- Email: [email protected]
How is the Xpert MRSA Gen 3 assay (Cepheid) performing on pooled eSwab medium?
How is the Xpert MRSA Gen 3 assay (Cepheid) performing on pooled eSwab medium? Stijn Jonckheere, Kristien Van Vaerenbergh, An Boel, Anne Vankeerberghen, Hans De Beenhouwer PII: DOI: Reference:
S0732-8893(15)00232-1 doi: 10.1016/j.diagmicrobio.2015.07.006 DMB 13864
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
22 April 2015 15 June 2015 7 July 2015
Please cite this article as: Jonckheere Stijn, Van Vaerenbergh Kristien, Boel An, Vankeerberghen Anne, De Beenhouwer Hans, How is the Xpert MRSA Gen 3 assay (Cepheid) performing on pooled eSwab medium?, Diagnostic Microbiology and Infectious Disease (2015), doi: 10.1016/j.diagmicrobio.2015.07.006
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 Title: How is the Xpert MRSA Gen 3 assay (Cepheid) performing on pooled eSwab medium?
T
Running title: Xpert MRSA Gen 3 on pooled eSwab
RI P
Authors: Stijn Jonckheere, Kristien Van Vaerenbergh, An Boel, Anne Vankeerberghen, Hans De Beenhouwer
MA NU
Corresponding author: Hans De Beenhouwer.
SC
Institution: Clinical Laboratory of Microbiology, OLVZ Aalst, Moorselbaan 164, 9300 Aalst, Belgium
Email: [email protected], Tel: +3253724272, Fax: +3253724588
Word count abstract: 50 Word count body text: 1168
PT
Abstract
ED
Conflicts of interest: none
The performance of the Xpert MRSA Gen 3 was compared to the Xpert MRSA on pooled eSwab
CE
media from nose, throat and perineum using broth enriched cultured as gold standard. A lower
p<0.05).
Manuscript
AC
specificity was found for the Xpert MRSA Gen 3 compared to the Xpert MRSA (91.8% versus 97.9%,
Active surveillance and isolation policy have been recommended for controlling the spread of methicillin-resistant Staphylococcus aureus (MRSA) in healthcare facilities (Köck et al., 2014). To improve hospital infection control, a timely, affordable, and reliable screening method for MRSA is essential (Calfee et al., 2014). Currently, the standard method for MRSA detection is culture, which can require several days to generate a definitive result. Molecular tests have a considerable shorter turn-around-time and have shown to be cost-effective in patients at high risk for MRSA (Li et al., 2012). The Xpert MRSA Gen 3 assay (Xpert-G3) (Cepheid, Maurens-Scopont, France) is a molecular screening test for MRSA recently commercialized in Europe. Compared to the former Xpert MRSA assay, it includes additional primers and probes to detect a wider range of orfX-staphylococcal cassette chromosome mec element (SCCmec) junction sequences. Also, in order to improve the specificity, the mecA and mecC gene have become additional targets. A positive result needs both 1
ACCEPTED MANUSCRIPT amplification of the SCCmec-orfX junction and the mecA/C genes, thereby preventing false positive results caused by mec drop-out mutants. The Xpert-G3 has only been validated on nasal swabs using the Copan Dual Swab (package insert Xpert MRSA Gen 3, 2014). However, pooling samples of nose,
T
throat and perineum (NTP) is an attractive tool to increase MRSA detection without increasing
RI P
laboratory costs and has already proven to be successful for the Xpert MRSA (Blanc et al., 2013). This study evaluates the Xpert-G3 using pooled eSwab media (Copan, Brescia, Italy) from NTP as input sample. Results were compared to the Xpert MRSA and broth enrichment culture was used as gold
SC
standard.
MA NU
Separate nasal, throat and perineal swabs were collected from a total of 115 high-risk patients (previously MRSA positive or roommates of a newly diagnosed MRSA carrier). Samples from patients in a MRSA decontamination scheme or from patients receiving antibiotics active against MRSA were excluded. ESwab media were pooled and 150 µL of the pooled sample were tested both on Xpert MRSA and Xpert-G3, according to a previous clinical validation study using 150 µL pooled sample on the Xpert MRSA (Martens et al., 2009). Another 500 µL of pooled sample were enriched in 4 mL
ED
tryptic soy broth of which 10 µL was transferred to a mannitol salt agar with lecithin (home-made) and a BBL CHROMagar MRSA II (Becton Dickinson, Heidelberg, Germany) to detect MRSA and
PT
methicillin-sensitive S. aureus (MSSA). Identity and susceptibility was confirmed by MALDI-TOF (Claydon et al., 1996) and disk diffusion (European committee on antimicrobial susceptibility testing).
CE
For discrepant results, the pooled eSwab was analyzed on the BD Max StaphSR assay (Becton Dickinson, Le Pont de Claix, France), a molecular test for the detection and differentiation of S.
AC
aureus and MRSA (Silbert et al., 2015). Moreover, if a MSSA was cultured from a false positive XpertG3 sample, 75 µL 1/1000 dilution of a 0.5 McFarland MSSA suspension were tested on the Xpert MRSA, Xpert-G3 and Xpert SA Nasal Complete (Cepheid), which detects and differentiates S. aureus and MRSA. Also, the MSSA isolate was characterized using Staphylococcal protein A (spa) typing.
The Xpert MRSA and Xpert-G3 both missed one culture positive sample rendering an equal sensitivity of 94.4% (17/18). The false negative result was probably related to a low MRSA inoculum as only a few colonies were cultured after broth enrichment and both tests were positive on the MRSA isolate. It should be noted that the coverage of an extended spectrum of MRSA types by the new Xpert-G3 did not lead to an increased sensitivity in our study. On the contrary, a lower specificity was observed for the new Xpert-G3 compared to the Xpert MRSA. Of the 97 culture negative samples, Xpert MRSA was positive in 2 samples whereas the Xpert-G3 was positive in 8 samples, yielding a significantly lower specificity for the Xpert-G3 compared to the Xpert MRSA (91.8% versus 97.9%, p<0.05) (Table 1). Interestingly, MSSA was more frequently present in Xpert-G3 false positive samples compared to 2
ACCEPTED MANUSCRIPT true negative samples (6/8 versus 25/89, p<0.05). In one case, the false positive result was likely to be caused by a mec drop-out mutant (sample 4 in table 2). The other samples most probably also represent true false positives of the Xpert-G3 because (i) all samples were tested MRSA negative by
T
the BD Max Staph SR, (ii) 6 out of 7 samples were tested MRSA negative by the Xpert MRSA, (iii)
RI P
culture was negative for MRSA despite applying non-selective broth enrichment and using different culture plates, and (iv) samples were taken from patients that were not in a MRSA decolonization scheme or were not taking antibiotics active against MRSA, minimizing the possibility of false positive
SC
results due to nucleic acid from non-viable MRSA.
MA NU
A recent study evaluated the performance of the Xpert-G3 and demonstrated a sensitivity of 95.7% and a specificity of 100% (Lepainteur et al., 2015). This study, however, examined only samples taken from the nares and not from the throat or perineum, which could explain the difference in specificity. When interpreting our results, the following considerations should be taken into account. First, our data indicate a specificity problem related to the SCCmec-orfX junction as most false positives demonstrated high Ct values for this target. A more comprehensive panel of primers and probes in
ED
the Xpert-G3 together with a more complex matrix in pooled eSwabs may promote non-specific amplification. Apparently, samples containing MSSA are more prone to non-specific amplification
PT
and may directly contribute to a false positive result as suggested by the positive SCCmec-orfX signal on two MSSA isolates cultured from false positive samples (sample 8 and 9 in Table 2). Spa typing
CE
revealed a heterogeneous nature of these MSSA isolates, indicating that the problem was not restricted to a single strain of S. aureus (Table 2). The second factor that contributed to a lower
AC
specificity is the increased Ct cut-off for SCCmec-orfX on the Xpert-G3. Compared to the Xpert MRSA, the cut-off has been raised from 36 to 38. In our study, the specificity would raise from 91.8% to 95.9% without affecting the sensitivity, when decreasing the Ct cut-off from 38 to 36. Third, the Xpert-G3 contains primers and probes targeting the mecA/C genes. This additional target was of little added value in our study due to the high positivity rate in pooled eSwab samples (105/115, 91%, with a median (range) Ct value of 29.2 (20.3 to 35.4)). This is probably caused by the high prevalence of coagulase negative Staphylococcus spp. harboring a mec gene, as also suggested by the much lower Ct values for the mecA/C compared to the SCCmec-orfX target.
In our evaluation on pooled NTP eSwabs, a lower specificity was found for the new Xpert-G3 compared to the Xpert MRSA using broth enrichment culture as gold standard. This study emphasizes that off-label use of the Xpert-G3 should be approached with caution. Reevaluation of the Ct cut-off value and the establishment of a “grey zone”, as previously suggested for the Xpert
3
ACCEPTED MANUSCRIPT MRSA (Van Vaerenbergh et al., 2008), could be used to optimize the performance of the Xpert-G3 on pooled eSwabs.
T
Acknowledgments
RI P
We thank Cepheid for providing the Xpert MRSA and Xpert MRSA Gen 3 cartridges used in this study and for performing the Spa-typing. We also thank dr. Arjan De Jong (UMC St Radboud, Nijmegen,
SC
Nederland) for performing the BD Max StaphSR assays.
References
MA NU
Blanc DS, Nahimana I, Zanetti G, Greub G. MRSA screening by the Xpert MRSA PCR assay: pooling samples of the nose, throat, and groin increases the sensitivity of detection without increasing the laboratory costs. Eur J Clin Microbiol Infect Dis 2013;32(4):565-8. Calfee DP, Salgado CD, Milstone AM, Harris AD, Kuhar DT, Moody J, et al. Strategies to prevent methicillin-resistant Staphylococcus aureus transmission and infection in acute care hospitals: 2014 update. Infect Control Hosp Epidemiol 2014;35(Suppl 2):S108-32.
ED
Claydon MA, Davey SN, Edwards-Jones V, Gordon DB. The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol 1996;14(11):1584-6.
PT
European committee on antimicrobial susceptibility testing. Antimicrobial susceptibility testing EUCAST disk diffusion method. Version 5.0; 2015.
CE
Köck R, Becker K, Cookson B, van Gemert-Pijnen JE, Harbarth S, Kluytmans J, et al. Systematic literature analysis and review of targeted preventive measures to limit healthcare-associated
AC
infections by meticillin-resistant Staphylococcus aureus. Euro Surveill 2014;19(29). 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;53(6):1955-8. Li J, Ulvin K, Biboh H, Kristiansen IS. Cost-effectiveness of supplementing a broth-enriched culture test with the Xpert meticillin-resistant Staphylococcus aureus (MRSA) assay for screening inpatients at high risk of MRSA. J Hosp Infect 2012;84(4):227-33. Martens K, De Beenhouwer H, Frans J, Van Den Abeele A, Cartuyvels R, Coppens G. Evaluation of eSwab for surveillance of MRSA by Xpert MRSA and culture on pooled samples. Poster at 19th ECCMID, 16-19 May 2009, Helsinki, Finland. Package insert Xpert MRSA Gen 3. GeneXpert. Version 301-2815; 2014. Silbert S, Kubasek D, Galambo F, Vendrone E, Widen R. Evaluation of BD MAX StaphSR and BD MAX MRSAXT assays using eSwab collected specimens. J Clin Microbiol 2015;Epub ahead of print.
4
ACCEPTED MANUSCRIPT Van Vaerenbergh K, Boel A, Cartuyvels R, Coppens G, Frans J, Van den Abeele AM, De Beenhouwer H on behalf of the BILULU study group. Is there a need for ‘a grey zone’ for Xpert MRSA rapid testing?
AC
CE
PT
ED
MA NU
SC
RI P
T
Poster at 20th ECCMID, 10-13 April 2008, Vienna, Austria.
5
ACCEPTED MANUSCRIPT
Tables
PT
Table 1: Performance of the Xpert MRSA and Xpert MRSA Gen 3 on pooled eSwabs from nose, throat and perineum using broth enrichment culture as gold
2 95
Xpert MRSA Gen 3 Positive (SCCmec-orfX Ct < 38) Negative
17 1
8 89
NU S
17 1
MA
Xpert MRSA Positive (SCCmec-orfX Ct < 36) Negative
94.4 (72.7, 99.9)
Specificity (%) (95% CI)
PPV (%) (95% CI)
NPV (%) (95% CI)
97.9 (92.8, 99.8)
89.5 (66.9, 98.7)
99.0 (94.3, 99.9)
91.8 (84.4, 96.4)
68.0 (46.5,85.1)
98.9 (94.0, 99.9)
D
Negative
94.4 (72.7, 99.9)
AC
CE P
Positive
Sensitivity (%) (95% CI)
TE
Assay and result
culture after enrichment (no. of samples)
CR I
standard (n=115)
CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value
6
ACCEPTED MANUSCRIPT
Sample 2
Sample 3
Sample 6
Sample 7
Sample 8
Sample 9
Neg Neg
Neg Neg
Neg Neg
Pos (32,3) MRSA
Neg Neg
Pos (34,7) MRSA
Neg Neg
Neg Neg
Neg Neg
Result (Ct) of Xpert MRSA Gen 3 on pooled eSwab SCCmec-orfX junction mecA/mecC Conclusion
Neg Pos (26,8) Neg
Pos (36,3) Pos (29,3) MRSA
Pos (38,0) Pos (28,3) MRSA
Pos (31,1) Pos (25,2) MRSA
Pos (37,2) Pos (30,0) MRSA
Pos (33,9) Pos (25,7) MRSA
Pos (37,2) Pos (30,0) MRSA
Pos (31,2) Pos (31,2) MRSA
Pos (34,7) Pos (28,8) MRSA
Result (Ct) of BD Max StaphSR assay on pooled eSwab SCCmec-orfX junction mecA/mecC nuc Conclusion
Neg Pos (33,4) Neg Neg
Neg Pos (38,4) Neg Neg
Neg Pos (30,3) Neg Neg
Pos (19,6) Pos (31,6) Pos (18,2) MRSA
Neg Neg Pos (23,7) MSSA
Neg Pos (29,4) Pos (23,8) MSSA
Neg Pos (34,5) Pos (20,5) MSSA
Neg Neg Pos (22,5) MSSA
Neg Pos (33,8) Pos (13,5) MSSA
b
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Result of Xpert MRSA on MSSA isolates SCCmec-orfX junction Conclusion
N/A N/A
N/A N/A
N/A N/A
Pos MRSA
Neg Neg
Neg Neg
Neg Neg
Neg Neg
Neg Neg
Result of Xpert MRSA Gen 3 on MSSA isolates SCCmec-orfX junction mecA/mecC
N/A N/A
N/A N/A
N/A N/A
Pos Neg
Neg Neg
Neg Neg
Neg Neg
Pos Neg
Pos Neg
Growth of MRSA in sample Growth of MSSA in sample
MA
D TE
CE P
AC
Result (Ct) of Xpert MRSA on pooled eSwab SCCmec-orfX junction Conclusion
Sample 4
CR I
Sample 5
NU S
Sample 1
PT
Table 2: Overview of discordant samples between Xpert MRSA Gen 3 and broth enrichment culturea
Yes
7
ACCEPTED MANUSCRIPT
N/A
Result of Xpert SA Nasal Complete on MSSA isolates SCCmec-orfX junction mecA staphylococcal protein A (spa) Conclusion
N/A N/A N/A N/A
N/A N/A N/A N/A
N/A N/A N/A N/A
Staphylococcal protein A (spa) type of MSSA isolate
N/A
N/A
Neg
Neg
Neg
Neg
Neg
Pos Neg Pos MSSA
Neg Neg Pos MSSA
Neg Neg Pos MSSA
Neg Neg Pos MSSA
Neg Neg Pos MSSA
Pos Neg Pos MSSA
N/A
t177
t002
t002
t008
unknown
t216
Sample 1 is a false negative result, samples 2-9 are false positive results. When applicable, Xpert MRSA, Xpert MRSA Gen 3 and Xpert SA Nasal Complete were also performed
MA
a
Neg
PT
N/A
CR I
N/A
NU S
Conclusion
on MSSA isolates. b Only a few colonies were cultured after broth enrichment. Xpert MRSA and Xpert MRSA Gen 3 were both positive on MRSA isolate.
AC
CE P
TE
D
Ct, cycle threshold; N/A, not available since there was no growth of MSSA in sample.
8
S0732-8893(15)00232-1 doi: 10.1016/j.diagmicrobio.2015.07.006 DMB 13864
To appear in:
Diagnostic Microbiology and Infectious Disease
Received date: Revised date: Accepted date:
22 April 2015 15 June 2015 7 July 2015
Please cite this article as: Jonckheere Stijn, Van Vaerenbergh Kristien, Boel An, Vankeerberghen Anne, De Beenhouwer Hans, How is the Xpert MRSA Gen 3 assay (Cepheid) performing on pooled eSwab medium?, Diagnostic Microbiology and Infectious Disease (2015), doi: 10.1016/j.diagmicrobio.2015.07.006
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 Title: How is the Xpert MRSA Gen 3 assay (Cepheid) performing on pooled eSwab medium?
T
Running title: Xpert MRSA Gen 3 on pooled eSwab
RI P
Authors: Stijn Jonckheere, Kristien Van Vaerenbergh, An Boel, Anne Vankeerberghen, Hans De Beenhouwer
MA NU
Corresponding author: Hans De Beenhouwer.
SC
Institution: Clinical Laboratory of Microbiology, OLVZ Aalst, Moorselbaan 164, 9300 Aalst, Belgium
Email: [email protected], Tel: +3253724272, Fax: +3253724588
Word count abstract: 50 Word count body text: 1168
PT
Abstract
ED
Conflicts of interest: none
The performance of the Xpert MRSA Gen 3 was compared to the Xpert MRSA on pooled eSwab
CE
media from nose, throat and perineum using broth enriched cultured as gold standard. A lower
p<0.05).
Manuscript
AC
specificity was found for the Xpert MRSA Gen 3 compared to the Xpert MRSA (91.8% versus 97.9%,
Active surveillance and isolation policy have been recommended for controlling the spread of methicillin-resistant Staphylococcus aureus (MRSA) in healthcare facilities (Köck et al., 2014). To improve hospital infection control, a timely, affordable, and reliable screening method for MRSA is essential (Calfee et al., 2014). Currently, the standard method for MRSA detection is culture, which can require several days to generate a definitive result. Molecular tests have a considerable shorter turn-around-time and have shown to be cost-effective in patients at high risk for MRSA (Li et al., 2012). The Xpert MRSA Gen 3 assay (Xpert-G3) (Cepheid, Maurens-Scopont, France) is a molecular screening test for MRSA recently commercialized in Europe. Compared to the former Xpert MRSA assay, it includes additional primers and probes to detect a wider range of orfX-staphylococcal cassette chromosome mec element (SCCmec) junction sequences. Also, in order to improve the specificity, the mecA and mecC gene have become additional targets. A positive result needs both 1
ACCEPTED MANUSCRIPT amplification of the SCCmec-orfX junction and the mecA/C genes, thereby preventing false positive results caused by mec drop-out mutants. The Xpert-G3 has only been validated on nasal swabs using the Copan Dual Swab (package insert Xpert MRSA Gen 3, 2014). However, pooling samples of nose,
T
throat and perineum (NTP) is an attractive tool to increase MRSA detection without increasing
RI P
laboratory costs and has already proven to be successful for the Xpert MRSA (Blanc et al., 2013). This study evaluates the Xpert-G3 using pooled eSwab media (Copan, Brescia, Italy) from NTP as input sample. Results were compared to the Xpert MRSA and broth enrichment culture was used as gold
SC
standard.
MA NU
Separate nasal, throat and perineal swabs were collected from a total of 115 high-risk patients (previously MRSA positive or roommates of a newly diagnosed MRSA carrier). Samples from patients in a MRSA decontamination scheme or from patients receiving antibiotics active against MRSA were excluded. ESwab media were pooled and 150 µL of the pooled sample were tested both on Xpert MRSA and Xpert-G3, according to a previous clinical validation study using 150 µL pooled sample on the Xpert MRSA (Martens et al., 2009). Another 500 µL of pooled sample were enriched in 4 mL
ED
tryptic soy broth of which 10 µL was transferred to a mannitol salt agar with lecithin (home-made) and a BBL CHROMagar MRSA II (Becton Dickinson, Heidelberg, Germany) to detect MRSA and
PT
methicillin-sensitive S. aureus (MSSA). Identity and susceptibility was confirmed by MALDI-TOF (Claydon et al., 1996) and disk diffusion (European committee on antimicrobial susceptibility testing).
CE
For discrepant results, the pooled eSwab was analyzed on the BD Max StaphSR assay (Becton Dickinson, Le Pont de Claix, France), a molecular test for the detection and differentiation of S.
AC
aureus and MRSA (Silbert et al., 2015). Moreover, if a MSSA was cultured from a false positive XpertG3 sample, 75 µL 1/1000 dilution of a 0.5 McFarland MSSA suspension were tested on the Xpert MRSA, Xpert-G3 and Xpert SA Nasal Complete (Cepheid), which detects and differentiates S. aureus and MRSA. Also, the MSSA isolate was characterized using Staphylococcal protein A (spa) typing.
The Xpert MRSA and Xpert-G3 both missed one culture positive sample rendering an equal sensitivity of 94.4% (17/18). The false negative result was probably related to a low MRSA inoculum as only a few colonies were cultured after broth enrichment and both tests were positive on the MRSA isolate. It should be noted that the coverage of an extended spectrum of MRSA types by the new Xpert-G3 did not lead to an increased sensitivity in our study. On the contrary, a lower specificity was observed for the new Xpert-G3 compared to the Xpert MRSA. Of the 97 culture negative samples, Xpert MRSA was positive in 2 samples whereas the Xpert-G3 was positive in 8 samples, yielding a significantly lower specificity for the Xpert-G3 compared to the Xpert MRSA (91.8% versus 97.9%, p<0.05) (Table 1). Interestingly, MSSA was more frequently present in Xpert-G3 false positive samples compared to 2
ACCEPTED MANUSCRIPT true negative samples (6/8 versus 25/89, p<0.05). In one case, the false positive result was likely to be caused by a mec drop-out mutant (sample 4 in table 2). The other samples most probably also represent true false positives of the Xpert-G3 because (i) all samples were tested MRSA negative by
T
the BD Max Staph SR, (ii) 6 out of 7 samples were tested MRSA negative by the Xpert MRSA, (iii)
RI P
culture was negative for MRSA despite applying non-selective broth enrichment and using different culture plates, and (iv) samples were taken from patients that were not in a MRSA decolonization scheme or were not taking antibiotics active against MRSA, minimizing the possibility of false positive
SC
results due to nucleic acid from non-viable MRSA.
MA NU
A recent study evaluated the performance of the Xpert-G3 and demonstrated a sensitivity of 95.7% and a specificity of 100% (Lepainteur et al., 2015). This study, however, examined only samples taken from the nares and not from the throat or perineum, which could explain the difference in specificity. When interpreting our results, the following considerations should be taken into account. First, our data indicate a specificity problem related to the SCCmec-orfX junction as most false positives demonstrated high Ct values for this target. A more comprehensive panel of primers and probes in
ED
the Xpert-G3 together with a more complex matrix in pooled eSwabs may promote non-specific amplification. Apparently, samples containing MSSA are more prone to non-specific amplification
PT
and may directly contribute to a false positive result as suggested by the positive SCCmec-orfX signal on two MSSA isolates cultured from false positive samples (sample 8 and 9 in Table 2). Spa typing
CE
revealed a heterogeneous nature of these MSSA isolates, indicating that the problem was not restricted to a single strain of S. aureus (Table 2). The second factor that contributed to a lower
AC
specificity is the increased Ct cut-off for SCCmec-orfX on the Xpert-G3. Compared to the Xpert MRSA, the cut-off has been raised from 36 to 38. In our study, the specificity would raise from 91.8% to 95.9% without affecting the sensitivity, when decreasing the Ct cut-off from 38 to 36. Third, the Xpert-G3 contains primers and probes targeting the mecA/C genes. This additional target was of little added value in our study due to the high positivity rate in pooled eSwab samples (105/115, 91%, with a median (range) Ct value of 29.2 (20.3 to 35.4)). This is probably caused by the high prevalence of coagulase negative Staphylococcus spp. harboring a mec gene, as also suggested by the much lower Ct values for the mecA/C compared to the SCCmec-orfX target.
In our evaluation on pooled NTP eSwabs, a lower specificity was found for the new Xpert-G3 compared to the Xpert MRSA using broth enrichment culture as gold standard. This study emphasizes that off-label use of the Xpert-G3 should be approached with caution. Reevaluation of the Ct cut-off value and the establishment of a “grey zone”, as previously suggested for the Xpert
3
ACCEPTED MANUSCRIPT MRSA (Van Vaerenbergh et al., 2008), could be used to optimize the performance of the Xpert-G3 on pooled eSwabs.
T
Acknowledgments
RI P
We thank Cepheid for providing the Xpert MRSA and Xpert MRSA Gen 3 cartridges used in this study and for performing the Spa-typing. We also thank dr. Arjan De Jong (UMC St Radboud, Nijmegen,
SC
Nederland) for performing the BD Max StaphSR assays.
References
MA NU
Blanc DS, Nahimana I, Zanetti G, Greub G. MRSA screening by the Xpert MRSA PCR assay: pooling samples of the nose, throat, and groin increases the sensitivity of detection without increasing the laboratory costs. Eur J Clin Microbiol Infect Dis 2013;32(4):565-8. Calfee DP, Salgado CD, Milstone AM, Harris AD, Kuhar DT, Moody J, et al. Strategies to prevent methicillin-resistant Staphylococcus aureus transmission and infection in acute care hospitals: 2014 update. Infect Control Hosp Epidemiol 2014;35(Suppl 2):S108-32.
ED
Claydon MA, Davey SN, Edwards-Jones V, Gordon DB. The rapid identification of intact microorganisms using mass spectrometry. Nat Biotechnol 1996;14(11):1584-6.
PT
European committee on antimicrobial susceptibility testing. Antimicrobial susceptibility testing EUCAST disk diffusion method. Version 5.0; 2015.
CE
Köck R, Becker K, Cookson B, van Gemert-Pijnen JE, Harbarth S, Kluytmans J, et al. Systematic literature analysis and review of targeted preventive measures to limit healthcare-associated
AC
infections by meticillin-resistant Staphylococcus aureus. Euro Surveill 2014;19(29). 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;53(6):1955-8. Li J, Ulvin K, Biboh H, Kristiansen IS. Cost-effectiveness of supplementing a broth-enriched culture test with the Xpert meticillin-resistant Staphylococcus aureus (MRSA) assay for screening inpatients at high risk of MRSA. J Hosp Infect 2012;84(4):227-33. Martens K, De Beenhouwer H, Frans J, Van Den Abeele A, Cartuyvels R, Coppens G. Evaluation of eSwab for surveillance of MRSA by Xpert MRSA and culture on pooled samples. Poster at 19th ECCMID, 16-19 May 2009, Helsinki, Finland. Package insert Xpert MRSA Gen 3. GeneXpert. Version 301-2815; 2014. Silbert S, Kubasek D, Galambo F, Vendrone E, Widen R. Evaluation of BD MAX StaphSR and BD MAX MRSAXT assays using eSwab collected specimens. J Clin Microbiol 2015;Epub ahead of print.
4
ACCEPTED MANUSCRIPT Van Vaerenbergh K, Boel A, Cartuyvels R, Coppens G, Frans J, Van den Abeele AM, De Beenhouwer H on behalf of the BILULU study group. Is there a need for ‘a grey zone’ for Xpert MRSA rapid testing?
AC
CE
PT
ED
MA NU
SC
RI P
T
Poster at 20th ECCMID, 10-13 April 2008, Vienna, Austria.
5
ACCEPTED MANUSCRIPT
Tables
PT
Table 1: Performance of the Xpert MRSA and Xpert MRSA Gen 3 on pooled eSwabs from nose, throat and perineum using broth enrichment culture as gold
2 95
Xpert MRSA Gen 3 Positive (SCCmec-orfX Ct < 38) Negative
17 1
8 89
NU S
17 1
MA
Xpert MRSA Positive (SCCmec-orfX Ct < 36) Negative
94.4 (72.7, 99.9)
Specificity (%) (95% CI)
PPV (%) (95% CI)
NPV (%) (95% CI)
97.9 (92.8, 99.8)
89.5 (66.9, 98.7)
99.0 (94.3, 99.9)
91.8 (84.4, 96.4)
68.0 (46.5,85.1)
98.9 (94.0, 99.9)
D
Negative
94.4 (72.7, 99.9)
AC
CE P
Positive
Sensitivity (%) (95% CI)
TE
Assay and result
culture after enrichment (no. of samples)
CR I
standard (n=115)
CI, confidence interval; PPV, positive predictive value; NPV, negative predictive value
6
ACCEPTED MANUSCRIPT
Sample 2
Sample 3
Sample 6
Sample 7
Sample 8
Sample 9
Neg Neg
Neg Neg
Neg Neg
Pos (32,3) MRSA
Neg Neg
Pos (34,7) MRSA
Neg Neg
Neg Neg
Neg Neg
Result (Ct) of Xpert MRSA Gen 3 on pooled eSwab SCCmec-orfX junction mecA/mecC Conclusion
Neg Pos (26,8) Neg
Pos (36,3) Pos (29,3) MRSA
Pos (38,0) Pos (28,3) MRSA
Pos (31,1) Pos (25,2) MRSA
Pos (37,2) Pos (30,0) MRSA
Pos (33,9) Pos (25,7) MRSA
Pos (37,2) Pos (30,0) MRSA
Pos (31,2) Pos (31,2) MRSA
Pos (34,7) Pos (28,8) MRSA
Result (Ct) of BD Max StaphSR assay on pooled eSwab SCCmec-orfX junction mecA/mecC nuc Conclusion
Neg Pos (33,4) Neg Neg
Neg Pos (38,4) Neg Neg
Neg Pos (30,3) Neg Neg
Pos (19,6) Pos (31,6) Pos (18,2) MRSA
Neg Neg Pos (23,7) MSSA
Neg Pos (29,4) Pos (23,8) MSSA
Neg Pos (34,5) Pos (20,5) MSSA
Neg Neg Pos (22,5) MSSA
Neg Pos (33,8) Pos (13,5) MSSA
b
No
No
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
Yes
Yes
Yes
Result of Xpert MRSA on MSSA isolates SCCmec-orfX junction Conclusion
N/A N/A
N/A N/A
N/A N/A
Pos MRSA
Neg Neg
Neg Neg
Neg Neg
Neg Neg
Neg Neg
Result of Xpert MRSA Gen 3 on MSSA isolates SCCmec-orfX junction mecA/mecC
N/A N/A
N/A N/A
N/A N/A
Pos Neg
Neg Neg
Neg Neg
Neg Neg
Pos Neg
Pos Neg
Growth of MRSA in sample Growth of MSSA in sample
MA
D TE
CE P
AC
Result (Ct) of Xpert MRSA on pooled eSwab SCCmec-orfX junction Conclusion
Sample 4
CR I
Sample 5
NU S
Sample 1
PT
Table 2: Overview of discordant samples between Xpert MRSA Gen 3 and broth enrichment culturea
Yes
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N/A
Result of Xpert SA Nasal Complete on MSSA isolates SCCmec-orfX junction mecA staphylococcal protein A (spa) Conclusion
N/A N/A N/A N/A
N/A N/A N/A N/A
N/A N/A N/A N/A
Staphylococcal protein A (spa) type of MSSA isolate
N/A
N/A
Neg
Neg
Neg
Neg
Neg
Pos Neg Pos MSSA
Neg Neg Pos MSSA
Neg Neg Pos MSSA
Neg Neg Pos MSSA
Neg Neg Pos MSSA
Pos Neg Pos MSSA
N/A
t177
t002
t002
t008
unknown
t216
Sample 1 is a false negative result, samples 2-9 are false positive results. When applicable, Xpert MRSA, Xpert MRSA Gen 3 and Xpert SA Nasal Complete were also performed
MA
a
Neg
PT
N/A
CR I
N/A
NU S
Conclusion
on MSSA isolates. b Only a few colonies were cultured after broth enrichment. Xpert MRSA and Xpert MRSA Gen 3 were both positive on MRSA isolate.
AC
CE P
TE
D
Ct, cycle threshold; N/A, not available since there was no growth of MSSA in sample.
8