MINI REVIEW
TESTING FOR MARKER BACTERIA IN PROGRESSIVE
PERIODONTITIS: THE EUROPEAN EXPERIENCE
Georg Conrads
PERIODONTAL DISEASES (gingivitis and periodonti-
tis) are chronic bacterial infections with a remarkably
high prevalence and morbidity. Almost 100% of adults,
but also 90% of children at school age, are periodically
or occasionally affected by gingival bleeding, the most
objective sign of early gingivitis. Gingivitis, with some
exceptions, is a polymicrobial infection with no single
associated bacterial agent and is reversible by adequate
oral hygiene. In contrast, periodontitis (Figure 1) is
moderately to rapidly progressive, associated with cer-
tain bacterial species (called periodontal pathogens or
“marker bacteria”), and affects all of the soft tissue and
bone supporting teeth [1]. Moderate forms of peri-
odontitis are demonstrated by 50 to 98% of adults with
regional and age-based differences. Periodontitis de-
stroys the integrity of oral mucous membranes and is
one of the main reasons for tooth loss, especially
among people aged forty and older. The cost of ther-
apy in the United States resulting from periodontal dis-
eases is estimated to be as high as $5 to 6 billion an-
nually. In a relatively small country like Germany with
high standards in dentistry, patients and health insur-
ance companies spend $5 billion each year with $20 as
the minimum annual costs to treat the inflamed peri-
odontium of just one tooth.
Between 3 and 13% of the population are suscepti-
ble to rapid and advanced loss of periodontal attach-
ment. They may develop aggressive forms of peri-
odontitis, such as rapidly progressive periodontitis
(RPP), juvenile periodontitis (JP), or refractory peri-
odontitis (RP), which cause severe problems. These in-
dividuals require prosthetic treatment within a short
period of time. In addition, as periodontal diseases dis-
turb the integrity of oral mucous membranes, peri-
odontal pathogens can frequently be detected in blood
Infectious Diseases in Clinical Practice, 2001;10:481–487
0796/01/0
Copyright © 2001 by Lippincott Williams & Wilkins, Inc.
From the University Hospital (Operative Dentistry, Periodontology and
Preventive Dentistry and the Institute of Medical Microbiology), Rhine-
Westphalian Technical University (RWTH), Aachen, Germany
Address for correspondence: Georg Conrads, PhD, Associate Professor,
Institute of Medical Microbiology, University Hospital, Pauwelsstraße 30,
D–52057 Aachen, Germany (Email: gconradsphd@aol.com).
481
cultures. These frequent bacterial attacks may not only
cause bacteremia, but also (under some circumstances)
septicemia, organ abscesses, or endocarditis, as well as
cardiovascular disorders or low birth weight when oc-
curring during pregnancy.
As documented by numerous publications [2–4], peri-
odontal diseases are associated with a shift in the peri-
odontal bacterial flora, from the healthy to the diseased
status. Among the periodontal pathogens, species such
as Actinobacillus actinomycetemcomitans, Bacteroides
forsythus, Campylobacter rectus, Eikenella corrodens,
Fusobacterium nucleatum, Porphyromonas gingivalis,
Prevotella intermedia and several forms of uncultivable
spirochetes play a major role in the pathogenesis.
For diagnosis of the activity of the different forms of
periodontitis, clinical symptoms alone may not be suf-
ficient, as they provide a historical record only (pocket
formation, attachment loss, alveolar bone loss) or have
low predictive value (bleeding during probing). But
predictions of recurrence of disease and prognosis for
the patient can be significantly improved when the
presence or absence of periodontal pathogens is mon-
itored concurrently [4].
To monitor bacterial changes or shifts in the gingival
sulcus or the periodontal pocket, several techniques
have been introduced. Ten years ago, these procedures
were only available at university institutions, but nowa-
days, especially in Central Europe, most of them are
widely marketed and available for every dental practice.
The aim of this present review is therefore to give an
overview of conventional and molecular biological
techniques for testing periodontal pathogens. Further-
more, it summarizes some experiences from both the
dentists and the laboratories involved with these test
procedures.
Conventional and Molecular Techniques in
Routine Testing of Periodontal Pathogens
For the detection of periodontopathogens, microscopy,
culture, immunoassays, enzyme tests, and DNA-based
techniques have been introduced.
482 Infectious Diseases in Clinical Practice, Vol. 10, No. 9
FIGURE 1. Case of progressive periodontitis. Paper points
are used to sample an aliquot of subgingival plaque for
testing quality and quantity of bacterial agents (kindly
provided by H. Sellmann, DDS, Marl, Germany).
Darkfield and Phase Contrast Microscopy
The microscopic picture of native subgingival plaque
from healthy and diseased sites is strikingly different.
Whereas “healthy plaque” consists of mainly coccoid
cells or small to large rods with almost no active motil-
ity, the plaque of diseased sites presents cells of high
motion and mainly consists of rods or long filaments.
The higher the motility of bacteria in plaque, the more
inflamed is the periodontium, and the greater the likeli-
hood for further progression of the disease. Neverthe-
less, microscopic examination of freshly sampled plaque
is not particularly practical for routine diagnosis. This
technique needs special equipment, is time-consuming,
and in the end gives results only for a small portion of
the ecological system. However, chairside microscopic
assessments as a diagnostic parameter in periodontal
diseases have been reviewed by several authors [5,6] and
should not be ignored as a promising diagnostic tool, es-
pecially in an era of molecular techniques.
Culture Methods
For routine diagnostic in medical microbiology, culture
methods are still the gold standard for evaluating molec-
ular methods [7]. As a matter of fact, most periodontal
pathogens are strictly anaerobic and quite fastidious, and
some are still uncultivable [8]. However, in some special-
ized laboratories, the cultivation of periodontal pathogens
Conrads
is well established. A reference laboratory to mention in
this context is LABORAL in Houten (The Netherlands),
which analyzes more than 12,000 periodontal samples
per year by cultivation. For culture procedures of anaer-
obic periodontal pathogens, it is extremely important to
bear in mind that these bacteria are killed by oxygen very
rapidly. As a consequence, rapid transportation of the
samples from the dental practice to the diagnostic labo-
ratory is most important. Cultivation needs time for grow-
ing and isolating the bacteria, and for biochemical differ-
entiation of the dominant species. Therefore, the results
require 10 days or longer to reach the dentist. But culture
also has two major advantages: 1) resistance testing of
pathogens is possible; and 2) pathogens of secondary im-
portance (e.g. Eubacterium spp., Peptostreptococcus mi-
cros, Streptococcus milleri-group species), usually not in-
cluded in molecular approaches, will also be reported
(usually in “percent cultivable flora”) if they are dominat-
ing the disease-associated plaque.
Enzymatic Activity
A rapid but imperfect diagnosis might sometimes be bet-
ter than a perfect but delayed one. With this as rationale,
chairside tests were developed, based on the enzymatic
activity of periodontal pathogens. Pathogens like Tre-
ponema denticola, P. gingivalis or B. forsythus produce
trypsin-like proteases. If these enzymes are present in
the paper-immobilized plaque tested, special substrates
(benzoyl-DL-arginine-ß-naphthylamide, “BANA”) are
hydrolyzed and a color reaction occurs. According to
Loesche et al. [9], the sensitivity is between 90 and 96%
with a specificity between 83 and 92%. However, one
of the major pathogens, A. actinomycetemcomitans, is
negative in the trypsin protease-reaction, and important
cases must be diagnosed differently. The enzymatic tests
to routinely screen for periodontopathogens (PerioScan
[Oral B Laboratories, Redwood City, CA], Periocheck
[Colla Genex Pharmaceuticals, Newtown, PA]) were not
very successful on the European market and some are
no longer distributed.
Immunoassays
For the detection of periodontal pathogens, polyclonal
and monoclonal antibodies are available. Conjugated
with fluorescent-reporter molecules, these antibodies
can enhance the specificity and sensitivity of light-
microscopic methods. Additional immunological meth-
ods such as enzyme linked immunosorbent assay (ELISA)
or latex-agglutination tests were also designed for de-
tecting periodontal pathogens in plaque [10]. The sensi-
tivity of these tests is relatively low, requiring 104 to 105
Testing for Marker Bacteria
bacteria per sample. As a consequence, the commercial
product (Evalusite; Kodak, Switzerland) has not been
widely marketed.
It should be mentioned that serological tests for di-
agnosing periodontal diseases might have a brighter fu-
ture, as documented by several recent publications
[11–13], but specific antigens for all major marker bac-
teria have not yet been found.
Genetic Approach
As mentioned above, periodontal pathogens are difficult
to culture. Therefore, they were one of the first targets
for routine DNA probe testing in the field of medical mi-
crobiology. Two different main strategies have been in-
troduced, based on: 1) species-specific fragments of the
bacterial genome (“genomic” DNA probes), which were
in some cases cloned into a vector system; and 2) short
synthetic oligodeoxynucleotides with computationally
extended database searches and empirically proven
specificity, which hybridize to a short (18 to 35 b) se-
quence, mostly found on ribosomal genes.
Murray and French were the first investigators who
detected P. intermedia and P. gingivalis with the aid of
purified DNA fragments labeled with 32P or biotin-11-
dUTP by nick-translation [14]. The sensitivity of these ge-
nomic probes was increased, requiring only 102 to 103
bacteria. However, the same procedure was found to be
very difficult for the major periodontal pathogen A.
actinomycetemcomitans (A.a). Thus the authors had to
clone specific fragments of the genome into plasmids
and these recombinant DNA-probes were then used for
diagnosis. With this procedure, cross-hybridization be-
tween the A.a.-probe and other species of Pasteurellae
were reduced to the minimum of 1%. Other pathogens
were added into this system, including T. denticola (ge-
nomic probe), or F. nucleatum, E. corrodens, C. rectus,
and B. forsythus (recombinant DNA-probes). A similar
testing system was marketed by the American company
OmniGene Laboratory Services (Cambridge, MA), and is
frequently used in some European countries also (under
the name DMDx/PATHOTEK). The samples are cen-
trally analyzed in the ANAWA laboratories AG (Wangen,
Switzerland). In the year 2000, about 16,000 of these
tests were performed in Europe, with 14,000 tests sent
from German dental practices, 600 tests from Switzer-
land, and the remaining from practices in the rest of Eu-
rope. As a consequence, most experience with the test-
ing system comes from Germany.
In contrast to genomic probes, oligonucleotides are
synthetically produced, short, stable molecules and can
easily be introduced into automated systems, the future
of diagnostics.
Infectious Diseases in Clinical Practice, Vol. 10, No. 9 483
Chuba was the first to introduce oligonucleotide-
probes, directed against species-specific sequences of
the 16S rRNA to detect species like P. gingivalis, P. in-
termedia, and A. actinomycetemcomitans [15]. By way
of background, the 16S rRNA is a part of the small sub-
unit of bacterial ribosomes. This amazing molecule
consists of both highly conserved regions, appropriate
as primer targets for amplification, and species-specific
sequences, of major interest to molecular taxonomists
and, so far, an ideal target for deducing specific
oligonucleotide-probes. Using pure cultures, the speci-
ficity of oligonucleotides can be noted as 100%, but this
might be reduced when detecting bacteria in complex
samples like subgingival plaque. The sensitivity of
oligonucleotide-probes depends on the labeling sys-
tem, but can be minimized to detect 102 to 103 cells per
analysis. In the important publication by Dix et al. [16],
highly species-specific and 16S rRNA directed DNA-
probes of 24 b length and 32P labeled were used to de-
tect A. actinomycetemcomitans, B. forsythus, P. gingi-
valis, P. intermedia, E. corrodens, F. nucleatum, and C.
rectus. The authors found that the sensitivity and speci-
ficity of the probes were not reduced by using subgin-
gival plaque as samples, but this might be true only un-
der ideal conditions, which could be problematic for
routine testing. Meanwhile, oligonucleotide-probe based
test systems for periodontal pathogens have been in-
troduced in different formats into the European market.
For instance, the IAI Pado Test 4.5-system (Institute for
Applied Immunology, Zuchwil, Switzerland), which is
available in some European countries, namely Switzer-
land, Germany, and The Netherlands. Whereas the IAI-
test system uses radioactively labeled DNA probes, the
new LCL-system (LCL Biokey GmbH, Aachen, Germany)
uses chemiluminescence-generating oligonucleotide-
probes (Figure 2 with representative report of the re-
sults in Figure 3). Both companies perform around
10,000 tests per year in Europe. In the case of the LCL-
system, it is known that the demand for such tests is
rapidly growing among practitioners.
To further enhance specificity, polymerase chain re-
action and combinatory molecular genetic techniques
were also introduced for the routine diagnosis of peri-
odontal pathogens [17]. After amplification of the 16S
rRNA-gene, specific DNA-probes used in a reversed hy-
bridization procedure are able to quantify the bacteria
in the amplicon. This two-step testing system may also
be very sensitive, but it seems questionable whether
the ratio of different bacteria, a result which is an im-
portant factor in reaching therapeutic decisions (e.g.,
the most appropriate antibiotic therapy), is still the
same when obtained by in-vitro amplification prior to
484 Infectious Diseases in Clinical Practice, Vol. 10, No. 9
hybridization. However, a testing system distributed by
the company HAIN-Diagnostic (Nehren, Germany) is
frequently used in Germany. As an advantage, the sam-
ples are not analyzed only in a central laboratory, but
the whole test system is distributed to local medical
laboratories, making specimen transport easier.
In the future, mini molecular laboratories will be
available for chair-side DNA probe testing in an hour
or less (Periodontal Microbial Identification Test,
Saigene Corp, Bothell, WA) [18]. However, the advice
of a microbiologist might still sometimes be necessary
to avoid problems that may occur in the diagnostic
process. Some of these problems will be reviewed in
the next section.
Experiences in the Dental Practice
As mentioned above, German dentists are confronted
with various test systems for periodontal pathogens.
With this in mind, we carried out a survey among these
Conrads
FIGURE 2. Laboratory procedure for
demonstrating periodontopathic bacteria in
plaque samples by chemiluminescence-labeled
oligonucleotide probes (kindly provided by LCL
biokey GmbH, Aachen, Germany).
practitioners. Among the approximately 50,000 dentists
practicing in Germany, 3 to 6% are specialized in the
diagnosis and treatment of periodontitis and were thus
chosen to report about their experiences.
Initially, these dentists seemed to be very satisfied by
the current test systems. Here are some representative
answers:
“The test gives me more certainty in the diagnosis
and therapy of periodontal diseases.” (M. Heilos,
Borchen, Germany)
“I feel supported in my decisions about the appropriate
therapy and especially the need for therapy in spouses.”
(P. Jedenak and Th. Heidrich, Goslar, Germany)
“By using new diagnostic procedures, we were able
to improve the results of periodontal therapy and were
able to avoid unnecessary treatments.” (P. and K.
Twesten, Hamburg, Germany)
“The tests avoid time being wasted and errors in
therapy. In addition, they reduce the number of surgi-
cal treatments performed in acute inflamed and in-
fected sites.” (J. Kromer, Minden, Germany)
Testing for Marker Bacteria Infectious Diseases in Clinical Practice, Vol. 10, No. 9 485

FIGURE 3. Representative laboratory report after DNA-probe analysis of subgingival plaque in a case of rapidly progressive
periodontitis (kindly provided by LCL biokey GmbH, Aachen, Germany).
486 Infectious Diseases in Clinical Practice, Vol. 10, No. 9
“Patients with acute infected sites receive special
anti-infective treatment simultaneously with their me-
chanical treatment. As a consequence, the permanent
success rate of periodontal surgery has increased in our
practice significantly.” (B. Pfäffle, Cologne, Germany)
“Diagnostic tests have improved our risk assessment
and the systematic and systemic antibiotic therapy, es-
pecially in cases of refractory periodontitis.” (K.
Schröder, Berlin, Germany)
“The number of recurrences in spite of optimized
oral hygiene and interval of recalls, could be reduced
by following the test results and their recommenda-
tions for antibiotic therapy.” (G.-R. Niestadtkötter,
Rheda Wiedenbrück, Germany)
“We avoid unnecessary antibiotic treatment.” (H.
Sellmann, Marl, Germany)
However, the information I got from numerous
lengthy discussions with dentists in a more private set-
ting is somewhat different. The problems “under the
surface” are interesting and are as follows:
Periodontal diseases are chronic infections and “heal-
ing” is almost impossible, even by using the most so-
phisticated diagnostic and treatment strategies. The pro-
longed treatment is definitely very time-consuming and
costly (health insurance covers only a portion from all the
expenses), and can be frustrating for both dentist and pa-
tient. As a consequence, some practitioners might give
up on these cases–unfortunately, sometimes too early.
Some practitioners, highly encouraged, use different
test formats over time (sometimes for one patient) in
order to select a favorite. To a certain extent, they get
different results from the different tests. Some of the dis-
crepancies are minor (e.g., different cell counts of an in-
dividual species), but still confusing, and others are ma-
jor (different species detected). According to recent
experiences [19], it is obvious that some testing proce-
dures have problems detecting A. actinomycetemcomi-
tans and report anaerobes only, with consequences for
the antimicrobial treatment. More validation is needed
for microbial test systems in periodontal diseases, to fur-
ther improve the acceptance of these useful tests.
Experiences From Laboratories
In our survey, we also asked the managers of labora-
tories about their experiences with dentists. Most of the
firms are very satisfied with the collaboration and are
very optimistic about the future of their testing systems
for periodontal pathogens. However, this review arti-
cle tries to concentrate on problems in communication
which can occur between dentists and microbiologists,
some of which are as follows:
Conrads
Some of the dentists are very over-enthusiastic at the
beginning and send samples from almost every patient
they see in their practice. Of course, this activity can
lead to confusion in the end. Testing periodontal
pathogens should for several reasons be concentrated
on a small spectrum of diseases and patients. Indica-
tions for microbial testing include:
1. Patients with advanced attachment and bone loss
before the age of 25 (juvenile periodontitis or pre-
pubertal periodontitis).
2. Patients, usually aged 25–35, with rapid destruction
of attachment and bone in a relatively short period
of time (rapidly progressive periodontitis).
3. Patients who continue to lose attachment despite
adequate mechanical treatment (refractory periodon-
titis).
To receive useful information, an appropriate me-
chanical treatment and an optimized oral hygiene reg-
imen are essential preconditions for subsequent test-
ing. Reducing the biofilm is necessary both for getting
access to the important, base located, subgingival
plaque for testing and supporting the diffusion of an-
timicrobial agents.
A fraction of the dentists seem to accept only “posi-
tive” results from the tests to support their (predeter-
mined) antibiotic therapy. But even in a patient with
serious clinical symptoms, the periodontal disease has
nonactive periods, when microbiological testing is truly
negative. Therefore, diagnosis needs both clinical and
microbiological data [20–22].
A diagnostic result can only be as representative as
the sample that was analyzed. This is true for medical
microbiology in general but especially true for peri-
odontal diseases. According to Mombelli [23], the
deepest periodontal pocket with the highest tendency
for bleeding must be chosen from each quadrant for
sampling. The dentist must get an overview about the
destructive processes in one session before sampling
in a second session to avoid provocation of bleeding.
It is extremely important that sampling is not per-
formed in a bleeding periodontal pocket because the
paper points, the sampling device in most cases, can
only absorb about 20
l (one drop) of fluid. If this vol-
ume is mainly blood or saliva or supra gingival plaque,
not much is left for the pathogen-containing deep
fluid. Several paper points should be used for sam-
pling and either tested separately or “pooled” (more
cost-efficient) [24].
To get the highest benefit from testing marker bacte-
ria in periodontal diseases, the education of the staff in-
volved must be improved. It is very important that the
Testing for Marker Bacteria
clinical microbiologists learn more about oral microbi-
ology, dentistry, and periodontology and that the peri-
odontologists keep in touch with microbiology, antibi-
otics, and the principles of modern diagnostic systems,
especially their predictive values and shortcomings.
In conclusion, microbiological testing in severe or ad-
vanced forms of periodontitis is a very promising tool to
determine active disease and predict future attachment
loss, ultimately improving treatment prognosis. How-
ever, controlling periodontitis needs optimal engage-
ment and communication by all concerned: patient, den-
tist or hygienist, and – sometimes – microbiologist.
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