Refractory periodontitis refers to chronic periodontal disease that responds poorly to conventional treatment such as scaling and root planing. About 10-15% of patients have refractory periodontitis. Several studies examined clinical, microbiological, and immunological parameters to better diagnose and treat refractory periodontitis. One study found that levels of certain bacterial species, percentage of sites with deep pockets, and number of bacterial species with high antibody levels could predict refractory cases. Another study found elevated antibody levels to specific bacteria correlated with refractory cases. Molecular studies identified higher expression of certain genes involved in inflammation and bone resorption in refractory patients. Microarray analysis found refractory patients had persistent pathogenic bacteria after treatment. Combin

1. REFRACTORY
PERIODONTITIS
Photo courtesy of: Dr. Darika
Saitawee

2. Refractory periodontitis
 periodontal disease is a chronic infectious
disease of the supporting tissue of the teeth
 Nowadays, a large majority of patients with
periodontitis respond well to conventional
therapies
 However, a small percentage of patients
respond poorly of treatment
“ Refractory periodontitis ”

3.  Characteristic of RP (Haffajee et al, 2004; Colombo et al,
2012)
 Poor responder or refractory of treatment
 AL and/or >3 sites
 AL >2.5 mm from the baseline visit to any
monitoring visit 1 year post-therapy
Refractory periodontitis
Photo courtesy of: Dr. Darika
Saitawee

4.  About 10–15% of US adults are ‘refractory’ to
therapy for chronic periodontitis
 Recently, studies suggest that these patients
have investigation was to identify of this
disease
“ proper treatment “
Refractory periodontitis

5.  Clinical and Laboratory
 Antibody response
 Real-Time Quantitative
Polymerase Chain
Reaction
 Human Oral Microbe
Identification
Microarray
Diagnosis for refractory
periodontitis
http://www.bio-rad.com/en-us/applications-technologies/what-real-time-pcr-
qpcr

6. Clinical and laboratory
parameters
 Colombo et al. 1999
 investigation was to use baseline clinical and
laboratory parameters to distinguish subjects
refractory to conventional periodontal therapy
 Baseline clinical
 microbial
 host parameters

7.  27 refractory subjects
 poor response after both SRP and surgery with systemic
tetracycline
Clinical and laboratory
parameters
clinic
• Att gain & no site
with new att loss
• Gingival redness
• BOP
• Suppuration
• Supragingival
plaque
accumulation
• PD
• AL
microbial
• Level of 40
subgingival texa
(subging sample)
• Checker board
DNA-DNA
hybridization
Immune
• Serum Ab to 85
subging species
(IgG)
• Checker board
immunoblotting

8. Clinical and laboratory
parameters

9. Clinical and laboratory
parameters

10. Clinical and laboratory
parameters
Ab↑-odd↑  Refractory

11.  selected by stepwise discriminant analysis:
 namely, number of species exhibiting serum
antibody ±50 mg/ml
 % of S. constellatus in plaque (similar previous study
Colombo 1998)
 % of sites with attachment level ±6 mm
Clinical and laboratory
parameters

12. Clinical and laboratory
parameters
 Discriminant analysis using these variables provided
 Sensitivity 0.66
 Specificity 0.92
 Positive predictive value 0.80
 Negative predictive values 0.85

13.  The relatively high sensitivities, specificities
and predictive values of the clinical,
microbiologic and immunologic variables
 these tests may distinguish a relatively
common form of refractory disease
 Refractory periodontitis subjects could be
distinguished using a subset of clinical,
microbiological and immunological parameters
Clinical and laboratory
parameters

14. Antibody-based diagnostic
Levine et al. 2002
 this study was to determine whether an
elevated IgG Ab response to lysine
decarboxylase, alone or with antibody to
bacterial Ag and baseline clinical
measurements  predict the ‘refractory’
patients
 Gingival microbiota were found to inhibit the
growth of cultured mammalian cells by
depleting them of “lysine”

15.  Eikenella corrodens and Capnocytophaga spp.
 Are major sources of the responsible enzyme,
lysine decarboxylase
 Lysine decarboxylase  Lysine
 inhibit the turnover of the most coronally situated,
dentally attached (DAT) cells and JE basal cells
that lie remote from capillaries
 cells stop proliferating  causing the intermediate
layer of JE to become attenuated and weaken the
epithelial barrier
Antibody-based diagnostic

16. Lysine
decarboxylase
activity
Effect of DAT cell
 High - Inhibit DAT cell turnover
 Low - DAT cell can turnover
- Maintaining oral hygiene and
- Low levels of bacteria in the
sulcus or pocket (retard accumulation)
- Stop attachment loss
Antibody-based diagnostic

17.  IgG antibody contents to a purified antigen
from
 Actinomyces spp. (A-Ab)
 Streptococcal d-alanyl glycerol lipoteichoic acid
(S-Ab)
  related in ‘refractory’ patients
Antibody-based diagnostic

18. Antibody-based diagnostic
↑
↑

19. Antibody-based diagnostic
*
*

20. Antibody-based diagnostic
E. Corrodens : major
sources lysine
decarboxylase
Similar of Colombo (1999)
study

21. Antibody-based diagnostic
A strong correlation between A-Ab and S-Ab
responses was noted in ‘refractory’

22. Antibody-based diagnostic
-Prediction of
‘refractory’ response
 moderate
periodontitis, 2–
4mm mean att loss at
baseline
-Sensitivity 86.4%
-Specificity 86.7%

23.  Subjects with advanced disease need for a
diagnostic test  the immunoassay is
proposed for use in subjects with moderate
periodontitis (mean attachment loss level of
2–4mm)
 HKL-Ab facilitated an accurate prediction of
therapeutic outcome in subjects with moderate
periodontitis
Antibody-based diagnostic

24. Real-Time Quantitative
Polymerase Chain Reaction
 Marconcini et al. 2011
 “Genetic risk factors” were proposed to influence
the natural history of periodontitis
 The study use this technology  evaluate the
expression levels of leader genes in the
leukocytes of refractory pt
 Blood samples

25.  PCR efficiencies were calculated with a relative
standard curve derived from a five cDNA dilution
series in triplicate
 The standard curves were obtained using
1. glyceraldehyde-3-phosphate dehydrogenase
(GAPDH)
2. growth factor bound protein (GRB2)
3. casitas B-lineage lymphoma (CBL)
4. nuclear factorKB1 (NFKB1)
5. REL-A (gene for transcription factor p65)
Real-Time Quantitative
Polymerase Chain Reaction

26. Real-Time Quantitative
Polymerase Chain Reaction
Results : levels of CBL
and GRB2 were statically
significant in patients with
periodontitis compared to
healthy patients
characteristics of GRB2,
CBL and NFKB1 : a
specific role during
inflammation and process
of bone resorption

27.  CBL oncogene  part of a transforming retrovirus
 GRB2  signal transduction pathwayThis gene has
never been associated with periodontitis
 Molecular studies, our hypothesis
 CBL played during the phase of bone resorption that can be
supposed to be the acute phase of the disease
 GRB2 might play a central role during the inflammation
process that gave the onset at the periodontitis acute phase
 Genes are important role in refractory chronic
periodontitis
 develop a treatment plan
 reduce risk factors and to focus of therapy
Real-Time Quantitative
Polymerase Chain Reaction

28. Human Oral Microbe
Identification Microarray
(HOMIM)
 Colombo et al. 2012
investigation evaluated the post-therapy
changes on the subgingival microbiota
of periodontitis patients who were RP or
GR as measured by using the HOMIM
technique
Tx: SRP + OHI + MWF + ABT (Amoxy
500 mg+Metro 250 mg)

29.  The HOMIM methodology used a total of 400 16S
rRNA-based, reverse-capture oligonucleotide probes
targeting >300 bacterial taxa
 16S rRNA genes were PCR amplified from DNA
extracts
 labeled 16S amplicons were hybridized overnight to
probes on the slides
 After washing, the microarray slides were scanned
and crude data were extracted using software for
microarray image analysis
Human Oral Microbe
Identification Microarray
(HOMIM)

30. Human Oral Microbe
Identification Microarray
(HOMIM)
*
*
*

31. Human Oral Microbe
Identification Microarray
(HOMIM)

32.  The majority of species evaluated decreased in
prevalence in both groups after treatment
 Species that increased or persisted in high frequency in
RP but were significantly reduced in GR included
Human Oral Microbe
Identification Microarray
(HOMIM)
Bacteroidetes sp.
Porphyromonas
endodontalis
Porphyromonas
gingivalis
Prevotella spp.
Tannerella forsythia
Dialister spp.
Selenomonas spp.
Catonella morbi
Eubacterium spp.
Filifactor alocis
Parvimonas micra
Peptostreptococcus sp.
OT113
Fusobacterium sp. OT203
Pseudoramibacter
alactolyticus
Streptococcus intermedius
/Streptococcus
constellatus
Shuttlesworthia satelles

33.  In contrast
 Capnocytophaga sputigena, Cardiobacterium
hominis, Gemella haemolysans, Haemophilus
parainfluenzae, Kingella oralis, Lautropia
mirabilis, Neisseria elongata, Rothia
dentocariosa, Streptococcus australis, and
Veillonella spp.
  associated with therapeutic success
Human Oral Microbe
Identification Microarray
(HOMIM)

34.  GR patients
 able to maintain low frequency and
proportions of these organisms
 RP patients
 rapidly colonized by these species
 incapable of maintaining low levels of
putative periodontal pathogens attributable to
host impairment and/or colonization by a more
virulent periodontal microbiota
Human Oral Microbe
Identification Microarray
(HOMIM)

35.  Haffajee et al. 2004
 Clinical and microbiological changes
associated with the use of combined
antimicrobial therapies to treat ‘‘refractory’’
periodontitis 
 Combine therapies
 SRP
 periodontal surgery
 locally delivered tetracycline at pockets ≥ 4 mm
 systemically administered amoxicillin (500 mg, t.i.d. for 14
days) metronidazole (250 mg, t.i.d. for 14 days)
 professional removal of supragingival plaque weekly
for 3 months
 f/u every 3 months post-therapy for 2 years
Treatment of refractory
periodontitis

36. Combined antimicrobial
therapies to treat ‘‘refractory’’
periodontitis
- 3 mo. plaque, gingival redness, BOP, suppuration
& PD ↓
- 6-24 mo.  AL improved

37. Combined antimicrobial
therapies to treat ‘‘refractory’’
periodontitis
members of the green, orange and red complexes as well as some of the
species in the other category were significantly elevated in the periodontitis
subjects compared with the refractory subjects

38. Combined antimicrobial
therapies to treat ‘‘refractory’’
periodontitis
- No sig this time
- Low level of

39. Combined antimicrobial
therapies to treat ‘‘refractory’’
periodontitis
3 mo Actinos & orange

40. Combined antimicrobial
therapies to treat ‘‘refractory’’
periodontitis
Mean count of

41.  conventional periodontal therapies could be
successfully treated using a combination of
mechanical and antimicrobial therapies
 combine therapies that were known to affect the
subgingival microbiota and provide a beneficial
clinical response (synergistic effects)
 Mean PD reduction was 0.83 ± 0.13 mm
 Mean attachment level gain was 0.44 ±0.12 mm
Combined antimicrobial
therapies to treat ‘‘refractory’’
periodontitis

42.  SRP  lower the biomass on the tooth surface
and PD
 Local drug delivery  directly diminish the
pathogen load in any residual pockets of ≥4 mm
 Systemically administered amoxicillin plus
metronidazole  rapidly lower the level of
pathogens throughout the oral cavity/poor
clinical access, and to attempt to control
pathogenic species that might have entered the
host’s tissues
 Repeated professional supragingival plaque
Combined antimicrobial
therapies to treat ‘‘refractory’’
periodontitis

43. conclusion
 The combined antibacterial therapy was
successful in controlling disease progression in
refractory periodontitis
 Proposed reasons for ‘‘refractory’’ disease have
included differences in the
 subgingival microbiota
 host response
 Environmental factors (smoking)
 All of these hypotheses were presented with the
understanding that proper home care procedures
and treatment of refractory patients