ACTIVE MMP 8 IN DIAGNOSIS OF PERIODONTAL DISEASE PROGRESSION. COLLAGENASE 2 (MMP8) IN PERIODONTAL DISEASE. PERIODONTAL DISEASE BIOMARKER. POC (POINT OF CARE) TESTING. CHAIR SIDE TESTS. HYBRID LABORATORY.

1. JOURNAL
CLUB
DR SHREEJA NAIR
GDCHA

3. WHAT IS aMMP 8 ?
WHY aMMP 8?
WHAT ARE THE
NORMAL VALUES?

14. aMMP-8 DIGITAL READER

15. PRODUCT DETAILS

20. MATERIAL AND METHODS
1. SUBJECTS
2. OUTLINE
3. CLINICAL MEASUREMENTS
4. RINSING SPECIMEN SAMPLING AND PROCESSING
5. BLINDING AND TRAINING OF INVESTIGATORS
6. SAMPLE SIZE CALCULATION AND STATISTICAL ANALYSIS

21. SUBJECTS
The prospective, mono-center, double-blinded, case–control study approved by the ethical review board of the Technische Universitat Dresden
(EK330112009)
INCLUSION CRIETERIA
– Males and females aged from 18 to 70 years
– Have at least 15 teeth excluding third molars
EXCLUSION CRIETERIA
– Severe systemic diseases and Under medications that affected the condition of the periodontal tissues and Pregnant and breastfeeding
– Received treatment with antibiotics less than 8 week before baseline or during the study.
According to the periodontal screening index (PSI) the participants were consecutively allocated to one of three groups:
• Group 1 : (n=35 ) PSI score of 0 in at least three sextants and scored 1 in no more than three sextants (periodontal healthy )
• Group 2 : (n=60) PSI scores of 1 or 2 ( gingivitis)
• Group 3 : (n=35) PSI scores of 3 or 4 in at least three sextants (periodontitis)
Patients were asked not to eat or brush their teeth less than 1 h before the rinsing samples were collected.

22. OUTLINE
– BASELINE VISITS (V1)
1. Baseline rinsing samples were collected.
2. Silness J, Loe H. plaque index (PI) , Loe H. gingival index (GI) and bleeding on probing (BOP) were assessed.
3. In patients from the periodontitis group, probing pocket depth (PPD) and clinical attachment level (CAL) were documented.
– 8 WEEK FOLLOW UP (V2)
1. Diagnosis-related periodontal treatment was performed.
2. Participants in group 1 were not treated. In group 2, participants underwent dental prophylaxis. Patients from group 3 received a
thorough scaling and root planing of all affected teeth under local anesthesia within one appointment by trained periodontists.
– FOUR WEEKS AFTER THERAPY (V3)
1. In groups 2 and 3, participants were recalled.
2. Participants of group 1 were recalled 4 wk after V1 without having any therapy.
3. The PSI was recorded again and aMMP-8 rinsing specimen collected.
– Clinical parameters and adverse events were documented.

23. CLINICAL MEASUREMENTS
– PI and GI were recorded at four sites per tooth at all teeth using scores 0–3.
– BOP was judged “yes” or “no” about 10 s after provocation at six sites per tooth.
– Clinical attachment level (CAL) and probing pocket depth (PPD) were measured at six sites per
tooth using a full-mouth approach in the periodontitis group only.

24. RINSING SPECIMEN SAMPLING AND
PROCESSING
Rinsed intensively with
15–20 mL tap water for
20 s to remove food
debris
Waited for 1 min, while
saliva was swallowed
Took 5.5 mL of rinsing
fluid (sterile purified
water) to rinse the oral
cavity for 30 s
Rinsing specimen was
spitted into a cup and 3
mL thereof were filled
into a syringe
Filter the sample, a
0.45 micron micro-filter
was placed on the tip
of the syringe
Filtrate was applied
drop by drop to the
sample port of the test
stick.
Remaining rinsing specimen was
subsequently filtered using a new filter
to receive an aliquot of 0.3 mL filtrate.
This filtrate was stored at 2–8°C and
utilized timely within 3 d for quantitative
aMMP-8 ELISA assessment as reference
standard (dentognostics GmbH, Jena,
Germany).
Active MMP-8 concentration was
measured via optical density. This value
served as a reference for the value
obtained with the POC test stick.
For POC
For ELISA

25. RINSING SPECIMEN SAMPLING AND
PROCESSING
– Active MMP-8 concentrations ≥ 25 ng/mL in mouth rinse specimens were evaluated as
positive.
– Samples were discarded when less than 3 mL were sampled, when less than three droplets
were gathered or when the micro-filter did not work properly.
– The POC test sticks were read after 5 min by one investigator (AF) that was trained before the
study start by a person from the evaluating laboratory.
– A valid test was scored “positive” when a clear, however, even faint test line and a control line
were visible. It was scored “negative” when no test line was visible and the control line was
visible.
– Owing to the outcomes of different pilot investigations, the cut-off of the POC test was set at
25 ng/mL.

26. BLINDING AND TRAINING OF
INVESTIGATORS
– The person who performed the POC aMMP-8 analysis and the person who conducted the ELISA
were blinded in terms of the allocation of the patient and sample to groups 1, 2 or 3.
– The patients were not informed about their test results until the study was finished.

27. SAMPLE SIZE CALCULATION
– Regarding the primary objective, a sample size of 100 was suggested by the Clinical and Laboratory Standards Institute
(CLSI) guideline covering the full range of diseases. In terms of clinical assessments, a recruitment minimum of 20–30 cases
per subgroup would be necessary.
– Assuming, 5% test positives (T+) for healthy, 60% test positives among patients with gingivitis, and 80% test positives among
periodontitis, both requests are approximately fulfilled when 30 healthy persons, 50 patients with gingivitis and 30
patients with periodontitis were included.
– Taking a drop-out rate of 20% into account a total of 35 healthy subjects, 60 subjects with gingivitis and 35 subjects with
periodontitis had to be recruited.
– According to CLSI guideline, AGREEMENT MEASURES were used to describe diagnostic accuracy.
– The positive percent agreement (PPA) is the proportion of patients with positive aMMP-8 POC tests among all patients
positively tested with aMMP8 ELISA, whereas the negative percent agreement (NPA) represents the proportion of patients
with negative aMMP-8 POC tests among all patients negatively tested with aMMP-8 ELISA.
– The ratio of agreeing test results divided by agreements expected by chance is presented as the kappa value(commonly
used statistics to test interrater reliability), whereby for interpretation the categorization as proposed by Altman is used.

28. STATISTICAL ANALYSIS
– Based on measurements at V1, test scores of the aMMP-8 POC test and test results
of the aMMP-8 ELISA were tabulated in a 2x2 table.
– PPA and NPA with their exact one-sided lower 95% confidence limits were calculated.
– Frequencies were calculated for all PI and GI scores.
– BOP was reported as the percentage of bleeding sites per patient.
– The relationship between clinical variables, test results and groups was investigated
in linear models.
– Estimates of the models with their 95% CI were reported. A significance level of 0.05
was defined before the study started, whereby no adjustment in terms of multiple
testing was performed.
– All computations were made with statistical software SAS 9.2 (SAS Institute SAS
Institute Inc., Cary, NC, USA).

29. RESULTS
1. DEMOGRAPHIC DATA
2. COMPARISON OF POINT-OF-CARE AND
ENZYME-LINKED IMMUNOASSAY TEST
RESULTS
3. POSITIVE TEST FRACTIONS FOR
DISEASE GROUPS
4. RELATION OF POINT-OF-CARE TEST TO
CLINICAL VARIABLES AND DISEASE

30. DEMOGRAPHIC DATA
– Of the 130 participants who were recruited, 111 finished the study.
– The drop-out rate thus was 14.6%.
– Drop-outs included persons who did not appear at V3 and who had insufficient
study compliance.
– Demographic data for each group are presented in Table 1.
– As expected, participants in groups 2 and 3 on average exhibited a higher age.
– In general, more females were recruited.
– A higher number of smokers were found in group 3.
– No adverse events occurred while performing the test.

31. COMPARISON OF POC AND
ELISA TEST RESULTS
– The PPA between aMMP-8 test results was above 70%
– The NPA was above 90% at both visits, V1 and V3
– The resulting kappa of 0.69 refers to a good agreement between the tests.

32. POSITIVE TEST FRACTIONS FOR
DISEASE GROUPS
– Positive test fractions in healthy participants (group 1) at V1 were
8.6% in the POC test and 5.7% measured with ELISA, while 6.9% for
this group were found with both methods at V3.
– In group 2 (gingivitis), 25.0% (V1) and 29.8% (V3) were identified as
positive by the POC test, 26.7% (V1) and 42.1% (V3) by ELISA.
– In group 3 (periodontitis), 40.0% (V1) and 48.1% (V3) were found
aMMP-8 positive with the POC method, compared to 42.9% (V1)
and 44.0% (V3) in the ELISA test.

33. RELATION OF POINT-OF-CARE TEST TO
CLINICAL VARIABLES AND DISEASE
– The dependency of positive test results of POC test on clinical variables was investigated in
general linear models using the log link function.
– Hereby, the sensitivity increased significantly or nearly significantly by factor = 2.54 (95% CI =
1.08–5.99), p = 0.03, for BOP and by factor = 1.63 (95% CI: 0.94–2.80), p = 0.08 for GI,
respectively, when these clinical parameters are elevated by one unit.
– Because of low numbers of false positives, the influence on specificity could not be evaluated.
– All clinical variables remained unchanged in the healthy participants, but decreased in groups 2
and 3 from visit 1 to visit 3 (results not shown).

34. RELATION OF POINT-OF-CARE TEST TO
CLINICAL VARIABLES AND DISEASE
– Four weeks after treatment the mean clinical attachment level decreased from 3.6±0.9 to 3.0±1.0
mm and the mean probing pocket depth decreased from 3.1±0.7 to 2.4±0.7 mm, in the group of
patients with periodontitis.
– Regarding the relationship between POC test results and clinical variables, differences were
obtained.
– Patients who had positive aMMP-8 test results at V1, i.e., before treatment, showed generally a
higher number of plaque and inflammation scores 2 and 3, and higher values for clinical attachment
level and probing pocket depth in the periodontitis group.

35. RELATION OF POINT-OF-CARE TEST TO
CLINICAL VARIABLES AND DISEASE
– Table 4 provides descriptive statistics for BOP, clinical attachment level and probing pocket depth in relation
on POC test result as well as results of statistical tests (p-values of U-tests, no adjustment for multiple
testing).
– Frequencies for PI and GI as well as statistical test results (p-values of Armitage trend tests) are depicted in
Table 5.

36. DISCUSSION
– While using the same antibodies comparable results between laboratory and chair-side devices were
obtained, while the immunofluorometric assay was more accurate than ELISA was (Leppilahti JM,
Hernandez-Rios PA, Gamonal JA et al. 2014).
– aMMP-8 concentrations were shown to be low in periodontally healthy sites and increase with advancing
inflammation and high inflammatory burden.
– In addition, the potential of aMMP-8 to predict treatment outcome in patients with chronic periodontitis
was reported.
– This study was undertaken to assess the diagnostic accuracy of a novel aMMP-8 POC test in comparison
to the ELISA reference standard. It revealed a PPA of > 70% and an NPA of > 90%. The overall agreement
of the new test device in comparison to the standard ELISA technique was about K = 0.7.
– According to Altman, this was considered a good strength of agreement, which was obtained not only at
baseline, but was confirmed also after treatment of the periodontal diseases, gingivitis and periodontitis.
– Therefore, the POC test can be used instead of ELISA to detect increased aMMP-8 levels in rinsing
specimens of patients with gingivitis and periodontitis.

37. DISCUSSION
– In patients with periodontitis, the number of positive aMMP8 test
results decreased after treatment, as did clinical attachment level,
probing pocket depth, plaque and inflammation and therefore
reflected a positive treatment outcome.
– Similar results were obtained in two other studies that recently
investigated the same test device. The POC test showed a
sensitivity of > 70% and a specificity of up to 96% when
periodontal inflammation (> 20% BOP positive sites, more than
two sites with ≥ 4 mm probing depth) was present in a group of
adolescents.
– A study in Nigeria revealed aMMP-8 POC test sensitivities
between 82% and 96% in patients who had poor oral hygiene, at
least two sites with periodontal pockets and positive BOP.

38. DISCUSSION
– Previous studies showed that MMP-8 concentrations are very low in healthy persons, are increased in
patients with gingivitis and are the highest in patients with periodontitis (Mantyla et al 2003, Prescher
N et al 2007, Leppilahti et al 2014). This pattern could be confirmed in the present study.
– In healthy participants, the number of positive aMMP-8 test results was small (5.7–8.6%) but increased
in patients with gingivitis (25.0–42.1%) and even more in patients with periodontitis (40.0–48.1%).
– Participants who had negative aMMP-8 test results presented less plaque and gingival inflammation
regardless of their disease status.
– In patients with periodontitis, the clinical attachment level and probing pocket depths were less in
participants that tested negative.

39. LIMITATIONS
– Regarding the demographic profile, it is known that the
occurrence of periodontitis is age dependent.
– Periodontitis is more prevalent in smokers .
– The current aMMP-8 test cannot evaluate site-specific values
per tooth, but only an overall aMMP-8 concentration for this
collagenase as released by the gingival tissue via gingival
crevicular fluid into the oral cavity.

40. CONCLUSION
– A POC test to detect aMMP-8 in patients with gingivitis and periodontitis before and
after treatment had proven to agree strongly with the standard method, ELISA.
– The test can be recommended for use in dental offices to support the clinical diagnosis.
– In addition, positive aMMP-8 test results in untreated patients are related to more
plaque and inflammation.
– Moreover, the test may enable medical physicians without specific knowledge and skills
in dentistry, for example medical specialists to detect patients that could be at risk for
periodontitis.