EDUCATIONAL COMMENTARY THROAT CULTURES LEARNING OUTCOMES. Upon completion of this exercise, the participant should be able to:

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1 EDUCATIONAL COMMENTARY THROAT CULTURES LEARNING OUTCOMES Upon completion of this exercise, the participant should be able to: distinguish three types of hemolysis produced by bacterial colonies. discuss two controversies involving the use of the bacitracin disc in throat cultures. identify three sources of error in performing the bacitracin disc test. Of the bacterial pathogens that can cause pharyngitis, Streptococcus pyogenes (group A strep) is the most common, especially in children. Left untreated, S. pyogenes infection can progress to acute rheumatic fever or acute glomerulonephritis. These two serious complications are more likely to occur in children than in adults. Hence, the primary goal of culturing throat specimens is to differentiate group A streptococcal pharyngitis, which can be treated with antibiotics, from viral pharyngitis, which cannot be treated with antibiotics. Patterns of Hemolysis To accurately assess growth on throat culture plates, you must be able to distinguish three types of hemolysis that bacterial colonies can produce on sheep blood agar (SBA). 1. Beta (β) hemolysis describes a zone of complete clearing around a colony which occurs when there is a complete lysis of the red blood cells. The presence of β hemolysis is a key characteristic in identifying suspected colonies of S. pyogenes as well as other pathogens such as Staphylococcus aureus, Listeria monocytogenes, and Arcanobacterium haemolyticum. Stabbing the SBA with the inoculating loop or incubating the culture anaerobically enhances hemolysis, thus making it easier to detect colonies that may be S. pyogenes. 2. In contrast to β hemolysis, alpha (α) hemolysis describes a zone of greenish discoloration surrounding a bacterial colony. This type of hemolysis, which is typical of many nonpathogenic streptococci found in normal mouth flora, occurs when the red blood cells are only partially lysed. 3. α-prime or wide-zone hemolysis describes a narrow zone of intact red blood cells adjacent to the colony surrounded by a wider zone of β hemolysis. This type of hemolysis, which can easily be mistaken for true β hemolysis, is produced by some members of the viridans group of streptococci

2 The correct technique for evaluating hemolysis consists of two crucial elements. 1. Only colonies on blood agar plates should be evaluated for hemolysis. Hemolysis should never be assessed on chocolate agar, because the red blood cells in chocolate agar are hemolyzed during the manufacturing process. Consequently, organisms that produce β hemolysis on blood agar often produce only a slight greenish tint on chocolate agar. 2. Transillumination should be used to view the colonies. This simply means that the light source should be behind the agar plate rather than directed down onto the surface of the agar. When you assess hemolysis, be careful to distinguish true β hemolysis from α-prime hemolysis. If necessary, a dissecting microscope or a scanning objective can be used for this purpose. Identification of S. pyogenes On SBA, colonies of S. pyogenes are 0.5 mm to 1.0 mm in diameter, grayish-white in color, convex, and surrounded by a wide zone of β hemolysis. However, because other bacterial colonies may resemble S. pyogenes, suspicious colonies should be confirmed as Streptococcus by a catalase test (streptococci are catalase-negative) and, if necessary, a Gram stain (streptococci appear as gram-positive cocci growing in pairs and chains). After the organism is confirmed as Streptococcus, it can be definitively or presumptively identified. Definitive identification is accomplished by determining the Lancefield serologic group to which the organism belongs. Commercial kits containing group-specific antisera are used to make this identification. S. pyogenes is the only streptococcal species in group A, so a positive result definitively identifies the organism. Most physician s office and small hospital laboratories perform presumptive identification because it is less costly, less time-consuming, and usually adequate for optimal patient care. The method most often used to presumptively identify S. pyogenes is the 0.04 U bacitracin disc. In a 2002 survey by Dale and colleagues of testing practices for group A streptococci at physician s office and small hospital laboratories, more than half of the respondents reported that they used this method. However, despite the popularity of the bacitracin disc, its use has been controversial, and experts have debated the best technique. This debate has centered on whether to place the disc on the primary plate or on a subculture plate and whether to use SBA or selective media. In surveys conducted by Dale and colleagues in 2002 and 2003, most laboratories that used the bacitracin disc reported that they placed the disc on the primary plate. Further, 51.3% of respondents used selective media as the primary plate, and 35.9% used SBA as the primary plate. Only 4.1% of respondents indicated that they applied the discs to subculture plates. Applying the disc to the primary

3 plate rather than a subculture plate reduces the turnaround time, but it also increases the risk of inaccurate results. This is because overgrowth of normal flora or other bacteria on a primary SBA plate can make it difficult to see growth inhibition around the bacitracin disc. In addition, other normal flora bacteria may be inhibited by the bacitracin and lead to a false positive interpretation of the test. On the other hand, although selective media will inhibit the growth of competing flora, inaccurate assessment of growth inhibition around the disc may result if too few colonies of β-hemolytic streptococci are present. Studies comparing the results of placing bacitracin discs on SBA primary plates or selective media primary plates have yielded conflicting results. For example, Dale and colleagues cite studies that show that accurate detection of S. pyogenes ranges from 26% to 96% with primary blood agar plates. Due to this large variation, they recommend that laboratories use a selective media plate rather than SBA if the bacitracin disc is to be placed on a primary plate. By contrast, Roddy and colleagues and Bisno and colleagues argue that, because studies have shown no clear benefit to using a selective media plate, a primary SBA plate is adequately sensitive for the bacitracin disc test. In addition to the controversies about the best technique, the bacitracin disc has inherent limitations that can cause inaccurate throat culture results. The use of the bacitracin disc to identify S. pyogenes is based on the fact that more than 95% of group A streptococci strains are sensitive to this drug and thus do not grow adjacent to the disc. Non group A β-hemolytic streptococci, on the other hand, are resistant to bacitracin and consequently do not show a zone of inhibition around the disc. However, rare strains of group A streptococci are resistant to bacitracin and thus will not be detected by this method. Also, about 5% to 10% of groups B, C, and G streptococci are sensitive to bacitracin and could cause a false-positive interpretation of the bacitracin disc test. For these reasons, many experts no longer recommend the bacitracin disc test and prefer instead the PYR (L-pyrrolidonyl-naphthylamide hydrolysis) test to presumptively identify S. pyogenes. Recommendations Although inherent limitations in the bacitracin disc method may occasionally yield erroneous results, unacceptable performance on proficiency test samples is more likely due to faulty technique. Three sources of error are: 1. Failure to correctly identify β-hemolytic colonies. Hemolysis should be evaluated only on blood agar, and only with the light source behind the plate. 2. Failure to confirm that suspicious β-hemolytic colonies are streptococci. Only β-hemolytic streptococci should be tested with the bacitracin disc.

4 3. Failure to incubate the culture plates 48 hours before finalizing a negative result. A 48-hour incubation time has been shown to substantially increase the likelihood of detecting S. pyogenes, thus avoiding false-negative results. Finally, detection of S. pyogenes requires not only proficient laboratory technique but also proper collection procedures and appropriate follow-up testing policies. Writing on behalf of the Infectious Diseases Society of America, Bisno and colleagues recommend three actions to improve the accuracy of throat culture results: 1. Obtain swab specimens only from the surface of both tonsils and the posterior pharyngeal wall. Avoid touching other areas of the oral pharynx with the swab, because this may result in a sample that does not truly represent the bacteria present at the back of the throat. 2. Obtain the throat swab before antibiotic therapy begins. Obtaining the sample after the patient has begun antibiotic therapy may cause a false-negative culture result. 3. Perform a throat culture to confirm a negative result from a rapid antigen detection test (RADT), at least in children and adolescents. The sensitivity of most RADT kits ranges from 80% to 90%, which means that a substantial number of group A Streptococcus infections may not be detected by this method. Although recent improvements in RADT technology may have improved the sensitivity, there has been insufficient research to justify relying exclusively on a negative RADT result. Conclusion Throat culture is the standard against which other methods of detecting S. pyogenes are evaluated. Performed correctly, throat cultures have a demonstrated sensitivity of 90% to 95%. However, studies have indicated that many laboratories do not follow generally accepted recommended practices for proper specimen collection and incubation time, and experts have debated the best technique for using the bacitracin disc. Laboratories should always follow consensus recommendations. In addition, if proficiency-testing results indicate that patient results may not be reliable, an investigation is warranted, and a change in method may be needed. First, ensure that testing personnel are proficient at recognizing β hemolysis and confirming that suspicious colonies are streptococci. Next, ensure that only β-hemolytic streptococci are tested with the bacitracin disc. Third, consider either placing the bacitracin disc on subculture plates or using selective media as the primary plate. Although the efficacy of these practices is disputed, many experts believe they yield more accurate results. Finally, if these steps fail to resolve the problem, consider using the PYR test instead of the bacitracin disc to presumptively identify S. pyogenes. ASCP 2011

5 References and Suggested Reading Bisno AL, Gerber MA, Gwaltney JM Jr, et al. Practice guidelines for the diagnosis and management of group A streptococcal pharyngitis. CID. 2002,35: Dale JC. Bacitracin testing for group A streptoccal pharyngitis [letter]. Arch Pathol Lab Med. 2003,127: Dale JC, Novak R, Higgens P, et al. Testing for group A streptococci: practices reported by 790 laboratories enrolled in the College of American Pathologists Excel Proficiency Testing Program. Arch Pathol Lab Med. 2002,126: Roddey OF Jr, Clegg HW, Martin ES, et al. Comparison of throat culture methods for the recovery of group A streptococci in a pediatric office setting. JAMA. 1995,274: