611
Development of a Rapid Latex Agglutination Test for Periodontal Pathogens Russell J.
Nisengard, Lynn Mikulski, *
*
Deborah
McDuffie, * and Paul Bronson*
studies reported here describe the development, characterization, and initial application of latex agglutination assays for periodontal pathogens. Latex reagents were prepared by sensitization of latex microspheres with rabbit IgG antibodies to either Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, or Prevotella intermedia. The protein concentration utilized for sensitization and microsphere size were optimized. One reagent was prepared to A. actinomycetemcomitans and a second combination reagent was prepared by mixing latex sensitized with antibodies to P. gingivalis and latex sensitized with antibodies to P. intermedia. The sensitivity of both latex reagents in the traditional wet and a dried format was evaluated. In addition, sensitivity and specificity with homologous and heterologous bacterial suspensions were evaluated. The reagents were found to demonstrate both specificity and sensitivity. Initial studies with subgingival human plaque demonstrated the ability of these reagents to detect the specific organisms in plaque. J Periodontol 1992; 63:611-617.
The
Key Words: Actinobacillus actinomycetemcomitans; votella intermedia; latex fixation test.
Porphyromonas gingivalis;
Pre-
The association of specific bacteria with active periodontal disease suggests a rationale for monitoring specific
bacteria in plaque samples.6 8 Their identification in high numbers may help in assessing disease activity, selecting individualized treatment modalities, monitoring effects of treatment, selecting appropriate recall intervals, and determining when retreatment is indicated.7 9 At present, culture is considered the "gold standard" or reference methodology for identifying subgingival bacteria. Its routine use in a clinical office, however, is limited because of a 1 to 2-week time period necessary for definitive test results, problems associated with maintaining viability and anaerobiosis during transportation of the plaque sample to the reference laboratory, and cost of the tests. An ideal assay for clinical use should be fast enough to provide results while the patient is in the office, simple enough to be performed by office personnel with minimal training or experience, and relatively inexpensive. An assay which has been extensively and successfully employed in a clinical setting is latex agglutination. This immunologie assay has been used as a screening test for a variety of bacterial infections including Clostridium difficile, C. perfringens, Escherichis, Neisseria gonorrhoeae, Haemophilus influenzae, Legionella Pneumophilia, Neisseria meningitidis, Salmo-
*State University of New York at Buffalo, Schools of Dental Medicine and Medicine, Departments of Periodontology and Microbiology, Buffalo, NY. 'School of Dental Medicine, Department of Oral Biology, Buffalo, NY.
pneumoniae, and Vibrio cholerae.10 Latex tests for these microrganisms have provided a faster therapeutic response by the clinician.
The role of plaque in the etiology of periodontal disease has long been established. Both quantitative and qualitative differences in the microbial flora occur in different forms of periodontal disease. Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans have frequently been associated with active forms of periodontal disease. Their prevalence and percent of the total microflora increases in progressive, adult Periodontitis; in refractory Periodontitis; and in generalized juvenile Periodontitis.14 One or more of these three microorganisms occur in 99% of progressive lesions, but in only 40% of non-progressive sites where their numbers are greatly reduced.4 Localized juvenile Periodontitis (LJP) is often considered an A. actinomycetemcomitans infection since it is usually characterized by an increased prevalence of A. actinomycetemcomitans which accounts for an increased percent of the total flora.5 Progressing sites in LJP patients have mean A actinomycetemcomitans levels of 2.3 x 105, while non-progressing sites have lower mean levels of 8.0 x
103
microorganisms.6
nella, Shigella, Staphylococci, Streptococci, Streptococcus
612
The research reported here describes the development of latex agglutination assays for the identification of specific periodontal pathogens including A. actinomycetemcomitans, P. gingivalis, and P. intermedia. MATERIALS AND METHODS
Preparation of Bacteria
Bacteria used as immunizing antigens or as sensitivity and specificity controls included Actinobacillus actinomycetemcomitans, serotype A (SUNYAB 75), serotype (UTSA Y4, U of JP-2), and serotype C (SUNYAB. 67, SUNYAB 463R); Actinomyces bovis (WVU 116); A. israelii (SUNYAB P62369-2); A. viscosas, serotype 2 (WVU 371); Bacterionema matruchotii (ATCC 14266); Bacteroides forsythus (SUNYAB P67759-4), B. loescheii (SUNYAB C5D-1A336); B. melaninogenicus (ATCC 25845); Capnocytophaga ochraceus (ATCC 27872); Eikenella corrodens (ATCC 23834); Fusobacterium nucleatum (Forsyth PM1-PH7, Forsyth JY-12, ATCC 25586); Haemophilus aphrophilus (ATCC 33389); Lactobacillus caseii (ATCC 393); Nocardia salivae (ATCC 19426); Peptostreptococcus micros (ATCC 33270); P. gingivalis (ATCC 33277, SUNYAB 2561, WEINE W50, ATCC 53977, SUNYAB A7A1-28); P. intermedia, serotype A (ATCC 25611); serotype B (NCTC 9336), and serotype C (SUNYAB 20-3, SUNYAB G8-9K3); Proprionumbacteria acnes, Rothia dentocariosa (ATCC 17931); Staphylococcus aureus (SUNYAB 49, SUNYAB 109.1); Streptococcus mutans (NCTC 10449, NCTC 10449S); S. salivarius (ATCC 13419); S. sanguis, serotype II (ATCC 10556, ATCC 10558); and Campylobacter rectus (formerly Wolinella recta) (ATCC 33238). Cultures of these microorganisms were grown aerobically or anaerobically, as appropriate, for 24 to 48 hours on tryptic soy agar supplemented with 5% sheep blood, 5.0 ug/ml hemin, and 0.5 ug/ml menadione.11 Bacteria were harvested and resuspended in 0.01 M phosphate-buffered saline, pH 7.0 (PBS), containing 0.1% sodium azide. Stock cultures were prepared by adjusting the suspensions to 1 x 109 cells/ml and stored at 4°C. of Antisera New Zealand white rabbits were immunized intraveneously with A. actinomycetemcomitans, P. gingivalis, or P. intermedia twice a week for 12 to 15 weeks with 1 mg of heatkilled, whole bacterial cells suspended in PBS. Antibody titers in the pre-immune and immune rabbit sera were assayed by indirect immunofluorescence.12 When antibody titers were > 320, the sera was collected and stored at 70°C until use. The IgG fractions of the antisera were isolated by precipitation with 100% saturated ammonium sulfate, pH 7.0 for 1 hour.13 The precipitates were washed twice with 50% saturated ammonium sulfate, pH 7.0, and resuspended in distilled water. After dialysis in PBS, the protein concen-
Preparation
-
J Periodontol July 1992
LATEX AGGLUTINATION TEST FOR PERIODONTAL PATHOGENS
tration was assayed by the Biuret method.14 The tions were stored at 70°C until use.
IgG frac-
-
Preparation of Latex Reagents
Latex reagents reactive to the bacteria were prepared by both passive sensitization and covalent coupling of rabbit anti-bacterial antibodies to latex microspheres. For passive sensitization, ammonium sulfate precipitated, antibacterial IgG antibodies diluted in glycine-buffered saline (GBS), pH 9.2 containing 0.1% sodium azide, were reacted with 2.5% solids-latex polystyrene microspheres* to a final latex solid concentration of 0.4%. After mixing for 1 hour at room temperature, the sensitized latex reagents were stored at 4°C.10'15 For some latex preparations, non-reactive sites were blocked by a further incubation with 0.1 to 0.05% bovine serum albumin or Tween 20§ for an additional hour. Aliquots of the passively sensitized latex reagents were also dried on plastic plates in a stabilized format and stored in sealed mylar bags.11 For covalent coupling, a 2.5% solids-latex suspension of carboxylated microspheres* was washed once and resuspended in 0.2M borate buffer, pH 8.1. The microspheres were activated by incubation for 1 hour with l-Ethyl-3(3Dimethylaminopropyl) Carbodimide hydrochloride5 diluted to 8.3% in borate buffer. The latex was then washed and resuspended in borate buffer. Coupling was achieved by incubation of the ammonium sulfate precipitated, IgG antibacterial antibodies with the activated latex overnight at 4°C. The covalently bound latex centrifuged and resuspended in glycine-buffered saline (0.15M glycine, 0.1M NaCl), pH 9.2, containing 1% bovine serum albumin and stored at 4°C.16 Latex Agglutination Assay Latex agglutination tests for the periodontal pathogens were performed with suspensions of latex (wet latex) and with the latex reagents dried onto plates (dry latex). For latex agglutination tests with the wet reagents, sensitized latex was placed within 1 cm diameter circles on glass slides. Within separate circles, bacterial suspensions including a positive control or GBS as a negative control were added. The reagents were gently mixed on a plate rocker11 for 5 to 7 minutes and read for the presence and degree of agglutination. The volumes of the latex suspensions and samples were varied from 20 to 50µ1 with 20µ1 of latex and 38µ1 sample selected as optimal. For latex agglutination tests with stabilized, dry reagents, bacterial suspensions were added to dried, sensitized latex spots of P. gingivalis/P. intermedia or A. actinomycetemcomitans with separate pipet/stirrers.1 For each test, nega-
*Poly Science Inc., Warington,
PA. 5Bio-Rad Laboratories, Richmond, CA. "Ampcor, Bridgeport, NJ. 1Hillwood, Warminster, PA.
Volume 63 Number 7
NISENGARD, MIKULSKI, McDUFFIE, BRONSON
tive controls included incubation of GBS on sensitized latex and positive controls included incubation of specific bacteria with the sensitized latex. After thorough mixing of each well to obtain a homogenous milky solution, the plates were mixed for 5 to 7 minutes on an automatic rocker. Tests were read within 10 minutes for the presence and degree of uniform agglutination. The volumes of the latex suspensions and samples were varied from 30 to 40µ1 and 60µ1 to 75µ1 with 40µ1 of latex and 75µ1 sample selected as
optimal.
Immunofluorescence Assay Rabbit antibody titers to the immunizing antigens were assayed by indirect immunofluorescence.14 Serial 2-fold, doubling dilutions of the sera were reacted on 20 µ heatfixed smears of the immunizing organism for 30 minutes. The smears were washed in PBS for 15 minutes, incubated with fluorescein-labeled goat, anti-rabbit IgG (F/P molar ratio 3.2, diluted 1/32 1/4 units of antibody/ml) for 30 minutes and again washed in PBS for 1 hour. The smears were examined for intensity of staining with a Leitz Ortholux II microscope equipped with incident illumination and an HBO 200 mercury bulb. Titers were determined as the highest dilution demonstrating a 1 + reaction. =
=
Handling of Plaque Samples
Plaque
was obtained with sterile curets by inserting the instrument to the depth of the pocket and removing sufficient plaque to visibly cover the curet tips. After collection of samples examined by latex agglutination alone, the instrument tip was inserted into a screw-capped vial containing 0.3 ml GBS and the curet handle twirled back and forth until the sample was freed from the tip. For samples examined by both latex agglutination and culture, plaque was first suspended in 0.3 ml anaerobically incubated Ringer's solution and then dispersed in a dental ultrasonic cleaner for 15 seconds. From this suspension, a 0.1 ml aliquot was processed for culture and a 0.1 ml aliquot was processed for latex agglutination by dilution in GBS. Aliquots for latex agglutination were dispersed additionally by placing the vials containing the plaque suspensions into a dental ultrasonic cleaner for 5 minutes.
Culture and Identification of Plaque Samples Plaque samples were cultured for total counts and counts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. From the original plaque suspensions in Ringer's solutions, 0.1 ml aliquots were further diluted 1:20 in 1.9 ml anaerobically in pre-reduced full strength Ringer's solution. Isolated colonies were obtained by an anaerobic 10fold dilution. Total cultivable counts were determined by anaerobic growth (85% N2, 10% H2, and 5% C02) at 35°C for 5 to 7 days on tryptic soy agar supplemented with 5% rabbit blood, 5.0 µ,& hemin, and 0.5 µg/ml menadione. PorphyromonasIPrevotella were isolated anaeiobically on se-
613
lective media consisting of tryptic soy agar supplemented with 5% rabbit blood, 5.0 µg/ml hemin, 0.5 µg/ml menadione, and 40 µg/ml kanamycin following 7 days growth at 35°C.n Ten isolated black-pigmented colonies were picked and recultured for purity before performing further identification tests. Black-pigmented colonies exhibiting an absence of long-wave UV light fluorescence and consisting of Gram-negative rods that were indole positive and a-Dglucosidase negative were classified as P. gingivalis.11 Blackpigmented colonies exhibiting long-wave UV light fluorescence and consisting of Gram-negative rods that were indole positive and a-D-glucosidase positive were classified as P. intermedia.18 A. actinomycetemcomitans were isolated on selective media (tryptic soy agar supplemented with 10% horse serum, 0.0075% bacitracin, and 0.0025% vancomycin) and incubated in C02 for 48 hours at 35°C.U Star-shaped colonies consisting of Gram-negative rods a-D-glucosidase and catalase positive (which excluded H. aphrophilus) were classified as A. actinomycetemcomitans. RESULTS
Preliminary experiments were performed to compare passive sensitization with covalently coupled latex preparations. Latex passively sensitized and covalently coupled with rabbit antibodies to A. actinomycetemcomitans, strain Y4 were evaluated in specificity tests with bacterial suspension of A actinomycetemcomitans, strain Y4, P. gingivalis (ATCC 2561), and P. intermedia (SUNYAB 20-3). The passively sensitized latex to A. actinomycetemcomitans, strain Y4 reacted with 108 A. actinomycetemcomitans, strain Y4 per ml but did not react with 108/ml P. gingivalis or P. intermedia. The covalently coupled latex, however, not only yielded agglutination with 108 A. actinomycetemcomitans, but also with 10s P. gingivalis per ml and 108 P. intermedia per ml. In addition, the non-reactive, covalently coupled latex was less homogeneous and appeared weakly granular making interpretation more difficult. Because of the observed lack of sensitivity with the covalently coupled latex and the greater difficulty in interpretation, only the passively sensitized latex was further studied. Experiments were performed with the passively sensitized, wet, latex reagents to optimize conditions affecting sensitivity and specificity including optimal concentration of antibody protein used to sensitize the latex microspheres and microsphere size. Separate batches of 0.53 diameter latex microspheres were passively sensitized with rabbit anti-bacterial IgG antibodies at concentrations of 320, 240, 160, 120, and 80 µg protein/ml. Each latex batch was then reacted in chessboard titrations with doubling concentrations of the homologous bacterial suspensions. An example of such a chessboard titration is presented in Table 1. In general, maximal sensitivity was observed over a range of protein concentrations. With increasing antibody concentrations, a "plateau" titer was reached where increasing the concentration of antibod-
614
Relationship of Antibody Protein Concentration to Latex Sensitivity
Table 1.
Rabbit A.
Bacterial
Suspensions 1.6 3.2 6.3
1.3 2.5 5.0 Buffer
J Periodontol July 1992
LATEX AGGLUTINATION TEST FOR PERIODONTAL PATHOGENS
106/ml 107ml 107ml 107ml 107ml 107ml
+ w+
w+
2+ 3+
2+ 3+
W+ 1+ 2+ 3+ 3+
w
*
Latex reactions graded as degree ±) or positive (1 + 2 + 3 + ). ,
IgG Antisera to
Bacteria/ml Detected
actinomycetemcomitans (75)
Protein Concentration 160 240
320
of
Table 2. Sensitivity of I'orphyromonas gingivalisIPrevotella intermedia Latex
agglutination
(\Lglm\) 120
Bacteria/Strain 80
1+
w+
1+ 1+ 3+ 3+
1+ 2+ 3+ 3+
from
negative (-
,
ies did not further increase the sensitivity. At higher protein concentrations, an inhibition was often observed with reduced sensitivity. At the same time, ± latex reactions were sometimes observed with buffer alone at higher protein concentrations. Limited specificity tests with heterologous bacterial suspensions also indicated cross reactions or reduced specificity at higher protein concentrations. Therefore, chessboard titrations were routinely performed with each antiserum to determine the optimal protein concentrations by selecting the greatest protein dilution yielding a 3 + reactivity. Most chessboard titrations revealed optimal results when protein concentrations between 80 and 160 µg/ ml were utilized for sensitization. Several blocking agents were evaluated including bovine, human, and rabbit serum albumin, normal rabbit IgG, egg
albumin, Tween-20,§ Triton-X,# desoxycholic acid, sodium salt,** and lauryl sulfate.** Titrations with the blocking agents indicated that low concentrations, less than 1%,
improved specificity and slightly decreased sensitivity. Tween-20 (0.005%) was selected as blocking agent for further studies. The effects of latex microsphere size on sensitivity was also evaluated. Aliquots of latex microspheres 3.10, 1.43, 1.02, 0.73, 0.53 µ in diameter were passively sensitized with rabbit IgG antibodies to A. actinomycetemcomitans (Y4). In tests with A actinomycetemcomitans suspensions, higher titers and intensity of agglutination were observed with the smaller diameter latex microspheres. Latex microspheres, 0.73 µ in diameter, were selected for further study.
Two passively sensitized latex reagents in wet and dry formats were prepared for further study: 1) a combination reagent to detect P. gingivalis and P. intermedia prepared by mixing equal volumes of latex sensitized with rabbit antisera to P. gingivalti, strain 2561 with latex sensitized with rabbit antisera to P. intermedia, strain 20-3; and 2) a reagent to detect A. actinomycetemcomitans prepared by sensitizing latex with rabbit antisera to A. actinomycetemcomitans, strain Y4. Fisher Scientific Company, Fair Lawn, NJ. **Sigma Chemical Co., St. Louis, MO.
P.
gingivalis
2561* W50 A7A1-28
Wet Latex
Dried Latex
2.5 6.3 3.2
107 106 106
1.3 2.5 6.3
107 107 106
6.3 1.0 1.6 3.2
106 10s 106 106
1.3 5.0 2.5 5.0
107 107 107 107
P. intermedia
Serotype Serotype Serotype
A 25611 9336 C 20-3* G8-9K-3
*Antisera used in Table 3.
preparation of latex.
Sensitivity of Actinobacillus actinomycetemcomitans Latex Bacteria/ml Detected
Bacteria/Strain
Serotype A 75
Serotype Y4* JP-2
Serotype C 67 463R 33384
*Antisera used in
Wet Latex
Dried Latex
5.0
107
1.0
10s
3.2 1.6
106 106
3.2 1.6
106 106
3.2 3.2 6.3
106 106 106
7.9 3.2 3.2
105 106 106
preparation of latex.
The sensitivity and specificity of the passively sensitized latex reagents were determined in tests with doubling dilutions of bacterial suspensions from 108 to 104 organisms/ ml. The sensitivities of the latex reagents were evaluated with suspensions of the immunizing and heterologous associated bacterial strains (Tables 2 and 3). The combination P. gingivalis/P. intermedia latex reagent was evaluated on 3 strains of P. gingivalis and on 4 strains of P. intermedia representing the 3 serotypes. The P. gingivalis/P. intermedia latex reagent detected from 106 to 108 organisms/ml. This combination preparation which was prepared to P. intermedia, serotype C organism was less sensitive in detecting serotype organisms. The wet latex generally had slightly greater sensitivity than the dry latex. The A actinomycetemcomitans latex reagent was evaluated on 6 strains of A. actinomycetemcomitans representing the 3 serotypes. The A. actinomycetemcomitans latex detected from 105 to 108 organisms/ml. This latex reagent prepared to a serotype organism was generally 1 log weaker in reactivity with a serotype A organism. With few exceptions, both the wet and dry latex had similar sensitivities. The specificity of the 2 latex reagents was evaluated in tests with 107/ml suspensions of other oral bacterial species (Table 4). No cross reactions with heterologous bacteria were observed. The storage or shelf life of the latex reagents was eval-
Volume 63 Number 7 Table 4.
NISENGARD, MIKULSKI, McDUFFIE, BRONSON
Specificity of Dried
Latex
Table 6.
Reagents A. actinomycetemcomitans Latex
Bacteria* A.
actinomycetemcomitans Actinomyces bovis
P. gingivalis/ P. intermedia Latex
+
A.
A. israelii A. viscosas Bacterionema matruchotii Bacteroides forsythus Bacteroides loeschii Bacteroides melaninogenicus
Lactobacilli casei Nocardia salivae
Peptostreptococcus micros Porphyromonas gingivalis Prevotella intermedia
Proprionumbacteria acnes Rothia dentocariosa
Staphylococcus aureus Streptococcus mutans Streptococcus salivarías Streptococcus sanguis suspensions tested at 107 microorganisms/ml.
Table 5. Stability of Dried Latex Conditions
Reagents
actinomycetemcomitans Room
0 7 14 28
days days days days
14 weeks 28 weeks
at Different
Temperature
+* +
+
+
+
+ +
+
+
+ + +
ND*
+
ND
ND
+
ND
at
10s
Storage
P. gingivalis/ P. intermedia Room 37°C Temperature
37°C
'Bacterial suspensions tested not done. *ND
actinomycetemcomitans
Plaque
Seeded
Plaque*
0/3* 0/3
ND* 3/3
0/3 0/3
ND
3/3
=
Haemophilus aphrophilus
Time
intermedia Culture results Latex agglutination results
*Seeded with A. actinomycetemcomitans, P. gingivalis, and P. intermedia. *Number of positive plaque samples/number of negative plaque samples. *ND Not done.
Eikenella corrodens Fusobacterium nucleatum
A.
gingivalis/P.
Culture results Latex agglutination results
Campylobacter rectos Capnocytophaga ochraceus Corynebacterium xerosis
"Bacterial
Bacteria
Seeding of Culture Negative Plaque With Specific Native
P.
615
+ + + +
microorganisms/ml.
=
uated in accelerated tests with latex reagents stored at 37°C for up to 28 days and in "real time" tests with latex reagents stored for up to 28 weeks at room temperature (Table 5). For immunologie reagents, 1 day at 37°C is generally considered to be equivalent to 1 month's storage at 4°C. In tests with 106 bacteria/ml, both latex preparations maintained reactivity after 28 days at 37°C and 28 weeks (7
months) at room temperature. For positive controls, several methods of preparation were evaluated including heat-killed bacteria (treated for 1 hour at 60°C or autoclaved) and bacterial sonicates. Reactions to killed bacteria and bacterial sonicates were compared to washed, viable suspension of the bacteria which produced maximum, strong, positive agglutination reactions. Reactivity with either type of heat-killed bacteria was stable over time, however, bacterial killing by 1 hour incubation at
60°C yielded stronger agglutination than autoclaving. The sonicated preparations initially yielded positive reactions, but this decreased with time. The method of treatment selected for further studies was heat killing for 1 hour at 60°C. Several conditions for collecting and handling subgingival plaque samples from patients with gingivitis and Periodontitis were evaluated. These conditions included volume of collection buffer into which plaque samples were suspended, time of exposure of the clinical samples to the collection buffer, and methods of dispersion of the clinical samples. The protocol selected was to collect the plaque with a curet and suspend the plaque in a tube containing 0.3 ml of GBS buffer, pH 9.0. Partial plaque dispersion was achieved by immersing the tube containing the plaque suspension into a dental ultrasonic cleaner to a depth covering the GBS buffer and running the ultrasonic cleaner for 1 to 5 minutes. The plaque suspension could then be tested immediately with the latex reagents or saved and batch tested within 24 hours. Preliminary tests with human plaque samples were performed to evaluate if these latex reagents would be reactive with clinical samples. Results indicated that agglutination reactions occurred in some plaque samples with both latex reagents. These ranged from negative reactions to varying intensities of agglutination from 1 + to 4 +. As part of the laboratory evaluation of the latex agglutination tests for the periodontal pathogens, possible interference by the mixed flora in subgingival plaque was investigated. Native subgingival plaque from 3 sites where A actinomycetemcomitans, P. gingivalis, or P. intermedia could not be cultured were seeded or spiked with a suspension containing 5.0 x 107 A. actinomycetemcomitans/m\, 2.5 x 107, P. gingivalis/ ml, and 2.5 x 107 P. intermedia/ml (Table 6). These concentrations yielded 3 to 4 + agglutination reactions with the plaque samples which were similar to the intensity seen when the bacterial suspensions were reacted directly with the latex reagents. In addition, aliquots from 26 plaque samples initially found to be negative with both latex reagents were also seeded or spiked with a pooled suspension containing 1.6 x 107 A. actinomycetemcomitans'/ml, 6.7 x 106, P. gingivalis/mi, and 3.3 x 106 P. intermedia/ml (Table 7). In preliminary tests, these lower concentrations yielded 2 to 3 + reactions when tested with the latex reagents. After seeding, the plaque samples became positive.
616
LATEX AGGLUTINATION TEST FOR PERIODONTAL PATHOGENS
Table 7.
Seeding of Latex Negative Plaque With Specific Bacteria Native
P.
gingivalis/P.
Plaque*
intermedia
Latex results A.
Plaque
Seeded
0%
(0/26)f
100%
(26/26)
0%
(0/26)
100%
(26/26)
actinomycetemcomitans
Latex results
*Seeded with . actinomycetemcomitans, P. gingivalis, and P. intermedia. tNumber of positive plaque samples/number of negative plaque samples.
This
suggested that the presence of other bacteria in the subgingival plaque samples did not inhibit the latex agglutination reactions.
DISCUSSION The experiments reported here evaluated the development of latex agglutination as a rapid immunologie assay to detect
periodontal pathogens.
Two principle methods of sensitizing latex microspheres with antisera have been successfully utilized for in vitro diagnostic tests to identify antigens: passive (physical) adsorption of the antibodies and covalent coupling of the antibodies.19'20 The method of choice partially depends on the ease and strength of antibody binding. Passive sensitization by incubating antibodies directly with the latex microspheres is faster and simpler. Covalent coupling of the antibody chemically to the latex microspheres is often more stable and would be necessary if the antibody does not passively bind to the latex. In the experiments reported here, antibodies passively and covalently bound to latex microspheres reacted equally well with the sensitizing antigen. The passively sensitized reagent appeared to be more specific and was therefore selected for further development. Covalent reagents prepared with other antibody concentrations, or buffers with other molarities or pH may yield a covalent reagent with desired specificity. Furthermore, the shelf life stability of the passively sensitized reagents ap-
peared adequate (at least 6 months).
Performance of chessboard titrations for optimizing sensitivity has frequently been utilized in quantitating immunologie tests. It is recommended in indirect immunofluorescence assays21 and for latex assays for identifying antibodies.22 As observed here, selection of the antibody concentration influences the sensitivity and specificity. Experience gained with the latex reagents prepared to the 3 periodontal pathogens suggests that such an assay should be readily adaptable for identification of other oral bacteria with appropriate antisera. In fact, multivalent reagents to identify various groups or serotypes of bacteria could be considered. The reagent to detect P. gingivalti and P. intermedia was a multivalent reagent prepared by mixing 2 latex reagents, 1 sensitized with antiserum to 1 bacteria and 1 sensitized with antiserum to the other organism. Mixing the 2 latex preparations did not significantly affect the sensitivity. A finite number of different latex preparations
probably can be pooled into one reagent before tivity is adversely affected.
J Periodontol July 1992
the sensi-
Several different formats could have been considered to detect the 3 microorganisms studied. Three individual reagents or various combinations of reagents could have been developed. While individual reagents would offer benefits in terms of specificity of reactions, the clinical implications of high numbers of one black pigmenting organism over another is not completely clear. It was therefore decided to pool the P. gingivalis and P. intermedia latex preparations to simplify and reduce the testing time. In addition, these 2 organisms can be clinically reduced mechanically by scaling and root planing. Reduction in A. actinomycetemcomitans, however, necessitates an antibiotic.11 The sensitivity of the tests reported here with suspensions of cultured bacteria ranged from 7.9 x 105 to 1.0 x 108 organisms/ml depending on the organism tested. These results are similar to those reported for a latex reagent to detect E. coli with a sensitivity of 1.5 x 106 to 5.7 x 106 cells/ml.19 The sensitivity observed to the periodontal pathogens depended somewhat on the antigens shared by the serotypes and strains being tested. For example, the latex reagent prepared with antisera to A. actinomycetemcomitans, serotype , strain Y4 was at least one log less sensitive when the reagent was tested on serotype A, strain 75 compared to serotype , strain Y4. Such differences may be reduced by pooling latex reagents with other specificities. The need to improve sensitivity will depend on performance studies where subgingival plaque is evaluated by both culture and latex agglutination. To utilize latex agglutination for detection of periodontal microorganisms or their products in a clinical office setting, several parameters must be investigated. These include: 1) comparison of latex agglutination with another generally accepted assay such as culture; and 2) studies to demonstrate clinical relevance. Simultaneous examination of plaque samples by culture and latex agglutination tests is necessary to determine their relative sensitivities in identifying subgingival bacteria. The sensitivity of the latex agglutination tests for periodontal pathogens deteimined with laboratory strains appeared lower than culture where cultivable bacteria usually do not exceed 106 organisms/ml from a pocket. It is, however, unclear whether this is a significant difference. Latex agglutination identifies both viable bacteria and non-viable bacteria/bacterial fragments while culture only identifies viable, cultivable organisms. While the relative ratio in a pocket of cultivable bacteria to non-viable bacteria/bacterial fragments is unknown, it is quite probable the cultivable bacteria make up a smaller proportion. This proportion most likely also varies in different sites and at different times. For any assay including latex agglutination, it is most important to demonstrate its clinical relevance. Latex agglutination assays for microorganisms have routinely been utilized by physicians for many years where they have pro-
Volume 63 Number 7
NISENGARD, MIKULSKI, McDUFFIE, BRONSON
rapid, simple, screening assay allowing a rapid therapeutic response. Evaluation of latex agglutination tests for periodontal pathogens could include studies to demonstrate their benefits in diagnosis, treatment, and patient management. Any assay must provide the clinician with information that would ultimately benefit the patient. The clinical relevance of an assay for periodontal pathogens is not solely related to the degree of sensitivity. This vided
a
differs from other diseases where there is a monoinfection. In cases of sore throats caused solely by ß-hemolytic streptococci, the more sensitive the assay, the better it is for diagnosis. In periodontal disease, with both quantitative and qualitative changes in the flora, an assay with the greatest sensitivity may not be relevant. For example, A. actinomycetemcomitans cannot only be cultured from involved first molars of patients with localized juvenile Periodontitis, but also from non-involved sites in these patients and from periodontally healthy siblings.11 At present, low numbers of these pathogens in healthy sites does not appear an indication for therapy since these low numbers have not been demonstrated to have any predictive or prognostic value. The studies reported here suggest that latex agglutination can be utilized for detection of periodontal pathogens. Comparisons with cultural results and clinical studies of relevance in diagnosis, prognosis, and patient management are necessary to demonstrate the efficacy of this type of test.
Acknowledgments This research was supported by New York State Science and Technology Grant #SSF (87)-4 and IMMCO Diagnostics Inc. REFERENCES 1. Tanner ACR, Socransky SS, Goodson JM. 2.
Microbiota of periodontal pockets losing crestal bone. / Periodont Res 1984;19:279-291. Slots J. Bacterial specificity in adult Periodontitis A summary of
recent work. / Clin Periodontol 1986;13:912-917. 3. Zambón JJ, Bochacki V, Genco RJ. Immunological assays for putative periodontal pathogens. Oral Microbio! Immunol 1986;1:39-43. 4. Slots J, Listgarten MA. Porphyromonas (Bacteroides) gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. / Clin Periodontol 1988;15:85-93. 5. Zambón JJ, Christersson LA, Slots J. Actinobacillus actinomycetemcomitans in human periodontal disease: Prevalence in patient groups —
617
and distribution of biotypes and serotypes within families. / Periodontol 1983;54:707-711.
RL, Ebersole JL, Socransky SS. Clinical, immunologie and microbiologie features of active disease sites in juvenile Periodontitis.
6. Mandell
/ Clin Periodontol 1987;14:534-540. 7. Genco RJ, Zambón JJ, Christersson LA. Use and interpretation of microbiological assays for periodontal diseases. Oral Microbiol Immunol 1986;1:73-79. 8. Greenstein G. Microbiological assessments to enhance periodontal diagnosis. / Periodontol 1988;59:508-515. 9. Kornman KS. Nature of periodontal diseases: Assessment and diagnosis. J Periodont Res 1987;22:192-204. 10. Hechemy KF, Michaelson EE. Latex particle assays in laboratory medicine. Part I. Labor Med 1984; 22:27-40. 11. Slots J, Mashimo P, Levine MJ, Genco RJ. Periodontal therapy in humans. I. Microbiological and clinical effects of a single course of periodontal scaling and root planing, and of adjunctive tetracycline therapy. J Periodontol 1979;50:495-509. 12. Nisengard RJ, Stinson MW, Pelonero L. Immunologie cross-reactivity between Streptococcus mutans and mammalian tissues. Annal NY Acad Sci 1983;420:401^t09. 13. Beutner EH, Nisengard RJ. Defined immunofluorescence in clinical immunopathology. In: Beutner EH, Chorzelski TP, Bean SF, Jordon RD, eds. Immunology of the Skin: Labeled Antibody Studies. Stroudsburg, PA: Dowden, Hutchinson and Ross, Inc; 1973;65-66. 14. Beutner EH, Nisengard RJ. Defined immunofluorescence in clinical immunopathology. In: Beutner EH, Chorzelski TP, Bean SF, Jordon RD, eds. Immunology of the Skin: Labeled Antibody Studies. Stroudsburg, PA: Dowden, Hutchinson and Ross, Inc; 1973, pp. 68-70. 15. Polysciences, Inc. Data Sheet #238E. 1988. 16. Polysciences, Inc. Data Sheet #238C. 1988. 17. Duerden BI, Goodwin L, O'Neil TCA. Identification of Bacteroides species from adult periodontal disease. J Med Microbiol 1987;24:133137. 18. Slots J, Felk D, Rams TE. Actinobacillus actinomycetemcomitans and Bacteroides intermedius in human Periodontitis: Age relationship and mutual association. / Clin Periodontol 1990;17:659-662. 19. Hechemy K, Stevens RW, Gaafar HA. Detection of Escherichia coli antigens by a latex agglutination test. App Microbiol 1974;28:306311. 20. Bangs LB. Uniform Latex Particles. 1984. Seragen, Inc.: Indianapolis. 21. Beutner EH, Nisengard RJ. Defined immunofluorescence in clinical immunopathology. In: Beutner EH, Chorzelski TP, Bean SF, Jordon RD, eds. Immunology of the Skin: Labeled Antibody Studies. Stroudsburg, PA: Dowden, Hutchinson and Ross, Inc; 1973;50-52. 22. Hechemy K, Stevens RW, Gaafar HA. Antigen distribution in a latex suspension and its relationship to test sensitivity. / Clin Microbiol;
1976;4:82-86. Send reprint requests to: Dr. Russell Nisengard, SUNY at Buffalo, School of Dental Medicine, Department of Periodontology, 3435 Main Street, Buffalo, NY 14214. Accepted for publication January 27, 1992.
Development of a Rapid Latex Agglutination Test for Periodontal Pathogens Russell J.
Nisengard, Lynn Mikulski, *
*
Deborah
McDuffie, * and Paul Bronson*
studies reported here describe the development, characterization, and initial application of latex agglutination assays for periodontal pathogens. Latex reagents were prepared by sensitization of latex microspheres with rabbit IgG antibodies to either Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, or Prevotella intermedia. The protein concentration utilized for sensitization and microsphere size were optimized. One reagent was prepared to A. actinomycetemcomitans and a second combination reagent was prepared by mixing latex sensitized with antibodies to P. gingivalis and latex sensitized with antibodies to P. intermedia. The sensitivity of both latex reagents in the traditional wet and a dried format was evaluated. In addition, sensitivity and specificity with homologous and heterologous bacterial suspensions were evaluated. The reagents were found to demonstrate both specificity and sensitivity. Initial studies with subgingival human plaque demonstrated the ability of these reagents to detect the specific organisms in plaque. J Periodontol 1992; 63:611-617.
The
Key Words: Actinobacillus actinomycetemcomitans; votella intermedia; latex fixation test.
Porphyromonas gingivalis;
Pre-
The association of specific bacteria with active periodontal disease suggests a rationale for monitoring specific
bacteria in plaque samples.6 8 Their identification in high numbers may help in assessing disease activity, selecting individualized treatment modalities, monitoring effects of treatment, selecting appropriate recall intervals, and determining when retreatment is indicated.7 9 At present, culture is considered the "gold standard" or reference methodology for identifying subgingival bacteria. Its routine use in a clinical office, however, is limited because of a 1 to 2-week time period necessary for definitive test results, problems associated with maintaining viability and anaerobiosis during transportation of the plaque sample to the reference laboratory, and cost of the tests. An ideal assay for clinical use should be fast enough to provide results while the patient is in the office, simple enough to be performed by office personnel with minimal training or experience, and relatively inexpensive. An assay which has been extensively and successfully employed in a clinical setting is latex agglutination. This immunologie assay has been used as a screening test for a variety of bacterial infections including Clostridium difficile, C. perfringens, Escherichis, Neisseria gonorrhoeae, Haemophilus influenzae, Legionella Pneumophilia, Neisseria meningitidis, Salmo-
*State University of New York at Buffalo, Schools of Dental Medicine and Medicine, Departments of Periodontology and Microbiology, Buffalo, NY. 'School of Dental Medicine, Department of Oral Biology, Buffalo, NY.
pneumoniae, and Vibrio cholerae.10 Latex tests for these microrganisms have provided a faster therapeutic response by the clinician.
The role of plaque in the etiology of periodontal disease has long been established. Both quantitative and qualitative differences in the microbial flora occur in different forms of periodontal disease. Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans have frequently been associated with active forms of periodontal disease. Their prevalence and percent of the total microflora increases in progressive, adult Periodontitis; in refractory Periodontitis; and in generalized juvenile Periodontitis.14 One or more of these three microorganisms occur in 99% of progressive lesions, but in only 40% of non-progressive sites where their numbers are greatly reduced.4 Localized juvenile Periodontitis (LJP) is often considered an A. actinomycetemcomitans infection since it is usually characterized by an increased prevalence of A. actinomycetemcomitans which accounts for an increased percent of the total flora.5 Progressing sites in LJP patients have mean A actinomycetemcomitans levels of 2.3 x 105, while non-progressing sites have lower mean levels of 8.0 x
103
microorganisms.6
nella, Shigella, Staphylococci, Streptococci, Streptococcus
612
The research reported here describes the development of latex agglutination assays for the identification of specific periodontal pathogens including A. actinomycetemcomitans, P. gingivalis, and P. intermedia. MATERIALS AND METHODS
Preparation of Bacteria
Bacteria used as immunizing antigens or as sensitivity and specificity controls included Actinobacillus actinomycetemcomitans, serotype A (SUNYAB 75), serotype (UTSA Y4, U of JP-2), and serotype C (SUNYAB. 67, SUNYAB 463R); Actinomyces bovis (WVU 116); A. israelii (SUNYAB P62369-2); A. viscosas, serotype 2 (WVU 371); Bacterionema matruchotii (ATCC 14266); Bacteroides forsythus (SUNYAB P67759-4), B. loescheii (SUNYAB C5D-1A336); B. melaninogenicus (ATCC 25845); Capnocytophaga ochraceus (ATCC 27872); Eikenella corrodens (ATCC 23834); Fusobacterium nucleatum (Forsyth PM1-PH7, Forsyth JY-12, ATCC 25586); Haemophilus aphrophilus (ATCC 33389); Lactobacillus caseii (ATCC 393); Nocardia salivae (ATCC 19426); Peptostreptococcus micros (ATCC 33270); P. gingivalis (ATCC 33277, SUNYAB 2561, WEINE W50, ATCC 53977, SUNYAB A7A1-28); P. intermedia, serotype A (ATCC 25611); serotype B (NCTC 9336), and serotype C (SUNYAB 20-3, SUNYAB G8-9K3); Proprionumbacteria acnes, Rothia dentocariosa (ATCC 17931); Staphylococcus aureus (SUNYAB 49, SUNYAB 109.1); Streptococcus mutans (NCTC 10449, NCTC 10449S); S. salivarius (ATCC 13419); S. sanguis, serotype II (ATCC 10556, ATCC 10558); and Campylobacter rectus (formerly Wolinella recta) (ATCC 33238). Cultures of these microorganisms were grown aerobically or anaerobically, as appropriate, for 24 to 48 hours on tryptic soy agar supplemented with 5% sheep blood, 5.0 ug/ml hemin, and 0.5 ug/ml menadione.11 Bacteria were harvested and resuspended in 0.01 M phosphate-buffered saline, pH 7.0 (PBS), containing 0.1% sodium azide. Stock cultures were prepared by adjusting the suspensions to 1 x 109 cells/ml and stored at 4°C. of Antisera New Zealand white rabbits were immunized intraveneously with A. actinomycetemcomitans, P. gingivalis, or P. intermedia twice a week for 12 to 15 weeks with 1 mg of heatkilled, whole bacterial cells suspended in PBS. Antibody titers in the pre-immune and immune rabbit sera were assayed by indirect immunofluorescence.12 When antibody titers were > 320, the sera was collected and stored at 70°C until use. The IgG fractions of the antisera were isolated by precipitation with 100% saturated ammonium sulfate, pH 7.0 for 1 hour.13 The precipitates were washed twice with 50% saturated ammonium sulfate, pH 7.0, and resuspended in distilled water. After dialysis in PBS, the protein concen-
Preparation
-
J Periodontol July 1992
LATEX AGGLUTINATION TEST FOR PERIODONTAL PATHOGENS
tration was assayed by the Biuret method.14 The tions were stored at 70°C until use.
IgG frac-
-
Preparation of Latex Reagents
Latex reagents reactive to the bacteria were prepared by both passive sensitization and covalent coupling of rabbit anti-bacterial antibodies to latex microspheres. For passive sensitization, ammonium sulfate precipitated, antibacterial IgG antibodies diluted in glycine-buffered saline (GBS), pH 9.2 containing 0.1% sodium azide, were reacted with 2.5% solids-latex polystyrene microspheres* to a final latex solid concentration of 0.4%. After mixing for 1 hour at room temperature, the sensitized latex reagents were stored at 4°C.10'15 For some latex preparations, non-reactive sites were blocked by a further incubation with 0.1 to 0.05% bovine serum albumin or Tween 20§ for an additional hour. Aliquots of the passively sensitized latex reagents were also dried on plastic plates in a stabilized format and stored in sealed mylar bags.11 For covalent coupling, a 2.5% solids-latex suspension of carboxylated microspheres* was washed once and resuspended in 0.2M borate buffer, pH 8.1. The microspheres were activated by incubation for 1 hour with l-Ethyl-3(3Dimethylaminopropyl) Carbodimide hydrochloride5 diluted to 8.3% in borate buffer. The latex was then washed and resuspended in borate buffer. Coupling was achieved by incubation of the ammonium sulfate precipitated, IgG antibacterial antibodies with the activated latex overnight at 4°C. The covalently bound latex centrifuged and resuspended in glycine-buffered saline (0.15M glycine, 0.1M NaCl), pH 9.2, containing 1% bovine serum albumin and stored at 4°C.16 Latex Agglutination Assay Latex agglutination tests for the periodontal pathogens were performed with suspensions of latex (wet latex) and with the latex reagents dried onto plates (dry latex). For latex agglutination tests with the wet reagents, sensitized latex was placed within 1 cm diameter circles on glass slides. Within separate circles, bacterial suspensions including a positive control or GBS as a negative control were added. The reagents were gently mixed on a plate rocker11 for 5 to 7 minutes and read for the presence and degree of agglutination. The volumes of the latex suspensions and samples were varied from 20 to 50µ1 with 20µ1 of latex and 38µ1 sample selected as optimal. For latex agglutination tests with stabilized, dry reagents, bacterial suspensions were added to dried, sensitized latex spots of P. gingivalis/P. intermedia or A. actinomycetemcomitans with separate pipet/stirrers.1 For each test, nega-
*Poly Science Inc., Warington,
PA. 5Bio-Rad Laboratories, Richmond, CA. "Ampcor, Bridgeport, NJ. 1Hillwood, Warminster, PA.
Volume 63 Number 7
NISENGARD, MIKULSKI, McDUFFIE, BRONSON
tive controls included incubation of GBS on sensitized latex and positive controls included incubation of specific bacteria with the sensitized latex. After thorough mixing of each well to obtain a homogenous milky solution, the plates were mixed for 5 to 7 minutes on an automatic rocker. Tests were read within 10 minutes for the presence and degree of uniform agglutination. The volumes of the latex suspensions and samples were varied from 30 to 40µ1 and 60µ1 to 75µ1 with 40µ1 of latex and 75µ1 sample selected as
optimal.
Immunofluorescence Assay Rabbit antibody titers to the immunizing antigens were assayed by indirect immunofluorescence.14 Serial 2-fold, doubling dilutions of the sera were reacted on 20 µ heatfixed smears of the immunizing organism for 30 minutes. The smears were washed in PBS for 15 minutes, incubated with fluorescein-labeled goat, anti-rabbit IgG (F/P molar ratio 3.2, diluted 1/32 1/4 units of antibody/ml) for 30 minutes and again washed in PBS for 1 hour. The smears were examined for intensity of staining with a Leitz Ortholux II microscope equipped with incident illumination and an HBO 200 mercury bulb. Titers were determined as the highest dilution demonstrating a 1 + reaction. =
=
Handling of Plaque Samples
Plaque
was obtained with sterile curets by inserting the instrument to the depth of the pocket and removing sufficient plaque to visibly cover the curet tips. After collection of samples examined by latex agglutination alone, the instrument tip was inserted into a screw-capped vial containing 0.3 ml GBS and the curet handle twirled back and forth until the sample was freed from the tip. For samples examined by both latex agglutination and culture, plaque was first suspended in 0.3 ml anaerobically incubated Ringer's solution and then dispersed in a dental ultrasonic cleaner for 15 seconds. From this suspension, a 0.1 ml aliquot was processed for culture and a 0.1 ml aliquot was processed for latex agglutination by dilution in GBS. Aliquots for latex agglutination were dispersed additionally by placing the vials containing the plaque suspensions into a dental ultrasonic cleaner for 5 minutes.
Culture and Identification of Plaque Samples Plaque samples were cultured for total counts and counts of A. actinomycetemcomitans, P. gingivalis, and P. intermedia. From the original plaque suspensions in Ringer's solutions, 0.1 ml aliquots were further diluted 1:20 in 1.9 ml anaerobically in pre-reduced full strength Ringer's solution. Isolated colonies were obtained by an anaerobic 10fold dilution. Total cultivable counts were determined by anaerobic growth (85% N2, 10% H2, and 5% C02) at 35°C for 5 to 7 days on tryptic soy agar supplemented with 5% rabbit blood, 5.0 µ,& hemin, and 0.5 µg/ml menadione. PorphyromonasIPrevotella were isolated anaeiobically on se-
613
lective media consisting of tryptic soy agar supplemented with 5% rabbit blood, 5.0 µg/ml hemin, 0.5 µg/ml menadione, and 40 µg/ml kanamycin following 7 days growth at 35°C.n Ten isolated black-pigmented colonies were picked and recultured for purity before performing further identification tests. Black-pigmented colonies exhibiting an absence of long-wave UV light fluorescence and consisting of Gram-negative rods that were indole positive and a-Dglucosidase negative were classified as P. gingivalis.11 Blackpigmented colonies exhibiting long-wave UV light fluorescence and consisting of Gram-negative rods that were indole positive and a-D-glucosidase positive were classified as P. intermedia.18 A. actinomycetemcomitans were isolated on selective media (tryptic soy agar supplemented with 10% horse serum, 0.0075% bacitracin, and 0.0025% vancomycin) and incubated in C02 for 48 hours at 35°C.U Star-shaped colonies consisting of Gram-negative rods a-D-glucosidase and catalase positive (which excluded H. aphrophilus) were classified as A. actinomycetemcomitans. RESULTS
Preliminary experiments were performed to compare passive sensitization with covalently coupled latex preparations. Latex passively sensitized and covalently coupled with rabbit antibodies to A. actinomycetemcomitans, strain Y4 were evaluated in specificity tests with bacterial suspension of A actinomycetemcomitans, strain Y4, P. gingivalis (ATCC 2561), and P. intermedia (SUNYAB 20-3). The passively sensitized latex to A. actinomycetemcomitans, strain Y4 reacted with 108 A. actinomycetemcomitans, strain Y4 per ml but did not react with 108/ml P. gingivalis or P. intermedia. The covalently coupled latex, however, not only yielded agglutination with 108 A. actinomycetemcomitans, but also with 10s P. gingivalis per ml and 108 P. intermedia per ml. In addition, the non-reactive, covalently coupled latex was less homogeneous and appeared weakly granular making interpretation more difficult. Because of the observed lack of sensitivity with the covalently coupled latex and the greater difficulty in interpretation, only the passively sensitized latex was further studied. Experiments were performed with the passively sensitized, wet, latex reagents to optimize conditions affecting sensitivity and specificity including optimal concentration of antibody protein used to sensitize the latex microspheres and microsphere size. Separate batches of 0.53 diameter latex microspheres were passively sensitized with rabbit anti-bacterial IgG antibodies at concentrations of 320, 240, 160, 120, and 80 µg protein/ml. Each latex batch was then reacted in chessboard titrations with doubling concentrations of the homologous bacterial suspensions. An example of such a chessboard titration is presented in Table 1. In general, maximal sensitivity was observed over a range of protein concentrations. With increasing antibody concentrations, a "plateau" titer was reached where increasing the concentration of antibod-
614
Relationship of Antibody Protein Concentration to Latex Sensitivity
Table 1.
Rabbit A.
Bacterial
Suspensions 1.6 3.2 6.3
1.3 2.5 5.0 Buffer
J Periodontol July 1992
LATEX AGGLUTINATION TEST FOR PERIODONTAL PATHOGENS
106/ml 107ml 107ml 107ml 107ml 107ml
+ w+
w+
2+ 3+
2+ 3+
W+ 1+ 2+ 3+ 3+
w
*
Latex reactions graded as degree ±) or positive (1 + 2 + 3 + ). ,
IgG Antisera to
Bacteria/ml Detected
actinomycetemcomitans (75)
Protein Concentration 160 240
320
of
Table 2. Sensitivity of I'orphyromonas gingivalisIPrevotella intermedia Latex
agglutination
(\Lglm\) 120
Bacteria/Strain 80
1+
w+
1+ 1+ 3+ 3+
1+ 2+ 3+ 3+
from
negative (-
,
ies did not further increase the sensitivity. At higher protein concentrations, an inhibition was often observed with reduced sensitivity. At the same time, ± latex reactions were sometimes observed with buffer alone at higher protein concentrations. Limited specificity tests with heterologous bacterial suspensions also indicated cross reactions or reduced specificity at higher protein concentrations. Therefore, chessboard titrations were routinely performed with each antiserum to determine the optimal protein concentrations by selecting the greatest protein dilution yielding a 3 + reactivity. Most chessboard titrations revealed optimal results when protein concentrations between 80 and 160 µg/ ml were utilized for sensitization. Several blocking agents were evaluated including bovine, human, and rabbit serum albumin, normal rabbit IgG, egg
albumin, Tween-20,§ Triton-X,# desoxycholic acid, sodium salt,** and lauryl sulfate.** Titrations with the blocking agents indicated that low concentrations, less than 1%,
improved specificity and slightly decreased sensitivity. Tween-20 (0.005%) was selected as blocking agent for further studies. The effects of latex microsphere size on sensitivity was also evaluated. Aliquots of latex microspheres 3.10, 1.43, 1.02, 0.73, 0.53 µ in diameter were passively sensitized with rabbit IgG antibodies to A. actinomycetemcomitans (Y4). In tests with A actinomycetemcomitans suspensions, higher titers and intensity of agglutination were observed with the smaller diameter latex microspheres. Latex microspheres, 0.73 µ in diameter, were selected for further study.
Two passively sensitized latex reagents in wet and dry formats were prepared for further study: 1) a combination reagent to detect P. gingivalis and P. intermedia prepared by mixing equal volumes of latex sensitized with rabbit antisera to P. gingivalti, strain 2561 with latex sensitized with rabbit antisera to P. intermedia, strain 20-3; and 2) a reagent to detect A. actinomycetemcomitans prepared by sensitizing latex with rabbit antisera to A. actinomycetemcomitans, strain Y4. Fisher Scientific Company, Fair Lawn, NJ. **Sigma Chemical Co., St. Louis, MO.
P.
gingivalis
2561* W50 A7A1-28
Wet Latex
Dried Latex
2.5 6.3 3.2
107 106 106
1.3 2.5 6.3
107 107 106
6.3 1.0 1.6 3.2
106 10s 106 106
1.3 5.0 2.5 5.0
107 107 107 107
P. intermedia
Serotype Serotype Serotype
A 25611 9336 C 20-3* G8-9K-3
*Antisera used in Table 3.
preparation of latex.
Sensitivity of Actinobacillus actinomycetemcomitans Latex Bacteria/ml Detected
Bacteria/Strain
Serotype A 75
Serotype Y4* JP-2
Serotype C 67 463R 33384
*Antisera used in
Wet Latex
Dried Latex
5.0
107
1.0
10s
3.2 1.6
106 106
3.2 1.6
106 106
3.2 3.2 6.3
106 106 106
7.9 3.2 3.2
105 106 106
preparation of latex.
The sensitivity and specificity of the passively sensitized latex reagents were determined in tests with doubling dilutions of bacterial suspensions from 108 to 104 organisms/ ml. The sensitivities of the latex reagents were evaluated with suspensions of the immunizing and heterologous associated bacterial strains (Tables 2 and 3). The combination P. gingivalis/P. intermedia latex reagent was evaluated on 3 strains of P. gingivalis and on 4 strains of P. intermedia representing the 3 serotypes. The P. gingivalis/P. intermedia latex reagent detected from 106 to 108 organisms/ml. This combination preparation which was prepared to P. intermedia, serotype C organism was less sensitive in detecting serotype organisms. The wet latex generally had slightly greater sensitivity than the dry latex. The A actinomycetemcomitans latex reagent was evaluated on 6 strains of A. actinomycetemcomitans representing the 3 serotypes. The A. actinomycetemcomitans latex detected from 105 to 108 organisms/ml. This latex reagent prepared to a serotype organism was generally 1 log weaker in reactivity with a serotype A organism. With few exceptions, both the wet and dry latex had similar sensitivities. The specificity of the 2 latex reagents was evaluated in tests with 107/ml suspensions of other oral bacterial species (Table 4). No cross reactions with heterologous bacteria were observed. The storage or shelf life of the latex reagents was eval-
Volume 63 Number 7 Table 4.
NISENGARD, MIKULSKI, McDUFFIE, BRONSON
Specificity of Dried
Latex
Table 6.
Reagents A. actinomycetemcomitans Latex
Bacteria* A.
actinomycetemcomitans Actinomyces bovis
P. gingivalis/ P. intermedia Latex
+
A.
A. israelii A. viscosas Bacterionema matruchotii Bacteroides forsythus Bacteroides loeschii Bacteroides melaninogenicus
Lactobacilli casei Nocardia salivae
Peptostreptococcus micros Porphyromonas gingivalis Prevotella intermedia
Proprionumbacteria acnes Rothia dentocariosa
Staphylococcus aureus Streptococcus mutans Streptococcus salivarías Streptococcus sanguis suspensions tested at 107 microorganisms/ml.
Table 5. Stability of Dried Latex Conditions
Reagents
actinomycetemcomitans Room
0 7 14 28
days days days days
14 weeks 28 weeks
at Different
Temperature
+* +
+
+
+
+ +
+
+
+ + +
ND*
+
ND
ND
+
ND
at
10s
Storage
P. gingivalis/ P. intermedia Room 37°C Temperature
37°C
'Bacterial suspensions tested not done. *ND
actinomycetemcomitans
Plaque
Seeded
Plaque*
0/3* 0/3
ND* 3/3
0/3 0/3
ND
3/3
=
Haemophilus aphrophilus
Time
intermedia Culture results Latex agglutination results
*Seeded with A. actinomycetemcomitans, P. gingivalis, and P. intermedia. *Number of positive plaque samples/number of negative plaque samples. *ND Not done.
Eikenella corrodens Fusobacterium nucleatum
A.
gingivalis/P.
Culture results Latex agglutination results
Campylobacter rectos Capnocytophaga ochraceus Corynebacterium xerosis
"Bacterial
Bacteria
Seeding of Culture Negative Plaque With Specific Native
P.
615
+ + + +
microorganisms/ml.
=
uated in accelerated tests with latex reagents stored at 37°C for up to 28 days and in "real time" tests with latex reagents stored for up to 28 weeks at room temperature (Table 5). For immunologie reagents, 1 day at 37°C is generally considered to be equivalent to 1 month's storage at 4°C. In tests with 106 bacteria/ml, both latex preparations maintained reactivity after 28 days at 37°C and 28 weeks (7
months) at room temperature. For positive controls, several methods of preparation were evaluated including heat-killed bacteria (treated for 1 hour at 60°C or autoclaved) and bacterial sonicates. Reactions to killed bacteria and bacterial sonicates were compared to washed, viable suspension of the bacteria which produced maximum, strong, positive agglutination reactions. Reactivity with either type of heat-killed bacteria was stable over time, however, bacterial killing by 1 hour incubation at
60°C yielded stronger agglutination than autoclaving. The sonicated preparations initially yielded positive reactions, but this decreased with time. The method of treatment selected for further studies was heat killing for 1 hour at 60°C. Several conditions for collecting and handling subgingival plaque samples from patients with gingivitis and Periodontitis were evaluated. These conditions included volume of collection buffer into which plaque samples were suspended, time of exposure of the clinical samples to the collection buffer, and methods of dispersion of the clinical samples. The protocol selected was to collect the plaque with a curet and suspend the plaque in a tube containing 0.3 ml of GBS buffer, pH 9.0. Partial plaque dispersion was achieved by immersing the tube containing the plaque suspension into a dental ultrasonic cleaner to a depth covering the GBS buffer and running the ultrasonic cleaner for 1 to 5 minutes. The plaque suspension could then be tested immediately with the latex reagents or saved and batch tested within 24 hours. Preliminary tests with human plaque samples were performed to evaluate if these latex reagents would be reactive with clinical samples. Results indicated that agglutination reactions occurred in some plaque samples with both latex reagents. These ranged from negative reactions to varying intensities of agglutination from 1 + to 4 +. As part of the laboratory evaluation of the latex agglutination tests for the periodontal pathogens, possible interference by the mixed flora in subgingival plaque was investigated. Native subgingival plaque from 3 sites where A actinomycetemcomitans, P. gingivalis, or P. intermedia could not be cultured were seeded or spiked with a suspension containing 5.0 x 107 A. actinomycetemcomitans/m\, 2.5 x 107, P. gingivalis/ ml, and 2.5 x 107 P. intermedia/ml (Table 6). These concentrations yielded 3 to 4 + agglutination reactions with the plaque samples which were similar to the intensity seen when the bacterial suspensions were reacted directly with the latex reagents. In addition, aliquots from 26 plaque samples initially found to be negative with both latex reagents were also seeded or spiked with a pooled suspension containing 1.6 x 107 A. actinomycetemcomitans'/ml, 6.7 x 106, P. gingivalis/mi, and 3.3 x 106 P. intermedia/ml (Table 7). In preliminary tests, these lower concentrations yielded 2 to 3 + reactions when tested with the latex reagents. After seeding, the plaque samples became positive.
616
LATEX AGGLUTINATION TEST FOR PERIODONTAL PATHOGENS
Table 7.
Seeding of Latex Negative Plaque With Specific Bacteria Native
P.
gingivalis/P.
Plaque*
intermedia
Latex results A.
Plaque
Seeded
0%
(0/26)f
100%
(26/26)
0%
(0/26)
100%
(26/26)
actinomycetemcomitans
Latex results
*Seeded with . actinomycetemcomitans, P. gingivalis, and P. intermedia. tNumber of positive plaque samples/number of negative plaque samples.
This
suggested that the presence of other bacteria in the subgingival plaque samples did not inhibit the latex agglutination reactions.
DISCUSSION The experiments reported here evaluated the development of latex agglutination as a rapid immunologie assay to detect
periodontal pathogens.
Two principle methods of sensitizing latex microspheres with antisera have been successfully utilized for in vitro diagnostic tests to identify antigens: passive (physical) adsorption of the antibodies and covalent coupling of the antibodies.19'20 The method of choice partially depends on the ease and strength of antibody binding. Passive sensitization by incubating antibodies directly with the latex microspheres is faster and simpler. Covalent coupling of the antibody chemically to the latex microspheres is often more stable and would be necessary if the antibody does not passively bind to the latex. In the experiments reported here, antibodies passively and covalently bound to latex microspheres reacted equally well with the sensitizing antigen. The passively sensitized reagent appeared to be more specific and was therefore selected for further development. Covalent reagents prepared with other antibody concentrations, or buffers with other molarities or pH may yield a covalent reagent with desired specificity. Furthermore, the shelf life stability of the passively sensitized reagents ap-
peared adequate (at least 6 months).
Performance of chessboard titrations for optimizing sensitivity has frequently been utilized in quantitating immunologie tests. It is recommended in indirect immunofluorescence assays21 and for latex assays for identifying antibodies.22 As observed here, selection of the antibody concentration influences the sensitivity and specificity. Experience gained with the latex reagents prepared to the 3 periodontal pathogens suggests that such an assay should be readily adaptable for identification of other oral bacteria with appropriate antisera. In fact, multivalent reagents to identify various groups or serotypes of bacteria could be considered. The reagent to detect P. gingivalti and P. intermedia was a multivalent reagent prepared by mixing 2 latex reagents, 1 sensitized with antiserum to 1 bacteria and 1 sensitized with antiserum to the other organism. Mixing the 2 latex preparations did not significantly affect the sensitivity. A finite number of different latex preparations
probably can be pooled into one reagent before tivity is adversely affected.
J Periodontol July 1992
the sensi-
Several different formats could have been considered to detect the 3 microorganisms studied. Three individual reagents or various combinations of reagents could have been developed. While individual reagents would offer benefits in terms of specificity of reactions, the clinical implications of high numbers of one black pigmenting organism over another is not completely clear. It was therefore decided to pool the P. gingivalis and P. intermedia latex preparations to simplify and reduce the testing time. In addition, these 2 organisms can be clinically reduced mechanically by scaling and root planing. Reduction in A. actinomycetemcomitans, however, necessitates an antibiotic.11 The sensitivity of the tests reported here with suspensions of cultured bacteria ranged from 7.9 x 105 to 1.0 x 108 organisms/ml depending on the organism tested. These results are similar to those reported for a latex reagent to detect E. coli with a sensitivity of 1.5 x 106 to 5.7 x 106 cells/ml.19 The sensitivity observed to the periodontal pathogens depended somewhat on the antigens shared by the serotypes and strains being tested. For example, the latex reagent prepared with antisera to A. actinomycetemcomitans, serotype , strain Y4 was at least one log less sensitive when the reagent was tested on serotype A, strain 75 compared to serotype , strain Y4. Such differences may be reduced by pooling latex reagents with other specificities. The need to improve sensitivity will depend on performance studies where subgingival plaque is evaluated by both culture and latex agglutination. To utilize latex agglutination for detection of periodontal microorganisms or their products in a clinical office setting, several parameters must be investigated. These include: 1) comparison of latex agglutination with another generally accepted assay such as culture; and 2) studies to demonstrate clinical relevance. Simultaneous examination of plaque samples by culture and latex agglutination tests is necessary to determine their relative sensitivities in identifying subgingival bacteria. The sensitivity of the latex agglutination tests for periodontal pathogens deteimined with laboratory strains appeared lower than culture where cultivable bacteria usually do not exceed 106 organisms/ml from a pocket. It is, however, unclear whether this is a significant difference. Latex agglutination identifies both viable bacteria and non-viable bacteria/bacterial fragments while culture only identifies viable, cultivable organisms. While the relative ratio in a pocket of cultivable bacteria to non-viable bacteria/bacterial fragments is unknown, it is quite probable the cultivable bacteria make up a smaller proportion. This proportion most likely also varies in different sites and at different times. For any assay including latex agglutination, it is most important to demonstrate its clinical relevance. Latex agglutination assays for microorganisms have routinely been utilized by physicians for many years where they have pro-
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NISENGARD, MIKULSKI, McDUFFIE, BRONSON
rapid, simple, screening assay allowing a rapid therapeutic response. Evaluation of latex agglutination tests for periodontal pathogens could include studies to demonstrate their benefits in diagnosis, treatment, and patient management. Any assay must provide the clinician with information that would ultimately benefit the patient. The clinical relevance of an assay for periodontal pathogens is not solely related to the degree of sensitivity. This vided
a
differs from other diseases where there is a monoinfection. In cases of sore throats caused solely by ß-hemolytic streptococci, the more sensitive the assay, the better it is for diagnosis. In periodontal disease, with both quantitative and qualitative changes in the flora, an assay with the greatest sensitivity may not be relevant. For example, A. actinomycetemcomitans cannot only be cultured from involved first molars of patients with localized juvenile Periodontitis, but also from non-involved sites in these patients and from periodontally healthy siblings.11 At present, low numbers of these pathogens in healthy sites does not appear an indication for therapy since these low numbers have not been demonstrated to have any predictive or prognostic value. The studies reported here suggest that latex agglutination can be utilized for detection of periodontal pathogens. Comparisons with cultural results and clinical studies of relevance in diagnosis, prognosis, and patient management are necessary to demonstrate the efficacy of this type of test.
Acknowledgments This research was supported by New York State Science and Technology Grant #SSF (87)-4 and IMMCO Diagnostics Inc. REFERENCES 1. Tanner ACR, Socransky SS, Goodson JM. 2.
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1976;4:82-86. Send reprint requests to: Dr. Russell Nisengard, SUNY at Buffalo, School of Dental Medicine, Department of Periodontology, 3435 Main Street, Buffalo, NY 14214. Accepted for publication January 27, 1992.
