, Harrison EM, Holmes MA. The emergence of mecC methicillin-resistant Staphylococcus aureus. Trends Microbiol. 2014 Jan;22(1):42–7. 7. Widen R, Healer V, Silbert S. Laboratory evaluation of the BD MAX MRSA assay. J Clin Microbiol. 2014 Jul;52(7):2686–8.
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8. Lepainteur M, Delattre S, Cozza S, Lawrence C, Roux A-L, Rottman M. Comparative Evaluation of Two PCR-Based Methods for Detection of Methicillin-Resistant Staphylococcus aureus (MRSA): Xpert MRSA Gen 3 and BD-Max MRSA XT. J Clin Microbiol. 2015 Jun;53(6):1955–8. 9. Kullar R, Vassallo A, Turkel S, Chopra T, Kaye KS, Dhar S. Degowning the controversies of contact precautions for methicillin-resistant Staphylococcus aureus: A review. Am J Infect Control. 2016 Jan 1;44(1):97–103. 10. Patel PA, Robicsek A, Grayes A, Schora DM, Peterson KE, Wright MO, et al. Evaluation of multiple real-time PCR tests on nasal samples in a large MRSA surveillance program. Am J Clin Pathol. 2015 May;143(5):652–8.
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ACCEPTED MANUSCRIPT
Cepheid result
BD MAX result
ChromID result
Positive
Negative
MSSA
Action Performed
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Molecular evaluation for presence of mecA gene Positive Negative MRSA Molecular evaluation of MREJ junction type and presence of mecC Negative Positive MSSA Molecular evaluation for presence of mecA gene Negative Positive MRSA Molecular evaluation of MREJ junction type and presence of mecC Table 1. Discordant Sample Analysis plan. Plan to analyze discordant results between BD MAX and Cepheid assays. Molecular evaluation performed by a PCR assay specific to the mecA or mecC genes, and by an in-house PCR assay and sequencing of the right SCCmec integration site within the orfX gene.
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ACCEPTED MANUSCRIPT Subject
MRSA XT
Xpert
TSB
CHROM
VITEK2
MREJ Type
1 2 3 4 5 6 7 8
A B C C C C D E
POS POS POS POS POS POS POS NEG
NEG NEG NEG NEG NEG NEG NEG POS
growth growth growth growth growth growth growth growth
MRSA NEG MRSA NEG MRSA NEG NEG NEG
MRSA SE MRSA SE MRSA SE SE MSSA
9 10 11 12 13
F G G H I
NEG POS POS POS POS
POS NEG NEG NEG NEG
NEG MRSA MRSA MRSA -
MSSA MRSA MRSA MSSA -
14 15
J K
POS NEG
NEG POS
MRSA -
MRSA -
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POS
NEG
growth growth growth growth no growth growth no growth growth
6 13 13 mecA DO 2 2 -
NEG
SE
Xpert Correct
Final ID
X X X X -
MRSA SE MRSA SE* MRSA SE* SE MSSA
X X X -
X
MSSA MRSA MRSA
13 -
X X
-
-
-
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MRSA XT Correct X X X X
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Discordant Specimen
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No MRSA MRSA No MRSA SE
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Table 2. Discordant Specimen Analysis. Results from analysis of specimens discordant between Xpert and MRSA XT assays. (CHROM= Chromagar, TSB= subculture to tryptic soy broth, SBAP= subculture to blood agar, VITEK2 = identification and sensitivity testing performed on subculture using The VITEK2 (bioMerieux) MRSA = methicillin resistant Staphylococcus aureus, MSSA = methicillin resistant Staphylococcus aureus, SE= Staphylococcus epidermidis, mecA DO= mecA gene dropout); Shaded columns = work performed at BD Research Lab in Québec; *other specimens from same subject did grow MRSA; 1 unable to interpret given discordance between Vitek2 and Chromagar.
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