ANNALS of Internal Medicine

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1 ANNALS of Internal Medicine MARCH 1979 VOLUME 90 NUMBER 3 Published Monthly by the American College of Physicians Streptococcal Pharyngitis: Diagnosis by Gram Stain GEORGE CRAWFORD, M.D.; FRANK BRANCATO, Ph.D.; and KING K. HOLMES, M.D.; Seattle, Washington Group A beta-hemolytic streptococci were isolated from 49 (10.4%) of 472 patients with pharyngitis. Throat culture results, interpreted by five observers of varying experience, showed the mean sensitivity, specificity, and predictive value of a positive Gram-stained smear of pharyngeal secretions as 73%, 96%, and 71%. Assignment to a high-risk group by clinical algorithm gave sensitivity, specificity, and predictive value of only 45%, 83%, and 23%. The Gram-stained smear is the most accurate method of early diagnosis of streptococcal pharyngitis. UPPER RESPIRATORY INFECTIONS are the most common cause of acute, short-term illness in the United States. The largest treatable subset of these infections are those attributable to group A beta-hemolytic streptococci {Streptococcus pyogenes) (1). It is important to distinguish treatable streptococcal infections, which have serious sequelae, from other infections for which no specific therapy is available. Despite difficulties in distinguishing streptococcal carriage from infection, the throat culture remains the recommended method of diagnosis (2). Because treatment on the basis of culture results necessitates delay of appropriate therapy, and a return visit for treatment entails additional time and cost, many attempts have been made to develop criteria for the diagnosis of streptococcal pharyngitis on initial presentation. Clinical criteria have been shown to be of no value in some reports (3), but other investigators have been able to assign patients into groups at significantly differing risks for streptococcal pharyngitis on the basis of clinical evaluation (4-6). Diagnosis by clinical criteria has suffered, however, from lack of sensitivity and specificity. Streptococcal pharyngitis correlates with increased numbers of polymorphonuclear cells on the Wright's-stained From the Departments of Medicine and Microbiology, University of Washington; and the U.S. Public Health Service Hospital; Seattle, Washington. smear of pharyngeal secretions (7). Pharyngeal secretions can be evaluated by Gram stain for both cellular content and microbial flora. This technique has been supported (8) and condemned (9) as an aid to the diagnosis of streptococcal pharyngitis. We report a prospective study of the Gram-stained smear of pharyngeal secretions for early diagnosis of streptococcal pharyngitis. Methods PATIENT POPULATION The study was conducted among outpatients with pharyngitis seen at the Seattle U.S. Public Health Service Hospital between 13 September 1976 and 16 February The clinic staff were requested to submit brief clinical summaries and smears of pharyngeal secretions with all throat culture requests. During this period, throat cultures were submitted on 535 consecutive outpatients with pharyngitis. Neither smear nor clinical information was available for 63 patients, leaving 472 evaluable patients for this study. CULTURE METHODS The recommended technique of throat culture (a cottontipped swab rubbed on the area of maximal erythema or exudate, or if none were present, on the tonsillar pillars and posterior pharynx) was demonstrated to the clinical staff, and they were encouraged, but not required to follow it. The material was smeared on a clean glass slide, and the swab placed in 2 ml of trypticase soy broth (Baltimore Biological Laboratories, Baltimore, Maryland) and transported to the laboratory. Upon receiving the swab in broth, the technician shook it vigorously for 30 sec to suspend the material and streaked one loopful onto trypticase soy agar (Baltimore Biological Laboratories) containing 5% human blood. A 1-cm undercut was made in the agar. The plate was incubated in 5% C0 2 for 24 h and the number of beta-hemolytic colonies recorded. These colonies were saved and confirmed as group A by fluorescent antibody staining (Difco Laboratories, Detroit, Michigan) at the conclusion of the study. No patient was excluded because of a "technically inadequate" smear or culture. GRAM STAIN The air-dried smears were successively flooded with crystal Annals of Internal Medicine 90: , American College of Physicians 293

2 Figure 1. Gram-stained pharyngeal secretions from a patient with nonstreptococcal pharyngitis, with intact polymorphonuclear cells and mononuclear cells. (Original magnification, x 100.) violet, Gram's iodine, acetone-alcohol, and 0.1 % basic fuchsin. The basic fuchsin counterstain provided better definition of cellular detail and fusospirochetal flora than did safranin counterstain. The slides were coded and read by five observers who were unaware of the culture results. The five observers varied in experience with the technique. They included a microbiologist who developed the technique (FB), his two technicians (PK and PB) who had extensive experience with the technique, a postdoctoral fellow in infectious disease (GC) with 20 h of experience, and a medical technologist (SR) with only 3 h of training in the technique. The Gram-stained smear was scanned at magnification X 100 for the presence of leukocytes. If none were found, the slide was read as negative. If leukocytes were present, the areas showing the greatest proportion of polymorphonuclear cells or showing "disruption" (loss of cytoplasmic integrity and cellular outlines) were located (Figures 1 and 2A). The microbial flora associated with the polymorphonuclear cells in these areas were examined at magnification X Spherical gram-positive cocci occurring singly and in pairs (structure typical of 5. pyogenes), were differentiated from other gram-positive cocci, which may be more elongated or encapsulated (pneumococci), form chains or clumps, or be of inappropriate size. If gram-positive cocci of typical structure were found in association with the polymorphonuclear cells, the slide was read as positive (Figure 2B). All observers independently graded the slides as presumptively positive or negative for 5. pyogenes. One observer (FB) noted the presence of "disruption," the number of polymorphonuclear cells present, and whether there was a predominance of either corynebacteria or fusospirochetal flora associated with the polymorphonuclear cells, suggestive of Corynebacterium hemolyticum infection, or Vincent's angina. CLINICAL EVALUATION The clinical summary for each patient included the presence or absence of cervical adenopathy, pharyngeal exudate, cough, fever of 38.3 C or greater, or a history of recent exposure to streptococcal pharyngitis, as determined by the examining clinic staff member. STATISTICAL METHODS The significance of the observed interobserver agreement was measured by kappa value and the variance of kappa value (10). All other significance levels were calculated by the chi-square method (11). Results THROAT CULTURE Beta-hemolytic streptococci inhibited by bacitracin disk (12) were isolated from 49 of 472 patients. Of these, nine patients showed 10 or fewer colonies on primary isolation, and 40 showed 11 or more colonies. At the conclusion of the study, 40 of these isolates were viable, and all were confirmed as group A by fluorescent antibody staining. PATIENT POPULATION The mean patient age was 17.6 years, and ranged from 1 to 85 years, with 58% aged 6 to 20 years. The 64 patients 6 to 10 years of age had the highest proportion of positive cultures (25%), in contrast to the 81 patients younger than age 6 and the 67 patients older than age 30, who had the lowest proportion of positive cultures (6%), which is consistent with the experience of others (13). Figure 2A. Gram-stained pharyngeal secretions from a patient with streptococcal pharyngitis showing "disrupted" polymorphonuclear cells characterized by loss of cytoplasmic integrity and cellular outline. (Original magnification, x 100.) B. Oil immersion field of figure 2A, showing gram-positive cocci singly and in pairs (structurally typical of Streptococcus pyogenes) in association with polymorphonuclear cells. (Original magnification, X 1000.) 294 March 1979 Annals of Internal Medicine Volume 90 Number 3

3 Table 1. Sensitivity, Specificity, and Predictive Value of Various Criteria for the Rapid Diagnosis of Streptococcal Pharyngitis Relative to Throat Culture Results Fever Cervical Pharyngeal Cough History of High Positive >38.3 C Adenopathy Exudate Absent Exposure Risk* Gram-Stain Sensitivity Specificity Predictive value P < P < P < P < P < 0.05 P < P < * As determined by the clinical algorithm of Walsh and associates (6). t Significance of agreement between presence of criteria and positive throat culture. GRAM-STAIN RESULTS The sensitivity (proportion of patients with positive cultures who had positive Gram stains), specificity (proportion of patients with negative cultures who had negative Gram stains), and predictive value (proportion of patients with positive Gram stains who had positive cultures) of the Gram stain were determined for the five observers. The results varied according to the experience of the observer, with SR having the poorest sensitivity, specificity, and predictive value (57%, 94%, 54%); GC an intermediate value (78%, 96%, 70%); and FB being the most accurate (92%, 96%, 74%). The mean sensitivity, specificity, and predictive value were 73%, 96%, and 71%, respectively. The agreement of Gram-stain interpretation and culture results was highly significant for each observer (P < 0.001). The five observers were unanimous in their interpretation of 87% of slides read. For the 10 possible pairs of observers, the mean overall agreement (proportion of slides read as positive or negative by both observers) was 94% (range, 90% to 97%), the mean specific agreement (proportion of slides agreed upon as positive to average proportion positive by that pair) was 71% (range, 57% to 83%), and the mean kappa value was 0.68 (range, 0.52 to 0.81). Kappa value is a measurement of agreement beyond that expected by chance and varies between +1 for perfect agreement, 0 for random agreement, and 1 for perfect disagreement. The interobserver agreement was highly significant (P < for each pair). Combining the results of all five observers, the sensitivity of the Gram stain was 79% for patients with 11 or more colonies on primary isolation, but only 47% for those having fewer than 11 (P < 0.001). Analysis of FB's scoring of polymorphonuclear cells and "disruption" showed that these were significantly greater in patients with exudate and positive cultures. The proportion of patients with moderate or many polymorphonuclear cells on smear was greater in the culturepositive group (86%) than in the culture-negative group (42%, P < 0.001) and was greater for the patients with pharyngeal exudate (81 of 117) than for those without exudate (138 of 355, P < 0.001). Among patients with leukocytes on smear, the proportion with "disruption" of leukocytes was greater for those with positive cultures (43 of 48) than for those with negative cultures (105 of 349, P < 0.001), and was greater for those with pharyngeal exudate (60 of 103) than for those without exudate (88 of 294, P < 0.001). Predictive value depends strongly on prevalence (14), and should be higher in groups with a higher prevalence of S. pyogenes isolation. The prevalence of positive cultures in the study group was 10.4%, and the mean predictive value of the Gram stain was 71% (range, 54% to 79%). In patients aged 6 to 10, the prevalence of positive cultures was 25%, and the mean predictive value of a positive Gram stain was 82% (range, 70% to 89%). CLINICAL CRITERIA Table 1 contrasts the sensitivity, specificity, and predictive value of the Gram stain with that of the individual clinical criteria, and a combination of clinical criteria associated with S. pyogenes isolation, as determined by the algorithm of Walsh and associates (6). Streptococcus pyogenes was isolated significantly more often from patients with fever of 38.3 C or greater, pharyngeal exudate, cervical adenopathy, or a history of exposure to streptococcal pharyngitis, and significantly less often from patients with cough. The sensitivity, specificity, and predictive value of a positive Gram stain were higher than that of any individual clinical criterion, or the assignment to a high-risk group by clinical algorithm. The algorithm assigns patients, on the basis of clinical criteria, into groups at high, moderate, and low risk for streptococcal pharyngitis. In the original description of this algorithm, a population of adults at high, moderate, and low risk had positive cultures in 28%, 15%, and 4% of cases, respectively. The results of this algorithm in our population could have differed, in that our population includes pediatric patients, and is more heterogeneous in age, and the clinical judgements by our staff may differ from those of Walsh and his group. However, the results in our patients were quite comparable, with our patients at high, moderate, and low risk having positive cultures in 23%, 12%, and 3% of cases, respectively. NONSTREPTOCOCCAL PHARYNGITIS The Gram stain may also have value in the diagnosis of nonstreptococcal pharyngitis. In our study group, three patients were noted by FB to have a predominance of gram-positive rods associated with the polymorphonuclear cells, and C. hemolyticum was isolated on culture in all of these cases. Thirteen patients had a predominance of fusospirochetal flora associated with the polymorphonuclear cells, as is seen in Vincent's angina. The latter patients were clinically distinct from other patients with negative cultures for S. pyogenes, having a higher propor- Crawford et al. Streptococcal Pharyngitis Diagnosis 295

4 Table 2. Pharyngitis Treatment Strategies: Laboratory Costs for 472 Patients* Laboratory Cost Patients Treated (Percent) Gram Throat Total Culture Positive Culture Stain Culture Cost Day 1 Total Negative A. Culture all patients $2950 $ B. Treat high risk patients, culture medium risk patients $1044 $ C. Gram-stain all patients, culture those with T > 38.3 C or cervical adenopathy $1534 $1279 $ D. Gram-stain all patients with a risk factor, culture those with fever > 38.3 C or adenopathy $949 $1279 $ * The throat culture was assigned a sensitivity and specificity of 100%. The sensitivity and specificity of the Gram stain is the mean value of findings of the five observers for that group. The cost of the Gram stain is $3.25, and the cost of a throat culture is $6.25 (mean cost for four Seattle hospitals). It is assumed that patients with positive Gram stains or cultures are treated, and patients with positive Gram stains are not cultured. Patients treated refers to the proportion of culture-positive or culture-negative patients in our study that would have been treated, using each strategy. Throat cultures were positive in 44 of 292 (17%) of the patients with at least one risk factor and in 40 of 242 (17%) of the patients with cervical adenopathy or fever of 38.3 C or greater, or both. tion of adenopathy (11 of 13 versus 181 of 410, P < 0.01) and pharyngeal exudate (7 of 13 versus 88 of 410, P < 0.02). Discussion Expertise in the interpretation of the Gram stain requires experience, but even a technician with only 3 h of training can outperform the clinical evaluation, and an observer with 20 h of experience can approach the performance of observers with long experience. The degree of skill required for interpretation of the Gram stain is higher than that required for interpretation of the throat culture, and thus the accuracy of observers who only occasionally do throat Gram stains may be low. However, with a sufficient volume of work to acquire and maintain proficiency, the principles given in this paper, together with a set of positive and negative teaching slides, should allow new observers to master the technique. In four instances slides were read as positive by four or five observers, but cultures were negative for 5. pyogenes. Three of these patients had a heavy growth of non-group- A beta-hemolytic streptococci. This may have clinical relevance, for both group C (15) and group G (16) betahemolytic streptococci may cause pharyngitis, with concomitant antistreptolysin O (ASO) titer rises. It is also possible that some of the negative Gram stains associated with negative cultures for 5. pyogenes represent falsenegative cultures and that some of the negative Gram stains associated with positive cultures for S. pyogenes represent streptococcal carriage, rather than infection. The lesser sensitivity of the Gram stain for cultures with less than 11 colonies is consistent with this possibility, as some investigators have found that patients with few streptococcal colonies on primary isolation are less likely to be infected, as defined by clinical findings (17) and serologic titer rises (18). Others have not found this correlation (19). Measurement of ASO titers and serologic grouping of non-group-a beta-hemolytic streptococci were not done in this study. Although delay of therapy, until culture results become available, does not compromise the prevention of rheumatic fever, there are several potential benefits of treatment on initial presentation of the patient. Some studies have shown a small decrease in the morbidity of streptococcal pharyngitis if treatment is given early (4, 20, 21). Treatment of the patient within the first 24 to 48 h of illness may decrease the chance of spread of streptococcal infection to other family members (22). The necessity of a return visit for treatment adds cost and inconvenience for the patient, and in some populations, follow-up may not be assured. At an Army medical center, Tompkins and colleagues (23) found that only 71% of patients with positive throat cultures for 5. pyogenes received antibiotic therapy. In this population, and in others with uncertain follow-up, a reliable method of early diagnosis of streptococcal pharyngitis may increase treatment rates of streptococcal pharyngitis. These considerations may account for the considerable popularity among practicing physicians of treatment based on clinical criteria. An optimal strategy for the treatment of streptococcal pharyngitis would maximize both the number of infected patients treated on initial presentation and the total number of infected patients treated, and minimize both the number of uninfected patients treated and the cost of the evaluation. Table 2 shows the laboratory costs, the proportion of the 49 culture-positive patients that would have been treated on Day 1, the proportion of culture-positive patients that would ultimately be treated, and the proportion of the 423 culture negative patients that would have been treated had four hypothetical treatment strategies been applied to our population. Strategy A (culture all patients and treat those with positive cultures) is the most specific, but is expensive, and does not lead to treatment on initial presentation. Furthermore, although the table shows 100% of culture-positive patients being treated, this undoubtedly overestimates the performance of strategy A in practice. It is well established that the sensitivity of throat cultures is no more than 90% (19), and, as noted above, not all patients with positive cultures ultimately receive therapy. Strategy B, based on the clinical algorithm, is lowest in cost, and allows some infected patients to be treated on presentation. However, 17% of the uninfected patients will be treated. Evaluation of the patient by a combination of clinical findings, Gram stain, and throat culture is presented in 296 March 1979 Annals of Internal Medicine Volume 90 Number 3

5 strategies C and D. Strategy C (obtain Gram stain from all patients, culture those with a negative Gram stain and either fever of 38.3 C or greater, or cervical adenopathy, and treat those with a positive Gram stain or culture) allows 73% of infected patients to be treated on presentation without increase in cost; as discussed above, the proportion of infected patients who ultimately received therapy would be at least as high as in strategy A. Algorithms with a combination of clinical and laboratory criteria could be used to further reduce cost. For example, in strategy D a Gram stain is done only in those patients with any one of the four major risk factors familiar to all clinicians (fever of 38.3 C or greater, cervical adenopathy, pharyngeal exudate, or a history of exposure to streptococcal pharyngitis), and only those with a negative Gram stain and either fever of 38.3 C or greater or cervical adenopathy are cultured. This would also reduce the proportion of culture-negative patients treated (on the basis of false-positive Gram stains) to 2%. Only 1.5% of the untreated patients would have positive cultures for 5. pyogenes. Tompkins, Burnes, and Cable (24) have shown that it is not cost effective to culture or treat a population with a prevalence of streptococcal infection less than 5%. These untreated patients should be at very low risk for rheumatic fever, because none would have either cervical adenopathy or fever of 38.3 C or greater, the only two clinical criteria that correlate with the risk of ASO titer rises in patients with positive cultures for S. pyogenes (19, 25). In a study of 75 patients with pharyngitis, Hedges (26) showed that the Gram stain of pharyngeal secretions could detect six of 15 with positive cultures for S. pyogenes. Our findings confirm this observation and show that in a larger population inexperienced observers can readily acquire this skill, with a high degree of inter-observer agreement on Gram-stain interpretation; our study also analyzes costs and outcomes for several clinical strategies. In addition to allowing early diagnosis of streptococcal pharyngitis, in this study the Gram stain allowed the detection and treatment of three patients with pharyngitis associated with C. hemolyticum (27), and 13 patients with microbiologic evidence of Vincent's angina (28). These patients are of particular interest in that culture on sheep blood agar is inefficient in the detection of C. hemolyticum and of no value in Vincent's angina. This study shows that the Gram-stained smear of pharyngeal secretions, evaluated by observers with a small amount of special training, allows the diagnosis of streptococcal pharyngitis to be made upon the initial presentation of the patient in most cases. The technique is applicable to many clinical settings and may be of special value when combined with the clinical evaluation and the throat culture. ACKNOWLEDGMENTS: The authors thank Ms. P. Kubota, Ms. P. Bohlander, Ms. S. Rajala, and Mr. S. Eng for technical assistance; Dr. R. Wood and Dr. R. Tompkins for valuable suggestions; and the staffs of the Pediatric Clinic, the Indian Health Clinic, and the Outpatient Department for participating in this study. Grant support: National Institutes of Health Program Project AI and a U.S. Air Force Institute of Technology postdoctoral training fellowship (GC). Requests for reprints should be addressed to George Crawford, M.D.; WHMC/SGHMI; Lackland Air Force Base, TX Received 26 June 1978; revision accepted 11 December References 1. MONTO AS, ULLMAN BM: Acute respiratory illness in an American community. The Tecumseh Study. JAMA 227: , KAPLAN EL, BISNO A, DERRICK W, FACKLAM R, GORDIS L, HOUSER HB, JACKSON WH, MILLARD HD, SHULMAN ST, TARANTA AV, WANNAMAKER LW (American Heart Association Committee Report): Prevention of rheumatic fever. Circulation 55:Sl-4, Ross PW: Accuracy of the clinical assessment of the microbial etiology of sore throat. Practitioner 207':659'-661, MERENSTEIN JH, ROGERS KD: Streptococcal pharyngitis: early treatment and management by nurse practitioners. JAMA 227: , BREESE BB, DISNEY FA: The accuracy of the diagnosis of beta-streptococcal infection on clinical grounds. J Pediatr 44: , WALSH BT, BOOKHEIM WW, JOHNSON RC, TOMPKINS RK. Recognition of streptococcal pharyngitis in adults. Arch Intern Med 135: , BAKER LH, HODGES GR: Examination of pharyngeal secretions to determine the etiology of pharyngitis. Am J Med Sci 272:89-93, BRANCATO FP, PARKER MJ: The stained direct smear of clinical material. Health Lab Sci 3:69-72, KEITEL HG: Pitfalls in laboratory tests. Pediatr Clin North Am 12:17-22, FLEISS JL, COHEN J, EVERITT BS: Large sample standard errors of kappa and weighted kappa. Psychol Bull 72: , COLTON T: Statistics in Medicine. Boston, Little, Brown and Company, 1974, pp MOODY MD: Old and new techniques for rapid identification of group A streptococci, in Streptococci and Streptococcal Disease, edited by WANNAMAKER LW, MATSEN JM. New York, Academic Press, 1972, p GLEZEN WP, CLYDE WA JR, SENIOR RJ, SHEAFFER CI, DENNY FW: Group A streptococci, mycoplasmas, and viruses associated with acute pharyngitis. JAMA 202: , VECCHIO TJ: Predictive value of a single diagnostic test in an unselected population. N Engl J Med 274: , BENJAMIN JT, PERRIELLO VA JR: Pharyngitis due to group C hemolytic streptococci in children. J Pediatr 89: , HILL HR, CALDWELL GG, WILSON E, HAGER D, ZIMMERMAN RA: Epidemic of pharyngitis due to streptococci of Lancefield group G. Lancet 2: , BREESE BB, DISNEY FA, TALPEY WB, GREEN JL: Beta-hemolytic streptococcal infection: the clinical and epidemiologic importance of the number of organisms found in cultures. Am JDis Child 119:18-26, MILLER JM, STANCER SL, MASSEL BF: A controlled study of beta hemolytic streptococcal infection in rheumatic families. Am J Med 25: , KAPLAN EL: Unsolved problems in diagnosis and epidemiology of streptococcal infection. See Reference 12, pp BRINK WR, RAMMELKAMP CH, DENNY FW, WANNAMAKER LW: Effect of penicillin and aureomycin on the natural course of streptococcal tonsillitis and pharyngitis. Am J Med 10: , HAIGHT TM: Erythromycin therapy of respiratory infections. I. Controlled studies on the efficacy of erythromycin and penicillin in scarlet fever. J Lab Clin Med 43:15-30, BREESE BB, DISNEY FA: Factors influencing the spread of beta-hemolytic streptococcal infections within the family group. Pediatrics 17: , TOMPKINS RK, WOOD RN, WOLCOTT BW, WALSH BT: The effectiveness and cost of acute respiratory illness medical care provided by physicians and algorithm-assisted physician's assistants. Med Care 15: , TOMPKINS RK, BURNES DC, CABLE WE: An analysis of the cost-effectiveness of pharyngitis management and acute rheumatic fever prevention. Ann Intern Med 86: , HONIKMAN LH, MASSELL BF: Guidelines for the selective use of throat cultures in the diagnosis of streptococcal respiratory infection. Pediatrics 48: , HEDGES JR, WAGNER DK: Pharyngeal Gram stains in the treatment of sore throats. J Am Coll Emergency Physicians 7: , RYAN WJ: Throat infection and rash associated with an unusual corynebacterium. Lancet 2: , UOHARA GI, KNAPP MJ: Oral fusospirochetosis and associated lesions. Oral Surg 24: , 1967 Crawford et al. Streptococcal Pharyngitis Diagnosis 297