Streptococci Utilizing a Two-Disk Technique

Transcription

1 JOURNAL OF CLINICAL MICROBIOLOGY, July 1979, p /79/ /05$02.00/0 Vol. 10, No. 1 Primary Plate Identification of Group A Beta-Hemolytic Streptococci Utilizing a Two-Disk Technique ELLEN J. BARONt* AND JOHN W. GATES' St. Marys Hospital Medical Center, and Dean Clinic,' Service Corporation, Madison, Wisconsin Received for publication 10 April 1979 A two-disk system is described which allows primary plate identification of group A beta-hemolytic streptococci. Group A beta-hemolytic streptococci could be visualized on primary throat culture plates by using trimethoprim-sulfamethoxazole to inhibit normal flora. In the heavily inoculated area of Trypticase soy agar plates containing 5% sheep blood, a 25-,tg/ml trimethoprim-sulfamethoxazole disk was placed contiguous to a 0.04-U bacitracin disk. A total of 259 throat specimens were examined with this two-disk system. The swabs from these throat specimens were incubated in Todd-Hewitt broth. The bacterial pellet from the broths was stained by fluorescent antibody as a control. Of the cultures that were determined to be positive on the plates, 75% could be read unequivocally after overnight incubation, whereas the remaining 25% required subculture. The plates recovered 91% of the cultures which were considered as true positives by the broth-fluorescent-antibody technique. This method provided a significant savings in time compared with standard plate methods and in cost of materials compared with broth-fluorescent-antibody methods. This technique is particularly valuable for producing rapid results in laboratories where fluorescence microscopy would not be cost-effective. One of the major priorities in clinical microbiology today is to rapidly report results to the physician. Although the fluorescent-antibody (FA) technique for identification of group A beta-hemolytic streptococci (BHS) has been shown to be accurate and reliable (3, 6, 9, 12), the initial expense of an ultraviolet microscope is not cost-effective for most small clinical laboratories. Bacitracin disks identify group A BHS with 95% accuracy when used on a pure culture (2, 5, 10), but this technique necessitates a second overnight incubation before results are available. We sought to overcome these problems by placing a trimethoprim-sulfamethoxazole (SXT) disk next to a bacitracin disk in the heavily streaked area of a primary throat culture plate. By inhibiting the normal flora in a zone around the SXT disk, visualization of small numbers of beta-hemolytic colonies within the zone was achieved. In addition, the zone of inhibition around the bacitracin disk was easily visible on those plates showing a heavy growth of group A BHS. MATERLALS AND METHODS Specimens. Pharyngeal cultures were obtained by simultaneously rubbing two Dacron-tipped swabs (Culturette II, Marion Scientific Corp., Kansas City, t Present address: Department of Medical Microbiology, University of Wisconsin, Madison, WI Mo.) over the inflamed throats of 259 pediatric patients who visited a large outpatient clinic over the course of several months. Both swabs were placed together in the provided plastic tube containing an ampoule of Carey-Blair transport medium. The ampoule was broken, and the swabs were maintained at room temperature until cultured. Plate cultures. Within 6 h of collection, each of the two swabs was rubbed over one-third of a plate containing Trypticase soy agar with 5% defibrinated sheep blood (BAP, GIBCO Diagnostics, Madison, Wis.). Isolated colonies were obtained by quadrant streaking. In the heavily inoculated area, a 0.04-U bacitracin disk (Taxo A, Difco Laboratories, Detroit, Mich.) and a p,g sulfamethoxazole with 1.25-,ug trimethoprim disk (SXT susceptibility disk, Difco) were placed side by side. The plates were then incubated overnight at 35 C, one plate in 5% CO2 and one in an air atmosphere. In the morning, the plates were examined for colonies surrounded by a zone of betahemolysis. After inoculating the BAPs, both swabs from the same patient were placed into a single screw-capped tube containing 5 ml of Todd-Hewitt broth. These cultures were incubated immediately, and all 259 broths were processed for FA staining as described below. FA test. Representative beta-hemolytic colonies from plates were emulsified in a drop of phosphatebuffered saline on a glass FA slide, allowed to air dry, and stained along with the slides prepared from Todd- Hewitt broth cultures. FA test from the 259 Todd- Hewitt cultures was performed by the procedure out-

2 VOL. 10, 1979 PRIMARY PLATE METHOD FOR THROAT CULTURE 81 lined by Facklam (4), except that the broth cultures were incubated for 6 h at 350C, rather than 3 h. Those slides not stained immediately were placed into a -72 C freezer (Revco) for storage. After staining with fluorescein-conjugated anti-group A streptococcal antiserum, slides were examined under a Zeiss microscope with a tungsten-halogen ultraviolet light source, * a fluorescein isothiocyanate exciter filter, and no. 50 and 53 barrier filters. Each smear was scanned for a minimum of 3 min, and the relative number of chains of cocci showing moderate to heavy fluorescence compared to the control slide was recorded. Piroduct control. All bacitracin disks used in the study were from a single, carefully quality-controlled Ak lot number (E. Baron Matcha, Abstr. Annu. Meet. Am. Soc. Microbiol. 1978, C90, p. 292). Other products were also monitored for performance quality according to standards required by the College of American V Pathologists Commission on Inspection and Accreditation (Inspection Checklist, College of American Pathologists, Traverse City, Mich.). The entire study was randomized and double-blind. RESULTS FIG. 2. Primary throat culture BAP with bacitra- Interpretation of disk-plate method. cim and SXT disks in heavily inoculated area after Plates which showed heavy beta-hemolysis overnight incubation. Heavy BHS not group A. within the zone of inhibition of normal flora surrounding the SXT disk and inhibition of growth around the bacitracin disk were interpreted as positive for group A BHS. This result is pictured in Fig. 1. Plates which showed heavy beta-hemolysis with growth of streptococci up to the bacitracin disk, as pictured in Fig. 2, were interpreted as heavy BHS, not group A. Those plates which grew only nornal flora and no visible BHS, as shown in Fig. 3, were interpreted _ as negative for BHS. FIG. 3. Primary throat culture BAP with bacitracin and SXT disks in heavily inoculated area after overnight incubation. No BHS seen. An additional group of plates exhibited few beta-hemolytic colonies within the zone of inhibition of normal flora surrounding the SXT disk, but growth was not sufficient to determine bacitracin susceptibility. In several cases, hemolytic colonies were visible only within the SXT zone, indicating that a culture made on a BAP without FIG. 1. Primary throat culture BAP with bacitra- the disk would have been called negative for cin and SXT disks in heavily inoculated area after BHS. A representative hemolytic colony from overnight incubation. Positive for group A BHS. each of these plates was transferred to one-half

3 82 BARON AND GATES of a second BAP with a bacitracin disk placed in the heavily inoculated area, and the plate was incubated overnight. The colony was also identified by the FA technique. In all of these cases, identification based on bacitracin susceptibility of the pure subculture agreed with the result obtained by direct FA stain of the colony. Small numbers of plates from both atmospheres grew beta-hemolytic colonies which were found to be staphylococci or neisseriae based on visual observation, Gram stain, and catalase test. FA stain. By FA staining from Todd-Hewitt broth, the control used in this study, 52 cultures were found to contain fluorescent chains of cocci indicative of group A BHS, as shown in Table 1. In nine of these smears, no more than a rare chain was seen after 3 min of screening. Todd- Hewitt broth cultures were incubated for 6 h during this study, which is longer than the time used in previous studies (6, 9) or the 2 to 4 h recommended by Moody (11). Thus, these specimens were given an enhanced opportunity for selection of small numbers of streptococci. As indicated in Table 1, there were 11 broth cultures found to be positive for group A BHS by FA whose corresponding plates were both negative. Of these 11 cultures, 7 of the smears showed only rare chains and probably represented either low-level carriers or artifact. If these 7 broth culture FA results are considered as carrier or artifact, the plate technique probably missed 4 significant positive cultures, but identified 41 of 45 cultures considered to be significantly positive for group A BHS by Todd- Hewitt-FA, yielding 91% agreement of the plate method compared to Todd-Hewitt-FA. Disk-plate method. As shown in Table 2, 44 plates incubated in either air or C02 were positive for group A BHS. About 75% of the plates from each atmosphere could be read unequivocally after overnight incubation, and an additional 25% of the plates showed BHS in numbers TABLE 1. Todd-Hewitt broth-fa results compared to disk-plate results for 259 throat cultures Method No. of cultures Broth-FA positive,' both plates' negative 11 (7c d) Broth-FA positive, only C02 plate positive 2 (1C) Broth-FA positive, only air plate positive.. 0 Broth-FA positive, both platesb positive 39 (19) a Positive indicates positive for group A BHS. b Plates incubated in both atmospheres, 5% C02 and air ċ FA smears showing rare fluorescent chains after 3- mm screening time. d Numbers in parentheses are included in the total number of cultures indicated. J. CLIN. MICROBIOL. TABLE 2. Disk-plate results for 259 throat cultures incubated in either 5% C02 or air atmosphere No. of cultures Results Air C02 Positive for group A BHS after overnight incubation Hemolytic colonies seen after 10 9 overnight incubation, positive for group A BHS on subculture plus bacitracin disk Heavy BHS, not group A, after 14 (la) 12 overnight incubation Hemolytic colonies seen after 0 2 overnight incubation, identified as non-group A on subculture plus bacitracin disk a A representative colony was identified as group A by FA smear directly from the colony. This falsenegative result has been included in the 14 plates listed in this category. See text for discussion. too small for interpretation of bacitracin susceptibility. These cultures required an additional subculture to determine whether the BHS seen were group A. It must be noted that one plate which had been incubated in air was interpreted as heavy BHS, not group A, by visual inspection; however, representative colonies were subsequently identified as group A BHS by direct FA stain from colonies on the plate. This may represent either a false-negative bacitracin result or a culture which contained both group A and nongroup A BHS. The corresponding plate from the same patient which had been incubated in C02 was correctly interpreted as group A BHS. Table 3 shows the numbers of disk-plate cultures which recovered group A BHS whereas the corresponding Todd-Hewitt broth cultures were negative by FA. These 6 cultures and the 52 cultures found to be positive by broth-fa give a total of 58 cultures found to be positive for group A BHS by at least one of the three methods (broth-fa, air-, and C02-incubated plates). DISCUSSION The disk-plate method enabled us to detect the presence of group A BHS in 75% of plated throat specimens examined after overnight incubation. This is a substantial shortening of the time required between receipt of the specimen and reporting of the results compared with conventional diagnostic methods utilizing plates. An additional subculture of individual colonies of BHS to a second BAP was necessary for 25% of the specimens. However, since two sub-

4 VOL. 10, 1979 TABLE 3. Disk-plate results compared with broth- FA results for 259 throat cultures Results PRIMARY PLATE METHOD FOR THROAT CULTURE No. of cultures Both plates positive," broth-fa negative... 2 C02-incubated plate positive only, broth-fa negative 1 Air-incubated plate positive only, broth-fa negative 3 apositive for group A BHS by disk-plate method, confirmed by direct FA stain of representative colony. cultures can be placed on a single BAP, less total BAPs are used. Pure culture bacitracin testing of all BHS as currently recommended (4) requires an additional day and additional plates for every culture suspected of having grown BHS. Using the combination of primary plate reading and subculturing, whether the primary plate was incubated in air or C02, we found 44 cultures positive for group A BHS. Of the 58 cultures found to contain group A BHS by any method, broth-fa identified 52, 45 of which are considered significant based on quantity of fluorescent chains seen. Thus, the total number of significant cultures found by broth-fa (45 cultures) and disk-plate (44 cultures) is remarkably similar Ẇe found one false-negative result with a plate incubated in air. One misidentification of BHS from the 58 (44 plates with group A BHS plus 14 plates with non-group A BHS) air-incubated plates shown to harbor BHS yields a falsenegative rate of 2%, which corresponds to the incidence of false identifications associated with bacitracin (2, 5). The C02-incubated plate from this patient correctly identified the BHS as group A. Although Murray and others reported an increased number of non-group A BHS recovered after C02 incubation of throat cultures compared with air incubation (14), our results showed little difference between the two atmospheres. The two non-group A BHS recovered from C02-incubated and not air-incubated plates may be a result of enhanced growth of non-group A BHS in C02; however, two specimens showed group A BHS only from the plates incubated in CO2 and not the corresponding plates from the same patients which were incubated in air. Gunn reported the first use of SXT and Taxo A disks for presumptive identification of group A BHS on pure cultures (7). His group subsequently reported enhanced recovery of group A BHS from a BAP medium which incorporated 25,ug of SXT per ml compared with standard BAP cultures (8). This medium is effective, but has shown performance variability in inhibition of normal flora by SXT with different lots of Trypticase soy agar base (Karl Leemkuil, GIBCO Diagnostics, and Bruce Gunn, Silver Spring, Md., personal communication). For this reason, GIBCO Diagnostics of Madison, Wis., has discontinued production of such a plate. Carr-Scarborough of Atlanta, Ga., currently produces an SXT-BAP commercially which has a shelf life of 3 to 5 weeks after receipt. Using prices provided for locally available products and 1979 College of American Pathologists workload recording unit values, a comparison of costs among the three methods utilized in this study showed the two-disk plate method to be similar to standard bacitracin techniques in materials expenses and considerably lower in technologist time. Table 4 illustrates these differences. Since microbiology is still primarily a labor-intensive operation, the two-disk plate method is beneficially cost-effective. Placement of a single bacitracin disk in the heavily inoculated area of a primary throat culture plate results in as many as 44% false-nega- TABLE 4. Cost comparison of three identification techniques for group A BHS from throat cultures with purchased materials and media Costs for a hypothetical laboratory' Mate- Tech- Identification technique I o ri nolo- Intial out- (dol- gist lay (dol- lars/ time lars) am! speci- (min/ speci- men) seci men) Todd-Hewitt-FA 3,835.00b 0.44c 4.25d Pure subculture with bac e 3.13f itracin disk Two-disk plate method h a Based on a total of 1,000 throat cultures received 20/day, from which 15% contain group A BHS and 10% contain non-group A BHS. b Transmitted-light microscope with mercury vapor lamp (AO series 120 Fluorestar). ' 1,000 cover slips ($22.00) FA slides ($2.00) + 1,000 Todd-Hewitt broths ($375.00) + 1,000 drops of FA conjugate ($40.00) = $439.00/1,000 = $0.44. d 25 min per staining set x 50 days [1,000/(20 specimens per day)] + 3-min scanning time per smear = e 1,000 primary BAPs ($282.00) subculture BAPs, 2 organisms per plate ($35.25) bacitracin disks ($6.25) = $323.50/1,000 = $0.32. f 2.5 min per plate x 1,250 plates = g 1,000 primary BAPs ($282.00) + 1,000 SXT disks ($23.00) + 1,000 bacitracin disks ($25.00) + 63 subculture BAPs, 2 organisms per plate ($9.02) + 63 subculture bacitracin disks ($1.58) = $340.60/1,000 = $0.34. h 2.5 min per plate x 1,063 plates =

5 84 BARON AND GATES tive results (13). These results were found to be due primarily to overgrowth of normal flora, too few streptococci present, or no visible zone of inhibition. The use of the SXT disk circumvents these pitfalls without the expense and inconvenience of an additional selective medium. In addition to the initial expense of an ultraviolet microscope, FA screening of throat cultures grown in broth has the disadvantages of (i) being unable to quantify the number of BHS in the specimen and (ii) requiring the microscopic screening of a large number of cultures, since both negative and positive slides must be examined (11). Breese and others (1) have demonstrated the significance, both clinically and epidemiologically, of quantifying the numbers of BHS in cultures. Thus, a plate method is preferred to a broth technique for initial isolation of these organisms (11). A Mayo Clinic study reported in 1977 showed recovery of group A BHS from 16.5% of 39,116 throat specimens (15). In our study, 44 plates were positive from either air or C02, either on initial examination or by subculture, yielding a 17% positive rate, which correlates well with the Mayo Clinic experience. This suggests that the two-disk plate method is reliable. The two-disk plate method provides a simple, cost-effective, and time-saving technique for determining the presence of group A BHS in throat cultures. Application of this method is particularly suited to small laboratories in which an ultraviolet microscope is unavailable and in which buying or making special media would be impractical. ACKNOWLEDGMENTS We gratefully acknowledge the assistance of Donald W. Smith for help with experimental design and Richard A. Proctor for comments during preparation of the manuscript. The work was supported by St. Marys Hospital Medical Center. Karen Ballweg's excellent secretarial assistance is also appreciated. LITERATURE CITED 1. Breese, B., F. Disney, W. Talpey, and J. Green J. CLIN. MICROBIOL. Beta-hemolytic streptococcal infection. Am. J. Dis. Child. 119: Coleman, D., D. McGhie, and G. Tebbutt Further studies on the reliability of the bacitracin inhibition test for the presumptive identification of Lancefield group A streptococci. J. Clin. Pathol. 30: Ederer, G., and S. S. Chapman Simplified fluorescent-antibody staining method for primary plate isolates of group A streptococci. App. Microbiol. 24: Facklam, R Streptococci, p In E. Lennette, E. Spaulding, and J. Truant (ed.), Manual of clinical microbiology, 2nd ed. American Society for Microbiology, Washington, D.C. 5. Facklam, R., J. Padula, L. Thacker, E. Wortham, and B. Sconyers Presumptive identification of group A, B, and D streptococci. Appl. Microbiol. 27: Freeburg, P Rapid fluorescent-antibody stain technique with group A streptococci. Appl. Microbiol. 19: Gunn, B SXT and Taxo A discs for presumptive identification of group A and B streptococci in throat cultures. J. Clin. Microbiol. 4: Gunn, B., D. Ohashi, C. Gaydos, and E. Holt Selective and enhanced recovery of group A and B streptococci from throat cultures with sheep blood agar containing sulfamethoxazole and trimethoprim. J. Clin. Microbiol. 5: Martin, A. J., and R. F. Bigwood, Jr Rapid fluorescent-antibody staining technique. Appl. Microbiol. 17: Maxted, W The use of bacitracin for identifying group A hemolytic streptococci. J. Clin. Pathol. 6: Moody, M Old and new techniques for rapid identification of group A streptococci, p In L. Wannamaker and J. Matsen (ed.), Streptococci and streptococcal diseases. Academic Press Inc., New York. 12. Moody, M., A. Siegel, B. Pittman, and C. Winter Fluorescent-antibody identification of group A streptococci from throat swabs. Am. J. Public Health 53: Murray, P., A. Wold, M. Hall, and J. Washington Bacitracin differentiation for presumptive identification of group A beta-hemolytic streptococci: comparison of primary and purified plate testing. J. Pediatr. 89: Murray, P. R., A. D. Wold, C. A. Schreck, and J. A. Washington II Effects of selective media and atmosphere of incubation on the isolation of group A streptococci. J. Clin. Microbiol. 4: Murray, P., A. Wold, and J. Washington Recovery of group A and nongroup A beta-hemolytic streptococci from throat swab specimens. Mayo Clin. Proc. 52:81-84.