Laboratory diagnostics for periodontal diseases

Perceptions about the pathogenesis of periodontitis have changed considerably in recent years. While previously it was considered a purely microbiological problem (plaque hypothesis), we now know that the individual immune response of a patient is critical for determining the consequences of the trigger stimulus (biofilm) for the patient. In most cases, progressive periodontitis is caused by a periodontal inflammation system that responds excessively to bacteria (referred to as high responders). In the remaining cases, immunodeficiencies, which are associated with reduced resistance of the mucous membrane, can also lead to persistent inflammations because the body’s defence against pathogens is rather too weak.

While the high responders (tendency to develop chronic inflammation) can respond successfully to anti-inflammatory therapy and for whom immunostimulation is rather contraindicated, for patients with a reduced defence against pathogens on the other hand measures that support the immune system have priority (antimicrobial therapy, if applicable, immunostimulation with interdisciplinary cooperation). An anti-inflammatory (= immunosuppressive) therapeutic approach would be contraindicated here.

To clarify the cause and make a prognosis as well as to determine the choice of accompanying therapy, the following laboratory tests are available:

1. Detection of high responders
   Determination of the genetic predisposition to inflammation (IL-1A, IL-1B, IL-1RN, TNF-A, where applicable also IL-6, IL-10)
2. Detection of reduced defence against pathogens   
   Serum IgA, MBL, granulocyte function
3. Microbiological investigation of the subgingival flora
   Detection of periodontitis-associated marker microbes
4. Assessment of periodontal tissue destruction
   aMMP-8

The stimulus that triggers periodontal inflammation is the subgingival (periodontitis) or supragingival (peri-implantitis) biofilm. The macrophages activated by this biofilm secrete pro-inflammatory cytokines, particularly interleukin-1 (IL-1), tumour necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6). These cytokines activate osteoclasts which leads to increased alveolar bone resorption. They also increase tissue destruction by stimulating granulocytes which release greater quantities of tissue-destroying matrix metalloproteinase 8 (aMMP-8). To prevent excessive (chronic) inflammation, the inflammatory response is normally later slowed down by the anti-inflammatory cytokines IL-1 receptor antagonist (IL-1RN) and interleukin-10 (IL-10).

Because the polymorphisms for the pro-inflammatory cytokines IL-1α, IL-1ß and TNF-α lead to an increased synthesis of these mediators, their presence results in an amplified inflammatory response. If the polymorphism in the gene for the IL-1 receptor antagonist (IL-1RN) is also present, this is released in smaller quantities, that is, inhibition of inflammation is also unfortunately absent.

The exceptional importance of the above cytokines for the individual inflammation susceptibility led to grading of the inflammation susceptibility using the genetic constellations described. The genetic degree of inflammation increases depending on the number of verified polymorphisms present from grade 0 (no polymorphism present) to grade 4 (all four polymorphisms tested are present).

Patients with a degree of inflammation of 0 and 1 are referred to as low responders because they have a normal inflammatory capacity. Patients in grades 2 to 4, the high responders, have a greatly elevated susceptibility to inflammation that is genetically determined.

Even if the extent of the initial inflammatory response is primarily determined by TNF-α, IL-1 and IL-1RN, it may be that genetic variants in the cytokines IL-6 and IL-10 lead to a high responder status.
The pro-inflammatory cytokine IL-6 is secreted by macrophages and epithelial cells amongst others and, like IL-1 and TNF-α, increases tissue destruction and bone resorption. On the other hand, IL-10 is an anti-inflammatory cytokine which, like IL-1RN, counteracts bone loss and tissue destruction caused by inflammation.

While the polymorphism in the IL-6 gene causes an increase in the release of IL-6, the polymorphism in the IL-10 gene is associated with reduced levels of IL-10. If both these genetic variants are present, similar to a patient with a degree of inflammation of grade 3 or 4, the result can be elevated pro-inflammation (increase in the release of IL-6) with lowered anti-inflammation (decrease in the release of IL-10).

For patients with chronic or aggressive periodontitis, the genetic degree of inflammation should first be determined if an excessive inflammatory pathogenesis is suspected. If the test indicates a degree of inflammation of 2, 3 or 4, an elevated inflammation susceptibility is considered as verified. If, however, the test indicates a degree of inflammation of 0 or 1, it is recommended to test the genetic IL-6/IL-10 variants in a second step. If the IL-6 and IL-10 polymorphisms are present, then there is an increased tendency towards inflammation despite a normal genetic degree of inflammation.

If the genetic inflammation susceptibility is verified as being elevated, this leads to the following risks and therapeutic consequences:

• For these patients, it is not uncommon that following antibiotic and/or manual therapy the marker microbes results temporarily decline but the tissue destruction marker aMMP-8 remains elevated and the clinical signs of inflammation persist. This is often followed by recolonisation by the marker microbes and thus the development of refractory periodontitis.
• As well as local manual therapy, a concomitant anti-inflammatory therapy should also be selected.
• What is important is that these patients do not receive immunostimulatory therapy.
• Sensitisations to dental restoration materials and other odontogenic interference factors should be specifically searched for and eliminated.
• An awareness of a genetic predisposition to a progressive course for periodontitis should help and be a reason to motivate the patient to stop smoking and maintain oral hygiene. Short intervals between recalls and prophylaxis are also recommended.

2 mL EDTA blood or 2 buccal swabs.
Transport to the laboratory is not time critical and can be done by post.
For the genetic tests we require a declaration of consent of the patient. All the polymorphisms indicated can be tested using a single sample.

For patients with reduced defence against pathogens, measures that support the immune system should have priority (antimicrobial therapy). In cases of chronic periodontitis in which there is no increased inflammation susceptibility, an additional immunostimulation (where applicable, with interdisciplinary cooperation) would be another possible therapeutic approach. On the other hand, an anti-inflammatory (= immunosuppressive) therapeutic approach would be contraindicated for these patients.

MBL and IgA are measured in the serum. One serum tube (10 mL) is sufficient for both tests.

For the granulocyte function test, we require 10 mL heparin blood. For this test, the laboratory must receive the sample within 24 hours of collection. The blood should be stored and transported at room temperature.

As well as the individual genetic inflammation disposition of the patient, which has a critical influence on the severity of the medical condition, the periodontitis-associated microbes in particular demonstrably shape the inflammatory process. The growth of these anaerobic or facultative anaerobic pathogens is favoured by the lack of oxygen in the periodontal pocket. The periodontal microbes of the local flora are displaced by these anaerobes. If the microbes indicated are present in the sulcus, concomitant antibiotic therapy may be necessary. Active substance and pharmaceutical form are determined by the composition and distribution of the subgingival flora (targeted antibiotic treatment) meaning that a microbial clarification before starting therapy is helpful.

While clinical diagnostics in the past were restricted to the most important key microbes Aggregatibacter actinomycetemcomitans, Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia, we are able to cost-effectively carry out semi-quantitative assessments of the equally important microbes Campylobacter rectus/showae, Eikenella corrodens, Capnocytophaga gingivalis/ochracea, Parvimonas micra, Eubacterium nodatum and Fusobacterium spp. using modern chip technology and to integrate these into the therapeutic approach.

The decisive advantage of molecular biology verification compared to bacterial culture methods is its sensitivity because anaerobic bacteria often die during transport to the laboratory. The PCR-based chip procedure is equally able to detect the DNA of living and dead pathogens, however.

The anaerobic marker microbes mentioned represent the causal factor (inflammation stimulus) for periodontitis. Ultimately, however, the body’s own inflammatory response is responsible for the destruction of the tooth support structure and this varies enormously from patient to patient depending on the individual’s genetically determined inflammation susceptibility (see page 50). For patients with refractory periodontitis but also for patients who show clinical signs of persistent inflammation after elimination of verified marker microbes, an excessive inflammatory response is the cause in many cases. For these high responders (usually with a genetic degree of inflammation of grades 3 and 4), along with the elimination of the marker microbes (stimulus elimination), an anti-inflammatory but never an immunostimulatory therapy is indicated. In contrast, elimination of the trigger stimulus (including the marker microbes) is the top therapeutic priority for normal responders. For these pathogen-induced processes, additional immunostimulation would then make sense, unlike the situation for high responders.

The sample is collected from the dental pocket using paper strips which are included in the relevant collection set. The collection must be done prior to any mechanical treatment or antibiotic therapy. Collection sets are provided by the laboratory.

Literature

Periodontal pathogenic bacteria in the dental biofilm that are taken up by tissue macrophages trigger the inflammatory cascade in the pathogenesis of periodontitis. As described on page 49, the resultant activated macrophages initiate the inflammatory cascade as part of which infiltrated granulocytes release the enzyme MMP-8. As a collagenase, MMP-8 (previously referred to as collagenase-2) in its active form destroys the three-dimensional collagen network of the periodontium to facilitate infiltration of additional cells of the immune system.

The enzyme aMMP-8 is therefore primarily responsible for tissue destruction. Measuring the aMMP-8 level in the sulcus fluid therefore enables early detection of attachment loss and thus timely therapeutic intervention.

aMMP-8 determination provides information about the inflammatory status of the periodontium often before clinical signs become apparent. Because aMMP-8 is not only an inflammation marker but also a direct marker of tissue destruction, it can deliver precise information about the medium-term progression.

The aMMP-8 levels fall within 10–12 weeks back to the normal range after successful periodontal treatment. The aMMP-8 levels therefore also enable detection of refractory disease.

Determination of aMMP-8 levels in the peri-implant sulcus fluid (PISF) can be used for routine monitoring of the implant or early detection of mucositis or to promptly initiate therapeutic measures for suspected peri-implantitis / mucositis.

The aMMP-8 level in the PISF correlates in these cases with bone loss. With peri-implantitis the aMMP-8 levels are increased up to 1000-fold compared to a healthy implant. Another indication is for monitoring the local inflammatory status prior to placing an implant.

To detect the aMMP-8, a sampling strip included in the collection set is inserted into the sulcus and sent to the laboratory in a shipment tube by post.
The collection and shipping material is provided by the laboratory free of charge.

Literature