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A Novel Strep A Test for a Rapid Test Reader Compared with Standard
Culture Method and a Commercial Antigen Assay
Markku Parviainen,1,2 Markku Koskela,3 Irma Ikäheimo,4 Eira Kelo,5 Helena Sirola,6 Kati Ryynänen7 and Ilkka Mononen8 1. Department of Clinical Chemistry, Faculty of Health Sciences, School of Medicine, University of Eastern Finland; 2. Managing Director, Reagena Ltd; 3. Chief Physician; 4. Microbiologist, Microbiology Laboratory, Oulu University Hospital; 5. Research and Development Manager; 6. Quality Control Manager; 7. Marketing Manager, Reagena Ltd; 8. Chief Physician, Department of Clinical Chemistry and Haematology, Turku University Central Hospital and Department of Clinical Chemistry and Joint Laboratory, University of Turku Abstract
ReaScan® Strep A is a novel rapid test for Streptococcus pyogenes (Group A Streptococcus [GAS]), which is a common cause of curable
tonsillitis and pharyngitis. ReaScan Strep A is suitable for point-of-care testing for GAS with minimum training, and its performance is comparable to a standard laboratory method for GAS. The test performance was compared with streptococcal culture and with a commercial antigen test in parallel throat swab samples. ReaScan Strep A test performed in good concordance with the culture, showing sensitivity 92.1 % and specificity 96.9 %, as well as positive predictive value (PPV) 85.4 % and negative predictive value (NPV) 98.4 %, with a prevalence of 16.5 %. The corresponding values for a commercial antigen test were somewhat lower; still, the difference was not Keywords
Rapid antigen detection test, Group A Streptococcus, streptococcal pharyngitis
Disclosure: Markku Parviainen, Eira Kelo, Helena Sirola and Kati Ryynänen are employed by Reagena. The remaining authors have no conflicts of interest to declare.
Acknowledgements: We thank the doctors and nurses at the Oulu University Hospital joint emergency department for the skilled sampling of the throat swab samples of
this study and for performing the rapid antigen detection test (RADT) with the TestPack® Strep A test. We also thank the personnel at the Oulu University Hospital microbiology laboratory for performing the reference cultivations on the selective sheep blood agar for streptococci and for performing the RADT with the ReaScan Strep A test. We greatly appreciate the development work carried out by Ilpo Kuronen and Mikko Regina. We also thank Anne Lyytikäinen and Riikka-Liisa Miettinen for their Received: 4 August 2011 Accepted: 4 August 2011 Citation: European Infectious Disease, 2011;5(2):143–5
Correspondence: Markku Parviainen, Reagena Ltd, Takojantie 18, FIN-70900 Toivala, Finland. E:
Support: The publication of this article was funded by Reagena Ltd.
Streptococcus pyogenes (Group A Streptococcus [GAS]) is a significant recommend that the diagnosis of GAS infection should only follow cause of tonsillitis and pharyngitis, accounting for approximately 30–40 % laboratory confirmation and that for any rapid antigen test that is of cases, while the remaining 60–70 % are mostly viral.1,2 The standard negative for GAS infection, a follow-up culture should be carried out.6 method for establishing the aetiological diagnosis of GAS pharyngitis According to Armengol et al.,3 the measured sensitivity of RADT is culture of a pharyngeal specimen obtained with a throat swab and depends to some degree on the culture method with which they are cultivated on selective sheep blood agar for streptococci. The throat compared, but yet they conclude that a negative RADT result in their culture requires 24–48 hours to interpret, thus delaying the diagnosis.3 paediatric practice still requires a confirmatory throat swab culture.
Rapid antigen detection tests (RADT), e.g. Strep A rapid tests, help in Materials and Methods
early diagnosis of and immediate intervention in, GAS infection, since RADT provide results much more quickly than the standard throat The ReaScan® rapid test reader (Reagena International Ltd, Toivala, swab plate culture does. RADT generally have specificity of 95 %, and Finland) is based on the measurement of light reflection from a therefore false-positive results are rare. The primary advantage of test membrane at the maximum intensity level (background) and RADT over throat swab cultures is that results can be available in only the intensity of the test line. Reflected light is measured by a 5–10 minutes and the tests are inexpensive. Treatment, if needed, can photodetector at a 45° angle. The measurement time is five seconds be started before the patient leaves the doctor’s office.4 and the ReaScan reader automatically subtracts the background intensity R from the test-line intensity T. The net intensity of the test The sensitivity of RADT has been questioned since rapid antigen tests line (R-T) appears as a numerical value on the display. This numerical have shown a significant percentage of false-negative results, resulting value is the basis for the final result that is determined to be positive, in recommendations to back up negative rapid antigen tests with a weakly positive or negative. The reference value ranges are given on culture.5 Guidelines from the American Academy of Pediatrics the lot-specific ReaScan result labels. The measurement is performed Table 1: Test Validation Data with the ReaScan Strep A Test
GAS positive control included in one kit lot* 1 mm x 1 mm GAS colony on blood agar dispensed in 1 ml PBS 1 mm x 1 mm Streptococcus group B** colony on blood agar dispensed in 1 ml PBS * The positive control is prepared to a level corresponding to 1.2 x 105 CFU/swab or more (most commercial RADT tests show positive GAS at this level).
** Similarly, Group C, F and G streptococci, (
S. milleri, S. anginosus, S. constellatus, S. intermedius), S. salivarius, Enterococcus faecalis, E. casseliflavus, Staphylococcus aureus, andArcanobacterium haemolyticum gave ReaScan values ≤5.0 (= negative).
*** The samples were confirmed as negative with the standard selective sheep blood agar for streptococci cultivation method.
Theoretical sensitivity: 1 x 104 CFU/sample. Specificity: only GAS has been found to be reactive in the test. CFU = colony-forming units; GAS = Group A
Streptococcus; N/A = not available; PBS = phosphate-buffered saline. Figure 1A: Outline of the Test Principle
Figure 1B: Outline of the Test Procedure
by the instrument and therefore the observer’s impaired vision or expectations do not interfere with the results. The ReaScan Strep A test (Reagena International Ltd, Toivala, Finland) is a modified immunochromatographic lateral-flow test for the detection with the TestPack® Strep A test (Inverness Medical, cranfield, UK), of GAS from a regular flocked nylon throat swab (microRheologics, according to instructions for use. Three parallel swab samples were Brescia, Italy). This rapid test detects streptococcal Lancefield group taken for each of the study methods from 231 volunteers visiting the A antigen, which is extracted from the specimen by the extraction outpatient clinic of Oulu University Hospital. The standard culture reagents included in the test kit. The extraction follows a previously method8 results were classified as negative or positive (+, ++ or +++).
well-described procedure.7 The extracted GAS antigen is allowed to In this method, sheep blood agar for streptococci is used (5 % sheep react with gold particles coated with GAS-specific antibodies. Then other blood agar, Müller–Hinton + tryptone–soya agar),  supplemented with membrane-bound GAS antibodies capture the gold particles with the colistin and nalidixic  acid (IS-ST-A, Abtek  Biological Ltd, UK) and with GAS antigen causing the appearance of a red test line (see Figure 1A).
supplemental carbon dioxide to enhance the growth of streptococci.
An outline of the test protocol is shown in Figure 1B. The intensity of the Streptococcal Grouping Reagent A (DR0586G, Oxoid Ltd, UK) was used.
test line is measured by a ReaScan reader, which gives a numerical value proportional to the amount of antigen in the sample. Since the test result Results and Discussion
is based on a numerical value, human errors are minimised, thus In the present study, we introduced a novel immunochromatographic increasing the diagnostic value of the ReaScan Strep A test. bacterial Strep A antigen test for the ReaScan rapid test reader. We evaluated the sensitivity, specificity and cross-reactivity of the novel The discrepant analysis of the ReaScan Strep A test was performed rapid test. Table 1 summarises the test validation data with the comparing the rapid test with the standard culture method and ReaScan Strep A test. No cross-reactions with cultured Group B, E U R O P E A N I N F E c T I O U S D I S E A S E Rapid Instrumental Testing of Bacterial Strep A Infections c, F and G streptococci (S. milleri, S. anginosus, S. constellatus, Figure 1C: Comparison of the Test Results Obtained by
S. intermedius), S. salivarius, Enterococcus faecalis, E. casseliflavus, the ReaScan Strep A Rapid Test and by Culturing
Group A Streptococcus
in the Present Study
Staphylococcus aureus or Arcanobacterium haemolyticum were observed, indicating the high specificity of the test. ReaScan values Reader value versus culture result (+, ++, +++) ≤5 (= negative) were observed with all these organisms. The results showed that the tests meet the requirements for diagnostic use.
The test performance was compared with streptococcal culture and with another commercial antigen test (TestPack Strep A test) for the parallel throat samples. The ReaScan cut-off value 20 was used in the discrepant analyses. A reading value below this cut-off value 20 represents a negative result and a value above this cut-off means a positive result. ReaScan Strep A test performed in good concordance with the culture method (see Table 2A) showing high sensitivity and specificity as well as high positive predictive value (PPV) and negative The reader values (mean ± standard error of the mean [SEM]), as measured and the predictive value (NPV). The corresponding values were somewhat corresponding culturing results (negative, +, ++ and +++) are shown. lower for the TestPack Strep A test (see Table 2B). The difference Table 2A: ReaScan Strep A Test against Culture,
between these two methods was not, however, statistically significant Test Reader Cut-off Value 20
when tested according to the procedures of Hawass,9 perhaps due to the limited number of patients tested. Figure 1C shows a comparison of the present test reader values with those of streptococcal culture.
A reasonable correlation line was achieved.
The test performance of ReaScan Strep A in the present study is very similar to that of the Lightcycler® polymerase chain reaction (PcR) against bacterial culture in the study of Uhi et al.10 Uhi et al. compared the performance characteristics of a realtime PcR method, the Lightcycler Strep A assay (Roche Applied Science, US), Table 2B: TestPack Strep A Test against Culture
with those of a rapid antigen immunoassay, the Directigen 1-2-3 Group A Strep test kit (BD Diagnostic Systems, US) and a standard culture method for detection of GAS, in 384 throat swabs. In this study, the Lightcycler PcR produced more positive results (n=58) than either culture (n=55) or the Directigen immunoassay (n=31).
The results of the Lightcycler PcR and the Directigen method were independently compared with the results of the accepted ‘gold standard’ bacterial culture. The sensitivities, specificities, PPVs and NPVs for this comparison were as follows: for the Directigen method, 55, 99, 97 and 93 %, respectively; for the Lightcycler PcR, which are directly proportional to the amount of GAS antigen in the Conclusion
In conclusion, ReaScan Strep A is a novel rapid test for reliable and
Objective interpretation of rapid test results with the aid of an quick diagnosis of GAS tonsillitis and pharyngitis. The ReaScan Strep instrument has not been widely used. Reagena International Ltd has A is suitable for point-of-care testing of GAS with minimum training now introduced the ReaScan reader to provide a more objective tool and its performance is comparable to the standard laboratory and to overcome hurdles of visual interpretation. The ReaScan reader method for GAS. An additional advantage of this novel instrumental is easy to use and inexpensive and together with dedicated tests, can Strep A assay is the possibility of receiving numerical test results be used in several clinically important applications.11 n Wannamaker LW, Perplexity and precision in the diagnosis of Roe M, Kishiyama c, Davidson K, et al., comparison of Murray PR, Baron EJ, Jorgensen JH, et al. (eds), Manual streptococcal pharyngitis, Am J Dis Child, BioStar Strep A OIA Optical Immune Assay, Abbott TestPack of Clinical Microbiology, 9th edition, Washington, Dc: Plus Strep A, and culture with selective media for diagnosis American Society for Microbiology Press, 2007;418.
Shaikh N, Leonard E, Martin JM, Prevalence of streptococcal of group A streptococcal pharyngitis, J Clin Microbiol, Hawass NED, comparing the sensitivities and specificities of pharyngitis and streptococcal carriage in children: A two diagnostic procedures performed on the same group meta-analysis, Pediatrics, 2010;126:e557–e64.
American Academy of Pediatrics committee on Infectious of patients, Br J Radiol, 1997;70:360–6.
Armengol cE, Schlager TA, Hendley JO, Sensitivity of a rapid Diseases, Group A streptococcal infections. In: The Red Book, 10. Uhl JR, Adamson Sc, Vetter EA, et al., comparison of antigen detection test for group A streptococci in a private Elk Grove Village, IL: American Academy of Pediatrics, Lightcycler PcR, Rapid Antigen Immunoassay, and culture pediatric office setting: answering the red book’s request for for Detection of Group A Streptococci from Throat Swabs, validation, Pediatrics, 2004;113:924–6.
Faclam RR, Specificity study of kits for detection of group A J Clin Microbiol, 2003;41:242–9.
Bisno AL, Gerber MA, Gwaltney JM, et al., Practice guidelines streptococci directly from throat swabs, J Clin Microbiol, 11. Parviainen M, Kelo E, Sirola H, et al., Detection of Acute for the diagnosis and management of group A streptococcal Hantavirus Infections Using Novel Instrument-readable pharyngitis, Clin Infect Dis, 2002;35:113–25.
Spellerberg B, Brand c, chapter 29: Streptococcus. In: Rapid Tests, Eur Infect Dis, 2011;5:35–7.
E U R O P E A N I N F E c T I O U S D I S E A S E


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