MAb. The production and characterization of the MAb. have been described earlier (12, 13). Briefly, B. fragilis

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Mar. 1988, p /88/ $02.00/0 Copyright «D 1988, American Society for Microbiology Vol. 26, No. 3 Detection of Bacteroides fragilis, Bacteroides thetaiotaomicron, and Bacteroides ovatus in Clinical Specimens by Immunofluorescence with a Monoclonal Antibody to B. fragilis Lipopolysaccharide MATTI K. VILJANEN,* LINNEA LINKO, AND OLLI-PEKKA LEHTONEN Department of Medical Microbiology, University of Turku, Kiinamyllynkatu 13, SF-20520, Turku, Finland Received 28 August 1987/Accepted 11 December 1987 A total of 1,897 clinical specimens (1,019 aspirates and 876 swabs) were studied by indirect immunofluorescence (IF) with a mouse monoclonal antibody (MAb) against a D-galactose oligomer of Bacteroidesfragilis lipopolysaccharide. The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about 50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobic organisms tested. The sensitivity of IF in comparison with culturing was 78.9% for all three species. Of the 32 strains originating from culture-positive, IF-negative specimens, 13 lacked the target determinant for the MAb. Sensitivity was highest with specimens taken from the perineal area (87.1%) and lowest with those taken from undefined sites (56.6%). Sensitivity was better with aspirates (86.8%) than with swabs (72.6%). The specificity of IF was 95.6% for all of the material. Positive and negative predictive values were 51.1 and 98.0%, respectively. Neither long transportation times of specimens nor antimicrobial therapy seemed to correlate with the occurrence of IF-positive, culture-negative specimens. This study shows that a single MAb can be used to establish an IF assay that can complement isolation in the detection of these three members of the B. fragilis group. Bacteroides fragilis is the anaerobic species most often isolated in clinically important mixed anaerobic infections (2). New antimicrobial drugs have greatly improved the outcome of infections with B. fragilis. Thus, it is important for clinicians to obtain rapid information on the possible involvement of this organism in an infection. Culturing is too slow a method even under optimal conditions, and rapid methods have to be based on the detection of either metabolic or antigenic substances of the bacterium. Only a few reports have indicated that the direct analysis of clinical specimens by gas-liquid chromatography can provide a reliable presumptive identification of anaerobes (3). In contrast, there are numerous reports on the use of immunofluorescence (IF) techniques in the diagnosis of infections with Bacteroides species (1, 4, 6, 8, 11, 15, 16, 18, 19), and commercial kits are even available for this purpose. However, despite promising preliminary results, IF techniques have not gained any wide use. All earlier IF techniques for B. fragilis detection have used polyclonal antibodies, and cross-reactions have been difficult to eliminate. We previously reported on the production and characterization of a monoclonal antibody (MAb) directed against the lipopolysaccharide (LPS) of B. fragilis (13). The target determinant is composed of a D-galactose oligomer in the core of LPS (12). The MAb has been shown to react with 96% of clinical B. fragilis isolates and with about 50% of Bacteroides ovatus and Bacteroides thetaiotaomicron isolates but not with other aerobic or anaerobic organisms tested (13). The purpose of this study was to investigate the sensitivity and specificity of an IF method with this MAb in the direct detection of the three members of the B. fragilis group (BFG) in clinical specimens. * Corresponding author. MATERIALS AND METHODS MAb. The production and characterization of the MAb have been described earlier (12, 13). Briefly, B. fragilis ATCC was grown on brucella blood agar plates supplemented with vitamin K (10,ug/ml) and hemin (1,g/ml) in anaerobic jars. Outer membranes were removed by treating the bacteria with heat and shearing in the presence of EDTA (9). Outer membranes were separated by ultracentrifugation and treated with Triton X-100 (no. 8603; E. Merck AG, Darmstadt, Federal Republic of Germany) at room temperature for 20 min. Insoluble material was separated by ultracentrifugation, and the supernatant was used for immunizations of BALB/c mice. The method of Kohler and Milstein (10) was used for the production of hybridomas. Culture fluids of the hybridomas obtained were tested for B. fragilis-specific antibodies by an enzyme immunoassay with bacterial sonic extract or LPS as the antigen (13). Nineteen stable clones reacting with both of the antigens were established, and one of them (designated BF5) was selected for this study. The selected MAb reacted with 96% of clinical B. fragilis isolates, with the type strain (NCTC 9343), and with one reference strain (ATCC 23745). Positive reactions were also obtained with five of nine B. thetaiotaomicron and two of five B. ovatus isolates (13). Only in vitro-produced MAbs were used throughout the study, since the preliminary experiments showed that ascitic fluids contained large numbers of polyclonal antibodies against various organisms of the normal flora, e.g., Escherichia coli and staphylococci. IF staining. IF staining was carried out concomitantly with the primary Gram staining of the specimens. Clinical material was smeared directly on slides; isolated colonies were suspended in saline solution before being smeared. Specimens were dried in the open air and fixed with cacodylateformaldehyde solution (Sigma Chemical Co., St. Louis, Mo.) at 4 C for 15 min. The slides were washed with distilled 448

2 VOL. 26, 1988 TABLE 1. Origins of specimens and comparison of isolation and IF in relation to the specimen groups No. of positive Origin of results in: Sensitivity specimen (n) Isolation IF (%)b only only Both Peritoneal cavity (369) il Perineal area (104) Urogenital area (95) Abdominal wall (104) Thoracic wall (73) Lower extremities (293) Upper extremities (88) Head and neck (96) Pleurae and lungs (110) Bronchi (23) Sinuses (49) Peritonsillar abscess (31) Brain abscess (115) 0 Cerebrospinal fluid (10) 0 0 Joint fluid (185) Bone (22) Miscellaneous or unknown (230) a MAb to B. fragilis LPS was used in the IF assay. b Calculated only in groups with over five specimens positive in both tests. The overall sensitivity was 78.9%. water and allowed to dry for 5 min. The MAb (undiluted culture medium) was incubated on the slides in a humid chamber at 23 C for 30 min. After the slides were washed and dried, fluorescein-conjugated rabbit antibodies [F(ab)2 fraction] to mouse immunoglobulin (Dakopatts a/s, Glostrup, Denmark) diluted 1:40 with phosphate-buffered saline supplemented with 1% normal sheep serum were added and incubated at 23 C for 30 min. After the slides were washed, cover slips were placed on the slides; buffered glycerol was used as the mounting solution. An SM-Lux UV microscope (H. E. Leitz Wetzlar GmbH, Wetzlar, Federal Republic of Germany) was used in the inspection of the preparation (magnification, 630x). A preparation was interpreted as negative if fewer than four fluorescing organisms per slide were found. The fluorescence should be an apple green outlining of the bacteria. Even if the fluorescing material mainly consisted of phagocytized or cell-associated bacterial particles, a prerequisite for positivity was the occurrence of some intact organisms in the specimen. If a specimen was positive by IF but did not yield any of the three BFG species, the organisms that were isolated were tested by IF for cross-reactivity. Conversely, if a BFG species was isolated but the specimen was negative by IF, the isolated strain was tested by IF to find out if it belonged to the strains not containing the target determinant for the MAb. During the present work it was found that the MAb also reacts with some group B streptococci (GBS). However, streptococci could be morphologically easily differentiated from bacteroides. The one specimen containing positive GBS was not included in the number of IF-positive specimens. Cultivation and identification of bacteria. The specimens were plated on chocolate agar (Columbia agar base; Oxoid Ltd., London, England) and blood agar (blood agar base, with 5% bovine blood; Oxoid) for aerobic culturing. The plates were examined after 24 and 48 h of incubation, and the isolates were identified by standard procedures. Anaerobic culturing was carried out on brucella blood agar plates supplemented with menadione and L-cysteine (MC plates), DETECTION OF B. FRAGILIS 449 MC plates supplemented with neomycin (0.2 mg/ml), and bacteroides bile esculin plates (14). In addition, the specimens were inoculated into 10 ml of thioglycolate broth supplemented with Fildes enrichment. The anaerobic plates were incubated in jars with a gas mixture containing 80% N2, 10%CO2, and 10% H2 at 35 C for 48 h. The API 20A system (API International, S.A., Geneva, Switzerland) supplemented with some tests listed in the Anaerobe Laboratory Manual (5) and the Wadsworth Anaerobic Bacteriology Manual (17) was used in the identification of anaerobic bacteria. Test tube modifications of trehalose, xylose, rhamnose, and mannitol utilization tests were used to confirm the differentiation of B. thetaiotaomicron and B. ovatus and in the differentiation of these species from the other indolepositive bacteroides, Bacteroides uniforms, Bacteroides asaccharolyticus, and Bacteroides eggerthii. Clinical specimens. A total of 1,897 random clinical specimens were tested. All specimens were from patients hospitalized in the Turku University Central Hospital or the Turku Municipal Hospital. The main categories of sampling sites are listed in Table 1. Of the specimens, 53.6% were pus aspirates or body fluids, and 46.4% were swabs. The aspirates were taken into syringes, capped tubes, or anaerobic transportation lagenae (Portagerm; BioMerieux, Charbonnieres-les-Bains, France). RESULTS Of the 1,897 specimens studied, 108 yielded B. fragilis, 41 yielded B. thetaiotaomicron, and 3 yielded B. ovatus in cultivation. Of the B. fragilis-positive specimens, 90 were positive by IF. The corresponding figure for the specimens growing either of the two other species was 30 (Table 2). Thus, the sensitivities of IF by comparison with culturing were 83.3% for B. fragilis, 68.2% for B. thetaiotaomicron and B. ovatus, and 78.9% for all three species (Table 2). IF of smears of BFG colonies isolated from the 32 IF-negative specimens revealed that 13 (40.6%) of these isolates were negative and, evidently, lacked the determinant recognized by the MAb (Table 2). A rough semiquantitative comparison showed that the sensitivity of IF correlated with the number of BFG colonies on the primary culture plate (Table 3). A majority of specimens strongly positive by IF yielded more than 50 BFG colonies on the primary culture plate. However, there were 53 specimens strongly positive by IF but negative for the three BFG species. Conversely, 21 specimens growing more than 50 BFG colonies on the primary culture plate proved IF negative. Nine of them proved negative in the staining of colonies (Table 3). The proportions of swabs in all of the material and among the specimens positive by IF were 46.2 and 48.9%, respec- TABLE 2. Comparison of isolation and IF with MAb to B. fragilis LPS in the detection of the three BFG species in 1,897 random specimens No. of specimens with Organism isolated indicated no. of organisms positive by IF: Sensitivity (%) Specificity (%) <4a 4 to 10 >10 B. fragilis 18 (7) B. thetaiotaomicron 14 (6) or B. ov'atus None or other 1, a IF was interpreted as negative. Numbers in parenthesis are numbers of strains without the target determinant for the MAb.

3 450 VILJANEN ET AL. TABLE 3. Semiquantitative comparison of isolation and direct detection of B. fragilis, B. thetaiotaomicron, and B. ovatus by IF with MAb to B. fragilis LPS No. of organisms positive by IF No. of specimens with indicated no. of bacteroides colonies on the primary culture plateau: 0 <10 10 to 50 >50 <4b 1,630 7 (4) 4 (0) 21 (9) 3 to > a The primary culture plate was a bacteroides bile esculin plate. Numbers in parenthesis are numbers of strains without the target determinant for the MAb. The sensitivities for the categories <10, 10 to 50, and >50 were 68.1, 71.4, and 81.9t%, respectively. b IF was interpreted as negative. tively (Table 4). The sensitivity of IF was higher with the aspirates (86.8%) than with the swabs (72.6%) (Table 4). A slightly higher sensitivity was also obtained with abscess and body fluid specimens (both 81.5%) than with wound specimens (74.1%) (Table 4). The overall specificity of IF was 93.4% (Table 2). There were 115 specimens among the 235 IF-positive specimens from which none of the three BFG species could be isolated. On the other hand, 1,630 specimens of the 1,662 IF-negative specimens also were negative in isolation. The positive and negative predictive values of IF were thus 51.1% and 98.0%, respectively. Of the 115 IF-positive, isolation-negative specimens, 53 were interpreted strongly positive in IF, since more than 10 fluorescing organisms were seen per slide. A total of 59.1% of the 115 specimens were from anatomical locations potentially infected with gastrointestinal microbial flora (i.e., the peritoneal cavity, perineal area, abdominal wall, and urogenital area); the corresponding figures were 75.0% for BFG isolation-positive specimens and 35.4% for all of the material (Table 1). The mean transportation time for the 115 specimens did not differ significantly from that for the other specimens (data not shown). Based on information on the submission form, the incidence of ongoing antimicrobial therapy was even lower among isolation-negative patients (50.4%) than among isolation-positive patients (61.2%). To get more reliable information on therapy, we inspected patient records for 57 of the 115 patients. In only 16 cases could the therapy be considered effective against bacteroides. The discordance between isolation and IF was particularly prominent with the specimens taken from peritonsillar ab- TABLE 4. Effects of the sampling method and infection type on the detection of B. fragilis, B. thetaiotaomicron, and B. ovatus by isolation and IF with MAb to B. fragilis LPS No. of positive results in: Parameter Sensitivity of (no. of specimens) Isolation IF only Both IF (%) only Sampling method Aspiration (1,019) Swabbing (876) Infection type Abscess (342) Wound infection (674) Infectious exudate (767) or body fluid Undefined (106) TABLE 5. Microbiological findings for the 115 specimens positive in IF with MAb but negative in isolation for B. fragilis, B. thetaiotaomicron, or B. ovatus Microbiological result specimens No. of Gram-negative rods in primary staining but not in isolation... 3 Totally isolation negative At least one aerobe At least one anaerobe Mixed infection Isolated microbes Escherichia coli Streptococcus faecalis Klebsiella sp... 7 Viridans group streptococcus... 7 Beta-hemolytic GBS... 3 Staphylococcus aureus... 2 Coagulase-negative staphylococcus... 3 Bacteroides vulgatus... 3 Bacteroides distasonis... 2 Unidentified bacteroides... 7 Fusobacterium sp... 4 Streptococcus intermedius... 3 Anaerobic gram-negative rod Anaerobic gram-positive coccus Anaerobic gram-positive rod... 3 Candida albicans... 7 a Only microbes isolated from more than one specimen are shown. scesses or the head and neck area (Table 1). None of the seven IF-positive peritonsillar abscess specimens and only one of the five IF-positive head and neck specimens yielded any of the three BFG species in isolation. All of the seven peritonsillar abscess specimens yielded one or more other bacterial species in isolation, and only one specimen from the head and neck area was totally isolation negative. Table 5 shows the microbiological findings for the 115 IF-positive specimens not yielding any of the three BFG species in isolation. In only three cases was a gram-negative rod found in the primary staining but not in isolation. In 44 cases, both aerobic and anaerobic organisms were isolated. E. coli was the most frequently isolated bacterial species. Staphylococcus aureus was isolated only from two specimens, and coagulase-negative staphylococci were isolated from three specimens. IF of colonies taken from pure cultures revealed that none of the organisms isolated from these specimens reacted with the MAb. Two main microscopic results were observed in the positive cases. If a positive specimen did not contain leukocytes, the bacteria remained intact and were easy to identify (Fig. 1A). When leukocytes were abundant, both intact organisms and phagocytized or leukocyte-associated fluorescing material were seen (Fig. 1B). As mentioned in Materials and Methods, a prerequisite for positivity by IF was the occurrence of intact bacteria, even in the leukocyte-rich specimens. No fluorescing large organisms resembling L-phase bacterial cells, tissue cells, or leukocytes were present in the direct smears. DISCUSSION J. CLIN. MICROBIOL. In this study, an in vitro-produced MAb with a wellcharacterized target determinant in the LPS of B. fragilis was used in an IF technique for detecting this microbe and two other BFG species, B. thetaiotaomicron and B. ovatus,

4 VOL. 26, 1988 FIG. 1. IF staining with the MAb against the D-galactose oligomer in B. fragiles LPS. (A) Intact B. fragilis organisms with typical fluorescent outlining. (B) Leukocyte-associated bacterial fragments in an isolation-positive specimen with abundant leukocytosis. directly in 1,897 clinical specimens. By comparison with cultivation, the sensitivity of the assay varied from 87.1 to 56.3%, depending on the anatomical location from which the specimen was taken. Earlier reported sensitivities of IF with polyclonal antibodies in the direct detection ofb.fragilis and related species varied between 80.9 (15) and 100% (8). Even with the same commercial reagents very different results have been obtained (80.9 to 98.5%) (1, 15). These results have been based mostly on relatively limited and selected materials. Perhaps the lowest sensitivity figure occurred with the largest amount of material (15). Conclusions about the performance of a diagnostic test must therefore be based on relatively long experience with defined material of representative clinical specimens. The differences may also reflect the efficacy of B. fragilis isolation in different laboratories. DETECTION OF B. FRAGILIS 451 The sensitivity of the assay was better with the aspirates than with the swabs. This result evidently was due to larger numbers of bacteria in aspirates, which could be taken only from infections with abundant pus production, and not to the superiority of aspiration as a sampling method. When considering the sensitivity figures obtained, it must be kept in mind that relatively strict positivity criteria were used. In specimens with abundant leukocytosis, large amounts of fluorescing material, both leukocyte associated and free, was a common finding. However, a prerequisite for positivity by IF was the occurrence of intact bacteria with a fluorescent outlining. Some specimens remained negative by IF since they yielded a BFG strain that did not contain the target determinant for the MAb. This was the case with 7 of the 18 B. fragilis strains and 6 of 14 B. thetaiotaomicron and B. ovatus strains isolated from IF-negative specimens. The sensitivity should be improved by developing MAbs against these strains and using antibody mixtures. However, our preliminary experiments indicate that the strains not reacting with the MAb used in this study do not contain a common epitope that could yield MAbs with sufficient coverage. MAbs obtained by immunization with some species not having the D-galactose oligomer have been strictly strain specific, even though some of them have been directed against LPS. The specificity of the present method, 93.4% in all of the material, is on the same level as in the reports with polyclonal antisera. However, almost as many IF-positive specimens (115 of 120) were found among both negative and positive specimens in isolation for the three BFG species, lowering the positive predictive value of the assay to 51.1%. In our opinion, this value does not do justice to IF. The specificity of IF was ascertained by many means. The MAb has proven specific for the three BFG species (13). The only exception is the reactivity with GBS strains observed in this study. However, this reactivity caused only one false-positive result and was not taken into account. The bacteria isolated from the IF-positive, BFG-negative specimens were tested for their reactivity with the MAb, and none of them proved reactive. Furthermore, several of these specimens were totally isolation negative. This result strongly suggests that the discrepancy between isolation and IF could not be caused by the poor specificity of IF. A more plausible explanation is that the specimens really contained killed BFG bacteria or bacteria with a low ability to grow on artificial media, making the use of isolation as a reference method questionable in the assessment of positive predictive values of IF and other antigen detection methods. In addition to antimicrobial therapy, inappropriate collection and transportation of specimens may affect adversely culture results. However, neither transportation time nor antimicrobial therapy could explain the discrepancy in this study. Overgrowth by other bacterial species and the resulting failure to recognize bacteroides colonies has been suggested as one possible explanation for negative cultures (1). In the present study, aerobic bacterial species were isolated from 76 and anaerobic species were isolated from 58 of the 115 specimens positive by IF but negative for the three BFG species in cultures. In these specimens, interference by other microbes may have been a factor impairing the recognition of bacteroides, although the selective bacteroides bile esculin plate largely overcame this problem. However, 25 of the specimens were totally isolation negative. Thus, overgrowth cannot be held as a general explanation for the discrepant results. With specimens taken from peritonsillar abscesses or the

5 452 VILJANEN ET AL. head and neck area, the discordance between IF and isolation was most prominent. None of the seven IF-positive peritonsillar abscess specimens yielded any of the three BFG species in isolation; only one specimen from the head and neck area was positive in both tests. With these specimens interference by other microbes may have taken place, since almost all of them were polymicrobial in nature. Such interference may occur in the infectious focus and, with some`local factors, may impair the viability of bacteroides. This problem clearly needs further exploration. DeGirolami and Mepani (1) have reported that GBS may cause a false-positive reaction in a commercial bacteroides IF test with polyclonal antibodies. Since the specimen in question in their study was not incubated anaerobically, the presence of B. fragilis could not be outruled. Our results suggest that this cross-reaction may be a true one, since 1 of the 12 GBS isolated in this study proved reactive with the MAb. Our preliminary studies have shown that ca. 12% of Streptococcus agalactiae strains isolated in our laboratory are positive. The staining of the streptococci was bright and resembled the membranous fluorescence of B. fragilis. This result suggests that the determinant is located on the surface of the organism. Studies on the nature of the cross-reactive antigen and its relation to type-specific antigens of GBS are under way. Leukocytes heavily loaded with fluorescing bacterial fragments were commonly found in leukocyte-rich specimens yielding B. fragilis in cultivation. Although phagocytized bacterial fragments could not be discriminated from those attached to the surfaces of cells, this phenomenon may be an expression of the low toxicity of B. fragilis LPS (7). On the other hand, we did not find fluorescing L forms or bacteria resembling eucaryotic cells, both of which have been reported to occur in some clinical specimens (16). An attempt to use the antibody in ascitic fluid led to cross-reactions with several organisms of the normal flora. Since absorption of ascitic fluids was not considered reasonable, only an in vitro-produced MAb was used throughout the study; such an MAb is generally recommended for diagnostic assays in bacteriology. Based on our experience, the use of undiluted culture medium was not any major drawback, since the hybridoma could be easily grown in large volumes of medium. This study shows that with a single MAb it is possible to establish an IF assay that detects ca. 79% of isolationpositive B. fragilis infections. The best sensitivities were obtained with specimens potentially infected with intestinal microbial flora. The specificity of IF was carefully ascertained, and the isolation-negative specimens that were positive by IF could not be considered false-positive, since IF can detect nonviable bacteria. ACKNOWLEDGMENTS This study was supported by grants from the Academy of Finland, the Emil Aaltonen Foundation, and the Research and Science Foundation of Laake Oy. We thank Maija Sarasniemi and Tarja Laine for technical assistance and Simo Merne for revising the language of the manuscript. LITERATURE CITED J. CLIN. MICROBIOL. 1. DeGirolami, P. C., and C. P. Mepani Evaluation of direct fluorescent antibody staining for rapid identification of members of the Bacteroidesfragilis group. Am. J. Clin. Pathol. 76: Gorbach, S. L., and J. G. Bartlett Anaerobic infections (second of three parts). N. Engl. J. Med. 290: Gorbach, S. L., J. W. Mayhew, J. G. Bartlett, H. Thadepalli, and A. B. Onderdonk Rapid diagnosis of anaerobic infections by direct gas-liquid chromatography of clinical specimens. J. Clin. Invest. 57: Griffin, M. H Fluorescent antibody techniques in the identification of the gram-negative nonsporeforming anaerobes. Health Lab. Sci. 7: Holdeman, L. V., E. P. Cato, and W. E. C. Moore (ed.) Anaerobe laboratory manual, 4th ed. Virginia Poly'technic Institute and State University, Blacksburg. 6. Holst, E., J. Oscarson, and P.-A. Mardh Evaluation of two fluorescent test kits for detection of selected Bacteroides species in clinical specimens. Curr. Microbiol. 3: Kasper, D. L Chemical and biological characterization of the lipopolysaccharide of Bacteroides fragilis subspecies fragilis. J. Infect. Dis. 134: Kasper, D. L., A. P. Fiddian, and S. Tabaqchali Rapid diagnosis of Bacteroides infections by indirect immunofluorescence assay of clinical specimens. Lancet i: Kasper, D. L., and M. W. Seiler Immunochemical characterization of the outer membrane complex of Bacteroides fragilis subspeciesfragilis. J. Infect. Dis. 132: Kohler, G., and C. Milstein Continuous cultures of fused cells secreting antibody of predicted specificity. Nature (London) 256: Labbe, M., N. Delamare, F. Pepersack, F. Crokaert, and E. Yourassowsky Detection of Bacteroides fragilis and Bacteroides melaninogenicus by direct immunofluorescence. J. Clin. Pathol. 33: Linko, L., A. Weintraub, P. Arstila, L. J. Pelliniemi, and M. K. Viljanen Characterization of the common immunodominant antigenic determinant in the lipopolysaccharide of Bacteroides fragilis by a monoclonal antibody. Scand. J. Immunol. 25: Linko-Kettunen, L. A. P., M. Jalkanen, H. Jousimies-Somer, O. Lassila, O.-P. Lehtonen, A. Weintraub, and M. K. Viljanen Monoclonal antibodies to the lipopolysaccharide of Bacteroides fragilis. J. Clin. Microbiol. 20: Livingston, S. J., S. D. Kominos, and R. B. Yee New medium for selection and presumptive identification of the Bacteroides fragilis group. J. Clin. Microbiol. 7: Slack, M. P., D. T. Griffiths, and H. H. Johnston The Fluoretec system for rapid diagnosis of bacteroides infection by direct immunofluorescence of clinical specimens. J. Clin. Pathol. 34: Stauffer, L. R., E. O. Hill, J. W. Holland, and W. A. Altemeier Indirect fluorescent antibody procedure for the rapid detection and identification of Bacteroides and Fusobacterium in clinical specimens. J. Clin. Microbiol. 2: Sutter, V. L., D. M. Citron, and S. M. Finegold Wadsworth anaerobic bacteriology manual, 3rd ed. The C. V. Mosby Co., St. Louis. 18. Weintraub, A., A. A. Lindberg, and D. L. Kasper Characterization of Bacteroides fragilis strains based on antigen-specific immunofluorescence. J. Infect. Dis. 147: Weintraub, A., A. A. Lindberg, and C.-E. Nord Identification of Bacteroides fragilis by indirect immunofluorescence. Med. Microbiol. Immunol. 167: