Oliver Schildgen reports to having received a travel grant by Roche Diagnostics, Germany. No other competing interests apply to this manuscript. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: VS OS FM. Performed the experiments: AA JL OK KS IW. Analyzed the data: VS OS FM SM. Wrote the paper: JL AA OS.
Nose/throat-swabs from 1049 patients were screened for MRSA using CHROMagar MRSA, LightCycler Advanced MRSA, and Detect-Ready MRSA. Results were compared to the CHROMagar MRSA results, which was set as reference system. MRSA was detected in 3.05% of the patients with CHROMagar MRSA. LightCycler MRSA Advanced showed a higher clinical sensitivity (84.38%) than Detect-Ready MRSA (57.69%).The negative predictive values were high for both tests (>98%). The specificity and the positive predictive value were higher for the Detect-Ready MRSA test than for the LightCycler MRSA test (99.59% and 78.95% versus 98.52% and 64.29%). For routine screening LightCycler MRSA Advanced proved to be more efficient in our clinical setting as the clinical sensitivity was much higher than the sensitivity of Detect-Ready MRSA. CHROMagar MRSA detected more MRSA positive samples than both PCR methods, leading to the conclusion that the combination of PCR with cultural screening is still the most reliable way for the detection of MRSA. LightCycler MRSA Advanced was faster and needed less hands-on time. The advantage of Detect-Ready MRSA was the additional identification of methicillin-sensitive
Nosocomial infections caused by methicillin-resistant
However, screening methodology is crucial for detecting MRSA and no method is tested the most sensitive, specific or cost effective, so far. Inexpensive culture methods have the disadvantage of a swab-to-result time of up to 48 hours and different commercially available media show even differing accuracy values
More expensive molecular-based test systems show more interesting turn-over-times but only few studies estimating accuracy values in a routine diagnostic setting are available so far
In our study we compared the Detect-Ready® MRSA Kit (MDI, Kent, UK) with the standard methods used in our laboratory, the LightCycler® MRSA Advanced Test (Roche, Mannheim, Germany) and the CHROMagar MRSA II (BD, Heidelberg, Germany). Our screening comprised all patients at admission to our emergency department or two intensive care units in accordance with our routine MRSA screening management.
The study was conducted at the Kliniken der Stadt Köln, a 1500 bed tertiary care facility and university affiliated teaching hospital in the City of Cologne, Germany. During a four month period from August to November 2011 all patients admitted to two intensive-care units or the emergency department of the hospital were screened for MRSA and included into the study, leading to 1049 patients included. Specimens were collected by the nursing staff using double-headed swabs with amies gel (Copan, Italy). In each patient a combined swab was taken from the throat and both nares, the most important sites for MRSA colonization
The BBL CHROMagar MRSA II (BD, Heidelberg, Germany) was used as screening agar for MRSA
Nasal/throat specimens were tested by Detect-Ready® MRSA Kit and by the LightCycler® MRSA Advanced Test. For testing, the double-headed swab was separated. One swab head was processed for directly plated culture on BD BBL™ CHROMagar™ MRSA Medium II, BD Columbia Agar with 5% sheep blood plates, and the LightCycler® MRSA Advanced Test. The other swab was used for the assay with Detect-Ready® MRSA Kit and directly plated culture on BD BBL™ CHROMagar™ MRSA Medium II, and BD Columbia Agar with 5% sheep blood plates. MRSA positive colonies onto the selective chromogenic agar were confirmed to be
In case of the absence of typical (mauve coloured) colonies on the chromogenic medium and colonies suspected to be
The LightCycler® MRSA Advanced Test targets the integration site of the SCC
Each PCR method contains an Internal Control (IC) to exclude or detect PCR inhibition and to monitor reagent integrity. The IC must be detected positive in all MRSA negative specimens otherwise the specimen is automatically set as “invalid” by the program software.
A true positive (TP) result was defined as positive result in the PCR and the corresponding culture. A true negative (TN) result was defined as a negative result in the PCR and the corresponding culture. A sample positive in the PCR and negative in the corresponding culture was defined as false positive (FP), a sample negative in the PCR and positive in the corresponding culture was defined as false negative (FN).
Cultured MRSA isolates from samples with negative PCR results were reanalyzed by PCR. Isolates with repeated negative result were genotyped with Identibac StaphyType (Clondiag, Jena, Germany). The analysis was conducted by Alere Technologies GmbH (Jena, Germany). In addition the SCC
The results of the LightCycler® MRSA Advanced test and the Detect-Ready® MRSA test were compared to the corresponding CHROMagar MRSA plates. The clinical sensitivity, specificity, and the negative and positive predictive values (NPV and PPV) were calculated for each PCR method. Samples with invalid PCR results were excluded from the calculations.
Of the 1049 patients, 499 (47.57%) were female and 550 (52.43%) were male. Overall, 214 (20.4%) patients were positive for
31 of the CHROMagar MRSA plates inoculated with the swabs used for the LightCycler® MRSA Advanced Test were positive for MRSA. Only 27 of the plates inoculated with the swabs used for the Detect-Ready® MRSA Kit were positive for MRSA. This difference is presumably due to improper sample collection technique leading to different amounts of material on the two heads of the swab. Only the samples tested positive for MRSA in the corresponding chromagar culture were included in the determination of true positive and false positive PCR results, except two cases with a positive MRSA result in both PCRs, but only on one of the culture plates. Those samples were defined as true positive results in the PCR as well, resulting in 32 culture positive samples. Of the 1049 samples, 21 samples could not be analyzed with the Detect-Ready® MRSA Kit, including two of the positive samples, due to a limited number of test kits.
The LightCycler® MRSA Advanced test detected MRSA in 42 samples, 27 of these samples were also positive on the CHROMagar, resulting in 27 true positive and 15 false positive results. Of the 1005 samples tested as MRSA negative in the LightCycler® MRSA Advanced test, 5 samples were positive on the CHROMagar and classified as false negative results (
LightCycler MRSA Advanced (Roche) | Detect-Ready MRSA (MDI) | |
|
1049 | 1028 |
culture results BBL CHROMagar MRSA II | ||||
|
29 | 2.76% | 24 | 2.33% |
|
31 | 2.96% | 25 | 2.43% |
|
1018 | 97.04% | 1003 | 97.57% |
PCR results | ||||
|
42 | 4.00% | 19 | 1.81% |
|
1005 | 95.81% | 989 | 94.28% |
|
2 | 0.19% | 20 | 1.91% |
result definition | ||||
|
27 | 2.57% | 15 | 1.46% |
|
15 | 1.43% | 4 | 0.39% |
|
5 | 0.48% | 11 | 1.07% |
|
1000 | 95.33% | 978 | 95.14% |
test performance (binary classification) | ||
|
84.38% | 57.69% |
|
98.52% | 99.59% |
|
64.29% | 78.95% |
|
99.50% | 98.89% |
The table lists the results of the MRSA screening with the LightCycler MRSA Advanced and the Detect-Ready MRSA test. True and false positive and negative values were determined by comparing the PCR results to the corresponding culture result. The results of the CHROMagar MRSA plates were set as gold standard. All positive cultural results were confirmed by the microbiological laboratory of our clinic. Two samples were tested positive in both PCRs and on one of the cultures and were treated as positive cultural samples for the determination of true und false positive values as well. Negative results of the Detect-Ready MRSA test are results not identified as MRSA by the test software.
With the Detect-Ready® MRSA Kit only 1028 samples could be processed. The test identified 19 samples as MRSA positive, 15 of those samples were positive on CHROMagar and therefore classified as true positive, the other four samples were classified as false positive. The other 989 samples were tested as MRSA negative; in detail 173 were identified as MSSA, 183 as a mixture of MSSA and coagulase negative staphylococci (CoNS), 324 as CoNS, and 309 as negative (
no. | % | |
|
1028 | - |
|
19 | 1,85 |
|
173 | 16,83 |
|
183 | 17,80 |
|
324 | 31,52 |
|
309 | 30,06 |
|
20 | 1,95 |
The lists the itemized results of the Detect-Ready MRSA assay. Additionally to the detection of MRSA the Detect-Ready MRSA PCR is able to differentiate the MRSA negative results into MSSA, MSSA+CoNS, CoNS, and negative results. MRSA: Methicillin-resistant
Overall 22 specimens lead to invalid results upon testing with the LightCycler® MRSA Advanced Test (2/1049) and the Detect-Ready® MRSA Test (20/1028). All specimens were negative on CHROMagar MRSA and were not included in the calculation of clinical sensitivity, specificity, PPV, and NPV of the respective test.
The LightCycler® MRSA Advanced Test achieved a better clinical sensitivity than the Detect-Ready® MRSA Kit (84.38% versus 57.69%) in our clinical setting. The low sensitivity of the Detect-Ready® MRSA Kit was basically due to a number of samples falsely identified as a combination of MSSA and CoNS. The negative predictive values were nonetheless high (>98.5%) for both tests. The specificity and the positive predictive value were higher for the Detect-Ready® MRSA Kit (99.59% and 78.95% versus 98.52% and 64.29% in the LightCycler® MRSA Advanced Test) (
Samples with MRSA growth on CHROMagar and negative PCR results were reanalyzed. The cultured MRSA isolates retrieved from the CHROMagar plates were used as templates for the respective PCR. In this approach three of five isolates from samples with false negative results in the LightCycler® Advanced MRSA-PCR were tested positive, one negative. One cultured sample was missing and could not be reanalyzed. The isolate tested negative was MRSA positive in the than performed Detect-Ready® MRSA Test. Concerning the Detect-Ready® MRSA PCR, the analysis of the isolates from 11 false negative PCR samples resulted in eight MRSA positive and three negative PCRs (one negative, one CoNS, one MSSA+CoNS). All three negative samples were tested positive in the LightCycler® MRSA Advanced Test. The four samples that failed to be detected in one of the PCR assays were sent to Alere Technologies GmbH for genotyping. All four MRSA strains belonged to the ST5/ST225-MRSA-II strain. SCC
MRSA is still a growing problem in health care settings leading to increased costs and patient risks. Identification of colonized patients is the first step in the containment of MRSA spreading. Different diagnostic tests are available for the identification of colonized patients so far.
Factors to consider for the choice of a MRSA screening platform include sensitivities, specificities, turnaround time, costs, and ease of interpretation, which had been shown to vary considerably
Here, we compared the Detect-Ready® MRSA Kit (MDI) with the methods used routinely for MRSA screening in our hospital (LightCycler® MRSA Advanced Test (Roche) and CHROMagar MRSA (BD)).
Costs and turnaround time play an important role in the decision which assay is the test of choice. Reagent and instrument costs are much higher and turnaround times shorter for the PCR assays. LightCycler® MRSA Advanced Test had the shortest turnaround time with less than 2 hours. Processing of the Detect-Ready MRSA Test was finished within 5 hours requiring a considerable hand-on time. Results from cultures were available after 24 hours of incubation at the earliest (94.78% of MRSA positive cultures could be identified after 24 hours (
Both test methods are based on the detection of the SCC
In this study both tests had high specificities (LightCycler® MRSA Advanced Test: 98.52%, Detect-Ready® MRSA Kit: 99.59%). LightCycler® MRSA Advanced Test was more sensitive for the detection of MRSA (84.38%) than Detect-Ready® MRSA Kit, but both tests had poorer sensitivities in our real-life study setting than reported in previous studies
While the Detect-Ready® MRSA PCR detected more false negative results, the LightCycler® MRSA PCR produced significantly more false positive results (LightCycler® MRSA: 15 FP, Detect-Ready® MRSA: 4 FP, p<0.05) resulting in a lower predictive positive value (64.29% versus 78.95%).
The detection of false positive and false negative results could have had several reasons. Recent studies reported a
The reason for false positive PCR results can be
In this study PCR based methods were compared to the direct plating on chromogenic agar. Since not performed under routine conditions neither, no broth-enriched culture was used in the study. Thus, our study is limited by the possibility that broth-enrichement would have lead to more culture positive results. PCR results defined here as false positive could also reflect a detection of non viable MRSA. The reason for choosing the routine culture method as standard was our demand to focus on patients colonised with a considerable amount of still viable MRSA, which determines its transmissibility.
Our study is further limited by the fact, that the false positive samples (amplicons) could not be characterized by molecular methods to examine whether other genetic abnormalities lead to a positive PCR result. No additional data concerning former antibiotic treatment was retrieved for the patients, so it could not be excluded that the detection of false positive samples was due to prior antibiotic treatment, nor to what extent. In contrast to our results, Peterson et al
In conclusion, our data show that the LightCycler® MRSA Advanced Test demonstrated a better clinical sensitivity compared to the Detect-Ready® MRSA Kit. We would recommend additional cultural testing in a clinical setting to close the diagnostic gap and to avoid false results. With the CHROMagar MRSA the majority of the positive results (95%) were achieved already after 24 hours, demonstrating this culture based test as a relatively fast, cheep and reliable screening method in situations where no immediate results are needed.,.