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Archs oral Bid. Vol.37, No. 2, pp. 9%104, 1992 Printed in Great Britain.All rightsreserved
LATEX AGGLUTINATION TEST FOR DETECTION OF MUTANS STREPTOCOCCI IN RELATION TO DENTAL CARIES IN CHILDREN I**T. FUJIWARA,’I. MORISAKI,’T. ~OSHIMA,*S. SOBUP~ T. TAKEI,“’ T. OGAWA,’S. ALALUUSUA, and S. HAMADA”~. Departments of ‘Oral Microbiology, ZPedodonticsand ‘Dentistry for the Handicapped, Osaka University Faculty of Dentistry, Yamadaoka, Suita-Osaka, 565 Japan (Accepted 22 August 1991) Summary-.A simple and rapid system based on a latex agglutination (LA) reaction was devised for the detection of mutans streptococci in dental plaque. Latex particles were sensitized with antibodies against whole cells of Streptococcusmutansstrains MT8148 (serotype c), MT703R (e) and OMZ175 (f) and Strep. sob&us strains B13 (d) and 6715 (g). These sensitized particles were agglutinated within a few
minutes after addition of l.O-long serotype-specific antigen from the homologous organisms or the nitrous acid extract of whole cells at 1OLlo6 c.f.u. The LA test specificallydifferentiated not only mutans streptococci from the other oral streptococci but also Strep.sobrinusfrom Strep.mutans.The LA test was also applicable to extracts of plaque from 206 human subjects who harboured mutans streptococci. In clinical trials, the outcome of the LA test correlated significantly with the number of mutans streptococci found in plaque (p < O.OOOl), which was quantified by the selective cultivation of mutans streptococci. Furthermore, the LA test discriminated between Strep. mutunsand Strep. sobrinusfrom human dental plaque. The sensitivity and the specificity of the LA test for detection of mutans streptococci were 78.9 and 100%. The degree of reactivity in the LA test correlated significantly with the number of decayed tooth surfaces (p < 0.0001) and decayed and filled tooth surfaces (p < 0.0001). These results suggest that the LA test could be useful clinically for the detection of mutans streptococci in dental plaque as well as serving as a caries-activity test. Key words: mutans streptococci, latex agglutination test. dental caries.
in saliva and on the application of these as cariesactivity test (Kohler and Bratthall, 1979; Newbrun et al., 1984; Alaluusua et al., 1984; Jordan et al., 1987; Jensen and Bratthall, 1989). However, these tests are relatively complicated and time consuming. The LA tests, in which latex particles are sensitized with a specific antibody, have been used for the clinical detection and identification of various pathogenic organisms including bacteria (Severin, 1972; Rench, Metzger and Baker, 1984; Facklam, 1987; Hopwood et al., 1987; Krambovitis et al., 1987; Lim
INTRODUCI’ION Mutans streptococci are strongly associated with the initiation of human dental caries and mutans strains induce caries in various experimental animals when infected and fed a high-sucrose diet (Hamada and Slade, 1980; Emilson and Krasse, 1985). Loesche and Straffon (1979) demonstrated a significant correlation between the number of mutans streptococci and prospective incidence of dental caries in man. As the number of mutans streptococci can be considered a useful indicator of caries risk, efforts have been focused on the development of microbiological tests for determining the number of mutans streptococci
and Fox, 1987; Burdick and Sottile, 1988; Moyer, Quinn and Showalter, 1990), viruses (Brandt et al., 1987; Grandien et al., 1987; Bodeus et al., 1988) and
*Present address: Department of Pedodontics and Orthodontics, Inst tute of Dentistry, University of Helsinki, Mannerheimintie 172, SF-00300 Helsinki, Finland. tAuthor to whom all correspondence should be addressed. Abbreviations:c.f.u, colony-forming unit; dfs, decayed and filled primary tooth surfaces; DFS, decayed and filled permanent tooth surfaces; ds, decayed primary tooth surfaces; DS, decayed permanent tooth surfaces; GBS, glycine-buffered saline (0.1 M, pH 8.2); LA, latex agglutination; MS13 agar, mitis-salivarius agar sup plemented with bacitracin (0.2 unit/ml) and sucrose (20%, final concentration); PBS, phosphate-buffered saline (0.05 M, pH 7.4); PBS-BSA, phosphate buffered saline containing 0.1% bovine serum albumin.
metabolic substances (Greenberg, Devine and McCrae, 1987; Lyerly et al., 1988; Sherman et al., 1988). These tests are becoming widely accepted for clinical examination because of the ease and speed of testing and their specificity. Mutans streptococci are now classified into eight serotypes, a-h. Among these, only serotypes c-g have frequently been isolated from human populations; other serotypes are only found sporadically in various populations in the world (Hamada and Slade, 1980; Hamada, Masuda and Kotani, 1980; Beighton et al., 1989; Coykendall, 1989). Current bacterial taxonomy indicates that serotype c/e/f strains belong to the Streptococcus mutans, while serotype d/g strains comprise the separate species, Strep. sobrinus 99
T. TAKEIel al.
(Coykendall, 1989). We prepared various antibodies specific for serotypes of mutans streptococci by immunizing rabbits. Our purpose now was to devise an LA system using antibodies for the detection of mutans streptococci and to examine the validity of the LA test with reference to dental decay in children. MATERIALS
Organisms Strep. mutans MT8148 (serotype c), MT703R (e) and OMZ175 (f), Strep. sobrinus B13 (d) and 6715 (g), Strep. cricetus E49 (a), Strep. rattus FA-1 (b), Strep. downei MFe28 (h), Strep. salivarius HHT, NCTC 8618 and NCDO 573, Strep. sanguis ST3, ST202, ATCC 10556, SK 4, SK 45 and SK 150, Strep. oralis ATCC 10557, SK 23 and NCTC 11427, Strep. gordonii ATCC 10558, SK 6 and SK 51, Strep. constellatus NCTC 10708, Strep. mitis SK 24, SK 96, SK 102 and SK 142, Strep. vestibularis MMl, Strep. pyogenes Sv and Enterococcus faecalis 476 were used in this study. These strains were kindly provided by Dr M. Kilian, Royal Dental College Aarhus, Denmark, and Dr R. A. Whiley, The London Hospital Medical College, London, U.K., or were selected from the stock culture collection at the Department of Oral Microbiology, Osaka University Faculty of Dentistry, Japan. In addition, 71 clinical isolates of mutans streptococci from 38 children who visited the Pedodontic Clinic, Osaka University Dental Hospital, Japan for regular oral examination were used for comparison. These isolates were serotyped by immunodiffusion test, as described by Hamada et al. (1980). Antigens
The organisms were cultured in TTY broth (Hamada and Torii, 1978), washed with distilled water and lyophilized. Cells were suspended in 20 mg (dry weight) per ml of saline and autoclaved at 120°C for 20min. They were centrifuged, and the supernatant was concentrated by a rotary evaporator. The clarified concentrate was applied to a column of DEAE-Sephadex A-25 (Pharmacia LKB Biotechnology, Uppsala, Sweden). The column was eluted with 0.05 M ammonium carbonate solution (pH 8.5). The unadsorbed fraction was further chromatographed on a Sephacryl S-200 (Pharmacia) column, which had been equilibrated with distilled water. The fractions containing serotype-specific polysaccharide antigen identified by the immunodiffusion test using serotype-specific antisera (vide infra) were combined and used as the serotypespecific antigen. Preparation of antiserum
Antisera to mutans streptococci were prepared by immunizing rabbits subcutaneously with whole cells of the streptococci emulsified with complete Freund’s adjuvant (Difco Laboratories, Detroit, MI). The immunoglobulin G (IgG) fraction was obtained from these antisera by salting out three times with l/3saturated ammonium sulphate, dissolved with 0.05 M PBS (pH 7.4) and stored at -20°C.
Sensitization of latex particles
Latex particles (0.876 pm dia, Immutex G2801, Japan Synthetic Rubber Co., Tokyo, Japan) were sensitized with partially purified antibodies against mutans streptococci by the method of Severin (1972), with some modifications. The latex suspension (0.5% w/v) in 0.1 M GBS (pH 8.2) was added to an equal volume of the IgG diluted to an appropriate concentration with GBS. After the mixture had been incubated at 37°C for 90 min, the sensitized latex particles were collected by centrifugation (3000 g), washed with 0.05 M PBS (pH 7.4) containing 0.1% BSA (Sigma Chemical, St Louis, MO) and 0.05% sodium azide, and resuspended with PBS-BSA to a concentration of 0.5% (w/v). Extraction of antigen for LA test
The polysaccharide antigen was extracted with nitrous acid from streptococcal cells with some modifications by the method of Slifkin and Interval (1980). Pellets of streptococcal cells ( 104-10’oc.f.u.) were suspended in a mixture of 50 ~1 of 8 M sodium nitrite and 50 ,ul of 2 M acetic acid, and incubated at room temperature for 5 min. The reaction mixture was neutralized with 1 M sodium hydroxide, and the extract was used for the LA test. Protocol of LA test
The solution (20~1) of serotype-specific polysaccharide antigen (0.01-100 ng carbohydrate) or the crude nitrous acid extract (20 ~1) of 103-lo9 c.f.u. of streptococcal cells was mixed with 20 ~1 of the sensitized latex reagent on a glass slide and allowed to stand at room temperature for 10 min with occasional shaking. The degree of agglutination reaction was evaluated as - , + and + + , based on both the quantity of clumps and the clarity of background: - , no agglutination; +, clump formation with some cloudy background; + + , heavy clumps with clear background. Subjects of clinical study
One-hundred and nineteen children, aged from 2 to 12 yr, who had visited the Pedodontic Clinic, Osaka University Dental Hospital for oral examination, and 87 children in eight nursery schools in the suburbs of Osaka, aged 3-5 yr, were included in this study. The childrens’ mouths were examined with a dental mirror and explored under appropriate light, and the number of ds and/or DS and dfs and/or DFS was determined according to the description of Msller (1966) (Table 1). Incipient occlusal lesions and visible decalcified spots on the enamel were not included. Bacteriological examination
Plaque samples were collected from the buccal and lingual surfaces of molars with a sterile excavator. After weighing, they were suspended in sterile saline and dispersed by ultrasonication for 10 s and IO-fold serial dilutions were made. One-hundred ~1 of the appropriate dilution were cultured on MSB agar (Difco Laboratories, Detroit, MI), supplemented with bacitracin (0.2 unit/ml; Sigma, St Louis, MO) and 0.58 M sucrose, final concentration) (Gold, Jordan and Van Houte, 1973). The agar plates were
LA test for mutans streptococci
Table 1. Subiects studied Patients of the University hospital (n = 119) Age (yr) ds and/or DS dfs and/or DFS The number of mutans streptococci in plaque (log,, c.f.u./O.1 mg)
6.9 & 2.7” 9.9 f 11.1 17.8 k 15.1 2.8 k 1.8
Children of nursery schools (n = 87) 4.3 + 6.6 * 9.5 + 2.4 +
1.0 9.5 12.6 2.1
Total (n = 206) 5.7 * 8.5 k 14.3 + 2.7 +
2.5 10.6 14.6 1.9
“Mean f SD.
incubated for one day in an atmosphere of 95% N, and 5% CO, at 37°C followed by incubation for one day in air at 37°C. Mutans streptococci were identified by their characteristic colonial morphology on MSB agar and the number of mutans streptococci was calculated (Table 1). Several typical colonies of Strep. mutans and Strep. sobrinus were picked up from each subject ;and cultured in Brain Heart Infusion broth (Difco L,aboratories, Detroit, MI). Speciation of mutans streptococci was confirmed by fermentation of m.annitol and sorbitoi. Serotyping was done by the immunodiffusion method using hot saline extract of lxhole cells and serotype-specific antisera (Hamada and Slade, 1980). LA test with plaque samples One mg (wet weight) of plaque sample was extracted in a mixture of 8 M sodium nitrite (50 ~1) and 2 M acetic acid (50 ~1) as described above. The extract was reacted with the sensitized latex reagents and the degree of latex agglutination reaction was recorded after 10 min of incubation at room temperature. Results of clinical trials are evaluated for sensitivity and specificity of the reaction. Sensitivity is defined as the percentage of infected subjects correctly identified as positive by the test; specificity is defined as the percentage of non-infected subjects correctly identified as negative. Statistical analysis Statistical analysis was done with contingency tables. Contingency coefficients were calculated as C = [x2/(x2 + N)]‘.“, where N is the total number of subjects in the study (Sharp, 1979). RESULTS
The latex reagents sensitized with antibodies against Strep. mutans (serotype c, e, or f)and Strep. sobrinus (d or g) produced positive agglutination when mixed with at least 10 ng of purified polysaccharide antigen of the homologous serotype or nitrous acid extract from at least 106c.f.u. of the homologous whole cells. Addition of nitrous acid extracts of homologous cells of mutans streptococci caused strong agglutination of the latex reagent sensitized with specific antibody, and some weak crossreactions were found among the strains of Strep. mutans or the strains of Strep. sobrinus and Strep. downei (Table 2). Forty-five strains of Strep. mutans (c), 17 strains of Strep. mutans (e), one strain of Strep. mutans (f),and one strain of Strep. sobrinus (d) and Strep. sobrinus (g) isolated from 38 patients of Osaka Universi1.y Dental Hospital were also used
for the LA test. The extracts of 62 strains of Strep. mutans had specific agglutination reactions with the latex particles sensitized against Strep. mutans MT8148 and/or Strep. mutans MT703R, although they did not cause an agglutination reaction with the other sensitized latex particles. A serotype f isolate only reacted with the latex particles sensitized against Strep. mutans 0MZ175. All the isolates of six strains of Strep. sobrinus have also positive reaction with the latex particles sensitized against Strep. sobrinus B13 and/or 6715 and negative with the other latex particles. However, there was no positive reaction with the nitrous acid extracts of the other oral and enteric streptococcal species (Table 2). Two-hundred and six plaque samples obtained from children with varying caries activities were examined for the presence of mutans streptococci by the LA test (Table 3). Mutans streptococci were not detected in 35 of these samples by culture on MSB agar. All these 35 plaque samples gave negative reactions against all of the latex reagents sensitized with antibodies specific for different serotypes of mutans streptococci; thus, the specificity of the LA test was 100%. In culture, we found that dental plaque samples from 171 subjects contained viable mutans streptococci, and 135 samples among them gave positive reactions with at least one of the five sensitized latex reagents, indicating that the sensitivity of the LA test was 78.9%. One-hundred and thirty-four out of 135 LA-positive samples also gave positive reactions with anti-Strep. mutans MT8148 (c) and/or MT703R (e) latex reagents, indicating that the sensitivity of the LA test using anti-Strep. mutans MT8148 (c) and MT703R (e) latex reagents was 78.4%. One-hundred and sixteen out of 150 plaque samples that contained Strep. mutans alone gave positive reactions with anti-Strep. mutans MT8148 (c), MT703R (e) and/or OMZ175 (f) latex reagents. One of two plaque samples that contained Strep. sobrinus alone gave a positive reaction with antiStrep. sobrinus B13 (d) latex reagent. Furthermore, both Strep. mutans and Strep. sobrinus were detected simultaneously by the cultural method in 19 plaque samples. Ten of them gave positive reactions with both anti-Strep. mutans and anti-Strep. sobrinus antibody-sensitized latex reagents. Eight of the 19 plaque samples agglutinated the latex reagents sensitized with anti-Strep. mutans antibodies; however, one
remaining plaque sample gave negative reactions with all of the latex reagents regardless of the nature of sensitization. Good correlation was found between the LA test and the number of mutans streptococci in dental plaque samples (Table 4). Seventy-one plaque
T. TAKEIet al.
Table 2. Specificity of the LA test sensitized with antibodies against mutans streptococci The latex reagent sensitized with antibodies against
Cell extracts with nitrous acid” Species
Strep. sobrinus Strep. Strep. Strep. Strep.
MT8148 (c) MT703R (e) OMZ175 (f)
cricelus rattus downei salivarius
Strep. constellatus Strep. mitis
Strep. vestibularis Strep. pyogenes E. fiecalis
MT8148 (c) MT703R (e) OMZl75 (f) B13 (d) 6715 (g) E49 (a)
FA-1 (b) MFe28 (h) HHT NCTC 8618b NCDO 573b ST3 ST202 ATCC 10556 SK 4’ SK 45’ SK 150’ ATCC 10557 SK 23’ NCTC 11427b ATCC 10558 SK 6c SK 51’ NCTC 10708 SK 24’ SK 96” SK 102’ SK 142’ MMlb sv 476
+ +d + -
+ ++ _
_ _ -
++ + + -
+ ++ -
“Nitrous acid extracts were prepared from 10’ c.f.u. of streptococcal cells. bObtained from Dr R. A. Whiley; see Whiley and Hardie (1988). ‘Obtained from Dr M. Kilian; see Kilian, Mikkelsen and Henrichsen (1989). dThe LA reaction was graded in three scales of - , + and + + samples extracted with nitrous acid did not produce agglutination with any of the latex reagents sensitized with antibodies against mutans streptococci. One plaque sample gave a single-positive reaction ( + ) with anti-Strep. sobrinus B13 (d) latex reagent only,
and the other 134 plaque samples gave positive reactions with either anti-Strep. mutans MT8418 (c) or MT703R (e) latex reagents. Statistical analysis showed a significant positive correlation between the degree of agglutination of the LA test and the number of mutans streptococci calculated by the cultural method (C = 0.594, p < 0.0001). When the LA test
was made with two types of latex reagents using antibodies against Strep. mutans MT8148 (c) and MT703R (e), a similar coefficient (C =0.589, p < 0.0001) was obtained. There was a significant positive correlation between the outcome of the LA test using the latex reagents sensitized with antibodies against Strep. mutans MT8148 (c) and MT703R (e) and the ds/DS (C = 0.483, p c 0.0001) (Table 5). Similarly, a positive correlation was found between the LA test and the dfs/DFS (C = 0.468, p < 0.0001) (data not
Table 3. Sensitivity and specificity of the LA test Detection of mutans streptococci Not detected Detected Strep. mutans alone Strep. sobrinus alone
Number of subjects found by the cultural method 35 171” 150 2
Both Strep. mutans and Streo. sobrinus
Number of positive subjects in the LA test 0 135a*b 116 lb 10
78.9 77.3 50.0
“Thirty-six subjects showing negative LA test had less than 1O’c.f.u. mutans streptococci/O.1 mg plaque when they were cultured on MSB agar plates. bAll subjects except one, who had a positive reaction only with anti-Strep. sobrinus B13 (d) latex reagent, showed positive reaction with anti-Strep. mutans MT8148 (c) and/or MT703R (e) latex reagents (sensitivity 78.4%, specificity 100%).
for mutans streptococci
Table 4. Relationship between the LA test and number of mutans streptococci in dental plaque Number of mutans streptococci (c.f.u./O. 1 mg) LA test + ++
Number of samples
71 71 64
35 0 0
17 15 8
102-104 >104 19 34 20
0 22 36
aN.D. = not detected.
II 0.01 Serotype
DISCUSSION In a series of preliminary experiments, latex particles were examined for their sensitization with various antibodies, e.g. rabbit polyclonal antibodies to whole cells of mutans streptococci, rabbit polyclonal and murine monoclonal antibodies to cell-surface protein antigen (F’Ac) (Okahashi et al., 1989), and rabbit polyclonal and murine monoclonal antibodies to glucosyltransferases (Hamada et al., 1989). When these monoclonal antibodies were used for sensitiz-
ation of the latex particles, the minimum quantity of the homologous antigen required was usually much greater than when. polyclonal antibodies were used for sensitization of the latex particles. Chromatographically purified IgG fractions of the polyclonal rabbit antibodies were also used for sensitization of latex particles; however, they caused non-specific agglutination without antigen. Various methods, such as nitrous acid (Slifkin and Interval, 1980) 0.05 M KOH, 0.:2 M NaCl, 0.02% EDTA, 6 M guanidine hydrochloride, 8 M urea, and detergents, including 1% sodium dodecyl sulphate, 1% 3-[(3dimethylammoniol-l-propanecholamidopropyl) sulphonate (CHAPS), 1% Tween-20, 1% Triton X-100, 1% n -octyl-/3-D-glucopyranoside, 1% n octyl-b-D-thioglucoside, 1% octanoyl-N-methylglucamide (MEGA-8), 1% nonanoyl-N-methylglucamide (MEG,4-9), and 1% deoxycholic acid (DOC), were attempted to extract polysaccharide and proteinaceous antigens from streptococcal cells and human dental plaque to improve the sensitivity and specificity of the L,A test (results not shown). These attempts showed that nitrous acid extracts and rabbit polyclonal whole-cell antibodies were the best combination for the LA test to detect mutans streptococci. The LA test using latex reagents sensitized with antibodies against whole cells of mutans streptococci could detect mutans streptococci at lo’-106c.f.u. of cell suspension (Fig. 1) and could also directly detect mutans streptococci in small amounts of plaque (0.1 mg wet weight) (Table 4). The sensitivity of our LA test was 78.9% and the specificity 100% in terms of the detection of mutans streptococci in the plaque samples examined (Table 3). A method simplified by using two latex reagents sensitized with antibodies Table 5. Relationship between the LA test and number of decayed tooth surfaces Number of decayed surfaces (ds and/or DS) LA test
37 9 5
22 16 13
7 24 15
6 21 31
Fig. 1. Correlation of LA reaction versus antigen concentrations. The latex reagents sensitized with antibodies against Strep. mutansMT8148 (c) or 0MZ175 m (0) and Strep. mutansMT703R (e), Strep. sobrinusB13 (d) or 6715 (g) (a) produced positive agglutination with homologous serotype-specific polysaccharide antigen (A) or the nitrous acid extract from whole cells of the homologous strain (B).
against Strep. mutans MT8148 (c) and MT703R (e) also showed a high sensitivity (78.4%) and specificity (100%) (Table 3). Furthermore, there was a positive correlation between the simplified LA test and the number of mutans streptococci in plaque, rather similar to that obtained when using five latex reagents (Table 4). These findings show that the latex reagents sensitized with partially purified antibodies against Strep. mutans MT8148 (c) and MT703R (e) were effective in demonstrating mutans streptococci. The simplified LA test can also be used to detect mutans streptococci in dental plaque and may serve as a quick ‘caries-activity test’ in dental clinics, which would in turn influence the management of patients by dental professionals. Several kits for rapid detection of group A streptococcal antigen based on LA reaction are now marketed. In this regard, Burdick and Sottile (1988) compared five LA tests for detecting group A streptococcal polysaccharide antigen. The sensitivity and specificity of these kits were reported as [email protected]
% and 91-98%. The minimum number of streptococcal cells needed for detection was 4.9 x lOl-4.6 x lo* c.f.u. of overnight broth culture. More recently, Moyer et al. (1990) reported that, in a sample of 327 children, their Group A Strep Test had a sensitivity of 75.0% and a specificity of 99.1%. In contrast to the highly sensitive LA tests for group A streptococci, our LA test for mutans streptococci is not so sensitive, although it is very specific. Our results, nonetheless, show that the amount of mutans streptococci antigen in plaque was usually enough to be detected by the LA test. Some epidemiological studies have shown that the number of mutans streptococci in saliva correlates with caries prevalence (Zickert, Emilson and Krasse, 1982) and Caries increment (Loesche and Straffon, 1979) and can be used for the evaluation of caries risk (Krasse and Newbrun, 1982). Recently, mass screening systems have been developed to estimate the salivary level of mutans streptococci, attempting to serve as a caries-activity test (Kiihler and Bratthall, 1979; Newbrun et al., 1984; Alaluusua et al., 1984; Jordan et al., 1987; Jensen and Bratthall, 1989). Although these cultural methods have been simplified, they take at least l-2 days to provide results. On the other hand, the LA test takes only
T. TAKEl et al.
about 15-20 min to give results and requires neither special facilities nor technical training. Furthermore, the LA test can be stored for at least 3 months in a refrigerator (4--8”(Z)(data not shown). Accordingly, the LA test would be more applicable than cultural methods in the clinical setting.
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