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RAPID DETECTION OF GROUP A STREPTOCOCCAL ANTIGEN WITH A NEW OPTICAL IMMUNOASSAY

1998; Lippincott Williams & Wilkins; Volume: 17; Issue: 4 Linguagem: Inglês

10.1097/00006454-199804000-00019

1532-0987

Patrick Supon, Sheila Tunnell, M I Greene, Rachel Ostroff,

Neonatal and Maternal Infections

Sore throat symptoms are the second most common illness complaint described as the reason for a physician office visit.1 Although most of the illnesses are viral in origin and would not benefit from antibiotic treatment, ∼30% of pediatric and 10% of adult pharyngitis cases are caused by group A streptococcal infections.2 These infections should be treated with antibiotics to prevent serious sequelae such as acute rheumatic fever and glomerulonephritis.3 The traditional method utilized for diagnosing group A streptococcal infections is the throat culture. However, the sensitivity of this method is variable, and results are available only after 1 or 2 days.4 This delay often results in treatment decisions based on symptoms alone, which may lead to unnecessary antibiotic therapy.5, 6 A number of rapid tests have been developed for the detection of group A streptococcal antigen. The specificity of these methods is very high but sensitivity varies widely.7 In this study we evaluated the newest optical immunoassay (OIA) test from BioStar®, the STREP A OIA MAX® assay. This assay is shorter and simpler than the original STREP A OIA® test, which previously was shown in some studies to be more sensitive than traditional blood agar culture.6, 8, 9 This study was undertaken to evaluate the clinical performance of the new STREP A OIA MAX® assay and traditional throat culture in comparison with broth-enriched culture for the detection of group A streptococcal pharyngitis. Materials and methods.Clinical material. Patients of any age who came in February and March, 1996, to the Pediatric Clinic, Adult Primary Care Clinic or the Emergency Room of Fitzsimons Army Medical Center with symptoms of acute pharyngitis were cultured for group A beta-hemolytic streptococcus (GABHS). The study population consisted of 252 pediatric patients (<18 years of age) and 151 adults. For this study a single throat culture swab (Culturette®-I; Becton Dickinson® Microbiology Systems, Cockeysville, MD) was collected from each of 413 patients presenting with pharyngitis and used for the primary throat culture and for testing for GABHS antigen with a new rapid test device (STREP A OIA MAX®, BioStar, Inc., Boulder, CO). Immediately after collection the ampule of Stuart's modified liquid media enclosed in the transport device was crushed to maintain swab moisture. This study was approved by the Institutional Review Board, Fitzsimons Army Medical Center. Laboratory methods. Primary culture. Throat swabs were processed for primary culture as soon as possible after collection (usually within 4 h). After being used to inoculate the primary plate, each swab was returned to the original collection device and held in a refrigerator (for no longer than 24 h) for subsequent testing by the STREP A OIA MAX® procedure. Primary culture involved inoculating one 5% sheep blood agar (SBA) plate (BBL®; Becton Dickinson, Cockeysville, MD) by standard microbiologic techniques and placing a bacitracin disk, 0.04 unit (Taxo A®, Becton Dickinson) at the junction of the first two streak quadrants. The plates were incubated in 5% CO2 at 35-37°C for 18 to 24 h and then evaluated for beta-hemolytic colonies and bacitracin sensitivity. Negative plates were reincubated for an additional 24 h. Betahemolytic colonies morphologically consistent with Streptococcus sp. (whether inhibited by bacitracin or not) were tested directly with a serologic grouping procedure (Pathodx®; Diagnostic Products Corp., Los Angeles, CA) or subcultured to obtain additional growth before serologic testing. Samples were determined to be primary culture-positive for GABHS when beta-hemolytic colonies isolated from the original SBA plate were determined to be group A by a commercial serologic test kit. Growth was recorded as 1+ if GABHS was found only in the first quadrant of the plate, 2+ if growth was in the first and second quadrant and so forth. Broth-enriched culture. After the swab was removed for testing with the STREP A OIA MAX® procedure, the cotton pledget from each sample was aseptically removed from the Culturette® and placed in 5 ml of Todd Hewitt broth (Becton Dickinson). After 12 to 18 h of incubation at 37°C, 10 μl samples of the broth were used to inoculate SBA and trimethoprim-sulfamethoxazole blood agar plates (Becton Dickinson). Bacitracin disks were placed on the plates and processed as described above. Broth-enriched culture-positive samples were those that yielded serologically confirmed GABHS on either subculture plate. Rapid Streptococcus antigen testing. The swabs were tested using the STREP A OIA MAX® test kit in accordance with the manufacturer's directions. The personnel reading the antigen detection test were blinded to the culture results. The kit differs from the original STREP A OIA® product8 in that the STREP A OIA MAX® kit has fewer reagents and total test time is reduced. Analytical comparison of the STREP A OIA MAX® and STREP A OIA®. A cell suspension of group A Streptococcus (ATCC 19615) was prepared in saline to an OD540 of 0.6 (3 × 108 colony-forming units (CFU)/ml, verified by plate counts). Dilutions were made in saline to yield concentrations of 300, 30, 5, 3 and 1.25 × 105 CFU/ml. Swabs were inoculated with 100 μl of each dilution. Testing was performed in duplicate for each assay. Data analysis. Sensitivities and specificities of the STREP A OIA-MAX® and primary culture were calculated in reference to the broth-enriched culture method with standard mathematical formulas. The McNemar test was used to compare the OIA test and SBA performance statistically. Results. One hundred seven of the 413 samples yielded GABHS from broth-enriched culture for a positivity rate of 26%. The sensitivities and specificities of the STREP A OIA MAX® test and primary culture vs. broth-enriched culture were calculated (Table 1). The sensitivities of the OIA test and SBA culture were 89 and 81%, respectively (P = 0.059), and the specificities were 93 and 100% (P < .001).TABLE 1: Comparison of STREP A OIA MAX® and SBA culture to enriched broth culture The STREP A OIA MAX® test was sensitive even for samples with low colony counts. The sensitivity of the rapid test for cultures having fewer than 25 CFU on the primary plate or yielding GABHS only after broth enrichment was 77%. Primary culture was positive for 62% of these same specimens. The STREP A OIA MAX® test also detected 13 positives confirmed by broth enrichment that were negative on the primary culture plate. Primary culture, however, detected five positive samples that were negative by the BioStar® test. Four of these positives were 1+ and one was 2+ for growth on the primary plate. No samples showing moderate to heavy growth on the primary culture plates were missed by the STREP A OIA MAX® procedure. Seven specimens were negative by both the OIA test and primary culture but positive after broth enrichment. Taken together these data demonstrate that the BioStar® test detected eight more true positives than primary culture plate. Examination of the 22 STREP A OIA MAX® devices for the samples with false positive results (i.e. positive STREP A OIA MAX® results with negative cultures) revealed that none of the reactions on the devices was strongly positive. One of the samples had been obtained from a patient who had been taking cephalexin, 250 mg 4 times a day, for the 5 days before throat swab collection. Non-group A hemolytic streptococci were identified in some cultures: 15 group B; 9 group C; 5 group F; and 4 group G. The STREP A OIA MAX® test showed no cross-reactivity with any of these non-group A streptococci. The analytical sensitivity of the STREP A OIA MAX® assay was compared with the original STREP A OIA® by testing dilutions of a group A Streptococcus cell suspension in each assay. The last positive dilution for both tests was 3 × 104 CFU/swab, indicating that both OIA tests have the same analytical sensitivity. Discussion. The STREP A OIA MAX® test is faster and easier to use than the original STREP A OIA® while maintaining similar clinical performance. The limit of detection of both the STREP A OIA MAX® and the original STREP A OIA® is fewer than 3 × 104 CFU/test. The sensitivity of the STREP A OIA® has repeatedly been shown to be equal to or higher than SBA culture.6, 8-13 A recent STREP A OIA® evaluation, encompassing more than 2,000 specimens, found the OIA test to be more sensitive than office-based culture.9 In this study the sensitivity of the STREP A OIA MAX® procedure calculated against enriched broth culture was 89%, compared with primary culture at 81% (P = 0.059). The majority of OIA false negative results also failed to grow any GABHS on primary culture. The OIA test detected 8 more true positives than SBA culture, all of which were positive only after broth enrichment. It is possible that these specimens represent chronic pharyngeal carriage rather than active GABHS infection. However, it has been shown that even patients with fewer than 10 GABHS colonies on traditional throat culture can have a serologic response, indicating true infection,14, 15 and should be treated. The specificity of the rapid test was 93%. One of the patients whose sample yielded a positive result with the STREP A OIA MAX® device but a negative culture had been receiving cephalexin. Although it is possible that the unreported self-administered use of antibiotics is responsible for the failure of GABHS to grow in culture from other samples, it was outside of the scope of this study to obtain this information from the patients. Our results are not inconsistent with previous observations that up to 12% of children may arrive at the physician's office having already taken antibiotics.12 A recent study comparing the STREP A OIA® test to agar and broth-enhanced culture demonstrated that 44% of samples positive by the OIA method but negative by culture were confirmed positive for group A streptococci by PCR.16 The false positivity rate of 7% in our study is probably acceptable in this setting and, even if all cases were treated, would lead to less inappropriate treatment with antibiotics than is currently experienced when clinicians rely predominantly on clinical signs and symptoms.6 The results of this study suggest that the STREP A OIA MAX® test is more sensitive than routine SBA culture. The calculated significance just missed the commonly accepted P < 0.05 value, a value that would have been reached if there had been one fewer false negative antigen test result. However, laboratory practice standards suggest that any new product should be evaluated in the setting in which it will be used to confirm performance in the specific patient population of the user. The American Academy of Pediatrics states that for new techniques such as optical immunoassay, "available data suggest that these tests are as sensitive as standard throat cultures on sheep blood agar and more sensitive than other rapid tests for GAS. However, additional corroborative information is needed before these tests can be recommended for routine use without a confirmatory throat culture in patients with negative test results."7 The STREP A OIA MAX® test is easy to use and interpret. Use of this test while the patient is still in the physician's office can provide for an immediate diagnosis. Appropriate therapeutic treatment could be initiated, reducing the duration of illness and communicability. Acknowledgments. We thank the staff of the Microbiology Department of Fitzsimons Army Medical Center for their assistance. This study was funded by a grant from BioStar. The opinions contained herein are the private views of the authors and are not to be construed as reflecting the views of the Department of the Army or the Department of Defense. Patrick A. Supon, Ph.D., M.T.(ASCP) Sheila Tunnell, M.D. Michele Greene, M.T., M.A.Ed. Rachel M. Ostroff, Ph.D. Departments of Pathology (PAS, MG) and Pediatrics (ST); Fitzsimons Army Medical Center; Aurora, CO BioStar, Inc.; Boulder, CO (RO)