Microbial Diagnosis—Test Methods

10.1055/b-0034-56512

Microbial Diagnosis—Test Methods

Microbiologic tests for the type and amount of marker bacteria are indicated primarily in aggressive periodontitis, refractory forms and patients manifesting severe periodontitis in conjunction with a systemic disease (e.g., diabetes, AIDS). Such tests can also provide clues concerning whether the mechanical therapy should be enhanced with antimicrobial supportive therapy. In addition, these tests can indicate the success of therapy.

The following will be presented:

  • Microscopic techniques—dark field, phase contrast

  • Bacterial cultures—non-specific/specific

  • Use of DNA or RNA probes—amplification of DNA using PCR

  • Immunologic methods—fluorescence microscopy, EIA-/ELISA tests

  • Enzymatic bacterial tests (BANA)

406 Subgingival Plaque Removal Left: Supragingival cleaning and drying of the harvest site. Middle: Inserting the paper point (average size, # 30–50) into the pocket. Right: Placing the paper point into the transport container.

Collection Technique

Various methods have been proposed for harvesting bacteria from the pocket (subgingival plaque). Most common today is collection using paper points of moderate strength. One can collect a single, site-specific probe, e.g., from the deepest pocket in a quadrant, or “pool” several probes from various pockets.

It is important to record the site and the date of the bacterial probe in the patient’s chart (or via photography).

The site to be tested must be cleaned of all supragingival plaque and then thoroughly dried (not with an air syringe!). Then the paper strip is inserted to the bottom of the pocket and left in situ for 10 seconds. During removal, the strip must not come into contact with saliva, pus or the oral mucosa.

Depending on the test method, the paper point is placed into the transport container with transport fluid (for RNA probes, e.g., IAI PadoTest 4.5) or without any fluid (DNA probes, DMDx/Anawa etc.). The container is then tightly closed and sent to the special laboratory for further processing.

Microbial Pocket Diagnosis—Dark Field and Phase Contrast Microscopy

These methods permit limited bacterial diagnosis directly at chairside. They do not require fixation or Gram-staining and are therefore quick and simple to perform. However, only the morphotypes can be identified, i.e., the shape of the bacteria and their motility. The determination of these criteria permits a limited conclusion about the pathogenicity of the microorganisms (Listgarten & Helldén 1978).

If the sample reveals primarily cocci and non-motile rods, it is an indication of only few active pathogens.

If the field exhibits numerous motile bacteria (e.g., rods and spirochetes) it is an indication of an active phase within the pocket, and elevated levels of potentially pathogenic pocket flora.

If the microbiologic picture is displayed on a screen and reveals numerous motile rods and spirochetes, it can provide a high level of motivation for the patient.

Even this simple and rapid diagnostic method is associated with costs (the microscope), and the clinician must determine the cost/benefit ratio in comparison to the classical examination techniques or other test procedures.

407 Dark Field Microscopy This method reveals the shapes of bacteria and their motility, but does not permit any identification of bacterial classification or species. The method is therefore limited in its informative potential: It permits differentiation between inactive and active pockets. Its greatest value is in patient motivation. Right: The identifiable vital bacteria as seen in dark field and phase-contrast microscopes are identified according to the criteria listed.
408 Bacteria from an Inactive Pocket—Cocci In this photomicrograph one observes a few cocci and some non-identifiable particles; virtually no motile rods or spirochetes (proportion of spirochetes to cocci ca. 1:40). Generally, in all segments of this microscopic field there is but a low number of bacteria from this inactive pocket.
409 Bacteria from an Active Pocket—Spirochetes! Motile rods and spirochetes predominate. Their relationship to cocci is about 4:1, with a massively increased bacterial count. In contrast to the inactive pocket, there has been a “shift” from cocci to rods, and from non-motile to motile microorganisms. Right: Comparative depiction and typical distribution (in %) of the morphotypes (see Fig. 407, right) in an inactive and an active pocket.

Microbial Pocket Diagnosis—Cultures

The preparation of bacterial cultures is one of the oldest “classic” diagnostic methods. For culturing, the bacteria must be maintained in the vital state.

Periodontopathic marker bacteria are usually anaerobic, which presents problems for the removal of the organisms and their transport to the laboratory (O2, temperature, culture medium). In addition, preparing cultures is time-intensive, demands microbiologic knowledge as well as adequate laboratory equipment. All of this leads to appropriate costs! Oral anaerobic bacteria grow very slowly, and a definitive result is often possible only after ca. 3 weeks.

Both selective and non-selective culture media can be used to demonstrate the bacterial strains of interest, e.g., Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivialis, Bacteroides species, Capnocytophaga, Eikenella corrodens, Fusobacteria etc. On the other hand, to date it has been virtually impossible to grow spirochetes on the usual artificial culture media. One advantage of the culture technique is the possibility to create an antibiogram, to test whether a certain marker organism is sensitive, or more importantly, resistant to specific antibiotics.

410 Anaerobic Bacterial Culture on a Blood Agar Plate Various bacterial colonies can be identified on a blood agar plate that was incubated anaerobically for 10 days. In addition to the colonies of black pigmented Bacteroides species, many others are observed. From the form of the colonies, their metabolism etc., the microorganisms can be determined and classified (Bergey’s Manual of Systematic Bacteriology 1984). Left: Magnified colonies.
411 Isolated Colonies Left: Porphyromonas gingivalis colony. Right: Actinobacillus actinomycetemcomitans colony in its typical star-shaped configuration. Courtesy A. Mombelli
412 Resistance Determination via Antibiogram The clear field surrounding each antibiotic-soaked pellet determines the extent of inhibition of bacterial growth, and therefore permits a targeted use of the most effective systemic medication. (Courtesy A. Mombelli) Left: Yellow? Red? Orange? Medicaments should not be empirically administered on the basis of their “colors”; rather, they should be selected only after more in-depth diagnosis (p. 287).

New Diagnostic Tests—Evaluation

The classical data collections, including the just-described bacteriological culture examinations, still represent the gold standard (GS), but these demonstrate only processes that have already occurred in the tissues. Because relatively imprecise, conventional diagnostic findings have often led to under- and over-treatment, new and improved diagnostic tests would, in the ideal situation, make possible a more definitive prognosis and an individually appropriate and targeted treatment.

New microbiological (and immunological, p. 186) tests in the field of periodontal diagnosis have already become valuable diagnostic aids. In certain “difficult” cases, these new tests represent important components of diagnosis, and especially in the selection of a therapeutic regimen, e.g., with aggressive (Type III) or refractory periodontitis, with periodontitis combined with systemic diseases (Type IV), with severely advanced CP-cases (Type II), and in dental implantology.

Such additional diagnostic methods provide information concerning:

  • The future progression of periodontitis

  • The identification of patients at risk

  • Criteria for an individualized recall program.

Periodontal Test Procedures

Modern test methods should be better, simpler, faster, more exact, non-invasive etc., and if at all possible low cost. In general they should provide more information than the old and often error-plagued gold standard, and must represent procedures that are acceptable to the practitioner! Attachment loss or attachment gain can only be precisely determined histologically, i.e., after extraction of the tooth!

Therefore, in routine clinical practice, this invasive standard must be replaced by a procedure that provides results that are equal to or better than those resulting from the gold standard (Müller 2001). The following must be evaluated:

  • Most important is the negative or positive predictive value

  • Sensitivity and specificity; these are defined in the Glossary (below)

  • The validity of the test results; this determines their practical use.

From a practical point of view, all of the test results are placed into a 2×2 schematic (“decision matrix”) and the desired parameters are calculated (Fig. 413).

413 Decision Matrix—Evaluation of a Diagnostic Test The parameters include • Sensitivity • Specificity • Positive predictor • Negative predictor Using this matrix, the validity of any test can be determined.

Glossary

In the example “diseased—healthy,” the following expressions must be defined and clarified (cf. also AAP 1995; Diagnosis of Periodontal Diseases; Pihlstrom 2001):

  • Gold standard: Measurement of the true condition–yes/no: disease present (= positive) or not present (= negative).

  • Sensitivity: Probability (in %) that an existing disease is scored as positive in the test. High sensitivity indicates that the test will provide only few false negative results. Thus true disease will not be overlooked. This is important with contagious conditions and life-threatening diseases (Hepatitis B etc.).

  • Specificity: The probability (%) that a healthy patient will be shown to be healthy by the test (= “disease negative”). High specificity indicates that no healthy person will be incorrectly shown as diseased (= false positive). This is important, for example, with severe diseases that would demand unbearable therapy in a healthy individual (carcinoma, HIV).

  • Periodontitis: A test with a sensitivity of, e.g., 70% (all diseased individuals were not detected!) and a specificity of, e.g., 90% (few healthy individuals were diagnosed as diseased) would suffice for this rarely contagious, non life-threatening disease, which is therapeutically not demanding of invasive procedures.

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Jul 2, 2020 | Posted by in Dental Hygiene | Comments Off on Microbial Diagnosis—Test Methods
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