Microbiological Investigation of Osteo-articular infections

1. Microbiological Investigations of the
joint and spine infections
Dr. Mahen Kothalawala MBBS, Dip in Microbiology, MD, MPH(NZ)
Consultant Clinical Microbiologist
Teaching Hospital Kandy

2. • Diagnostic aspect of of SA
• TB arthritis and TB spine
• PCR based molecular assays in finding
aetiology of osteo- artricular infections

3. A. Diagnosis of SA

4. Diagnosis of SA
• SA- Separate entity
• Purulent invasion of a joint/joints by infectious agent
– Heamatogenous or Post surgical/traumatic
• Only type of arthritis amenable to treatment with
Antibiotics
• Failure to prompt early aggressive therapy → lead to
– joint destruction
– septicemia/death

5. Diagnosis of SA - challenging
• Clinical signs and symptoms → poor sensitivity
and specificity
• Children – usually present with classical symptoms&
signs
• Adults/Pts with existing joint disorders - symptoms&
signs
• Lab based test-very essential

6. Diagnosis of SA – challenging….
• Made on Clinical Suspicion and confirmed
with laboratory tests
• Two types of tests are employed
1. Synovial Fluid Analysis
2. Routine blood tests/ tests for Inflammatory
markers

7. 1. Synovial Fluid Analysis
• If properly performed provide valuable
information to evaluate for Joint Disorders and
guide treatment

8. Specimen collection and transportation
• Specimen – Joint Fluid
• Container –
– Sterile Tube with Anti-coagulant (Clot interfere with
counts)
– Blood Culture Bottle
• Contact lab and request for early report

9. Specimen collection and transportation cont..
• Immediately dispatch for processing and culturing.
• Directly inoculating in to culture bottles, ↑ sensitivity of
detecting organisms
– Meticulous sterile technique is required to ↓ False Positives
• In clinical suspicion of SA, culture of synovial membrane ↑
isolation rates
• Concurrent specimens for Ix,
– Sputum,
– urine, and
– blood cultures required

10. Joint Fluid Analysis….. chemical analysis
Component in
Jt fluid
Normal Value Septic Episode likely with Comment
Glucose 80% of that of
Plasma
(Average of 10mg/dl
lower than plasma)
< 40mg
Greater than 20mg/dL drop
suggestive of infection
Likely-hood of SA is
higher with lower
values
Protein Usually within 25%
of the Plasma
glucose
1 to 3g/dL
> 3g/dL in SA
High protein levels seen in
•anky spond,
•SA,
•Gout,
•Reiters,
•Psoriatic Arthropathy
All types of plasma
proteins present in Jt
fluid
Lactate
dehydrogenase
>333 IU/L or >10mg/L May increase in
Rheumatoid Arthritis,
Infectious Arthritis
and in Gout

11. WBC Breakpoints
Cut off level Differential Interpretation
Up to 150/ml Mostly Mono nuclear Normal Cell Count
< 2000 /ml Unlikely to be of inflammatory origin
> 2000 /ml Likely to be inflammatory
< 5000 /ml with RBCS in
fluid
Likely to be following traumatic
5000 to 15,000/ml <25% are PMN Toxic Synovitis
10000- to 15000/ml Nearly 50% are PMN Acute Rheumatic Fever/ Early SA/ GC
> 50,000 /ml Nearly 75% PMN Likely to be septic arthritis
> 100,000 /l > 90% Septic Arthritis Likely
Low total count with
>90% DC
gout, pseudogout, acute rheumatic
fever, Reiter's disease, and
RA

12. Gram Stain and Culture
• Gram stains → Confirms septic arthritis.
• Helps to initiate therapy on empiric basis (Before AST
available)
• Sensitivity → from 29% to 50%
– Gram positive pathogens 50 to 75%
– Sensitivity for Gram Negative organisms –less
– Sensitivity for GC- <30%
• Specificity 100%
• Sensitivity of Culture – 82% - Higher for NON GC septic
arthritis

13. 2. Blood analysis
• Blood culture (Sensitivity 50%)
• Inflammatory markers (Supports ∆ of SA)
– CRP,
– Pro-calcitonin
• ESR and Full count

14. Detection of Septic insults to human body –
Which inflammatory marker is better?
False Positives
21/04/2013
Receiver observer characteristics
True positives

15. 21/04/2013

16. Joint WBC count(area under the
curve (AUC) 0.75, 95% confidence
interval (CI) 0.58 to 0.92),
ESR
WBC(AUC 0.69, 95% CI 0.57 to
0.80)

17. Causative organisms of SA
• Virtually all bacterial organism has been
reported
• Type of Organism largely dependent on host
factors
• Role of exotic organisms- Atypical Mycos,
Fungi emerged due to immuno-supression
and prosthetic implants

18. Organism Remarks
Staphylococcus aureus most frequent organism in all ages and risk groups except > 2years
Incidence of MRSA on the rise - >25% of cases are due to MRSA
37%-56%
of All
Streptooccus sp Streptococcus pyogenes most common, usually after a trauma, procedure
or Secondary to chronic skin condition
Group B – specially in elderly with chronic diseases such as DM,Cirrhosis,
CRF etc
Group C and Pneumococci- Less frequent
Gram Negatives Rods Escherichia coli, Proteus mirabilis, Klebsiella and Enterobacter - Rare
Mainly in very young and in elderly with co-morbid factors
at least
20%
Anerobes a history of diabetes, joint prosthesis implantation or following
penetrating trauma
Rare
causes
HIV associated
- SA, S pneumonia, Myco
and fungal sp
Are common causes, But difficult to diagnose
Gram Negative Cocci and
rods
N. gonorrhoeae and N. Meningitidis H. influenzae is uncommon in the
adult population, commonest <2yrs
20%
Causative organisms of SA- Adult SA

19. Causative organisms-pediatric age group
Age 5 years to 12
years
Major Pathogen MSSA
and MRSA
As in adults
Age 2 months to 5
years
methicillin-sensitive S.
aureus and MRSA
Major cause of septic arthritis
Streptococcus
pneumonia
Significant reduction of SA due to Strep noted
recently due to vaccination, but considerable
number of cases may be due to Strep. Pneumo
types which were not covered by vaccination
Haemophilus
influenzae
Kingella Kingae
in children between the ages of 2 months and
5 years major cause of gram negative organism
is K.kingi, may be due to HIB vaccination
Infants <2 months age S. aureus, Still major pathogen is Staph. aureus
S. agalactiae and
gram-negative enteric
bacteria.

20. Synovial Fluid
• Request for - Three “C”s
– Cell count
– Crystals &
– Culture
• Gram Stain

21. Infections of the spine
• History and Examination
• Imaging for evidence of infection at site and imaging
for possible primary site (Infective endocarditis etc)
• Specimens-
– Blood
– Needle aspiration
– Bone biopsy
– IV Disc Biopsy

22. B. Diagnosis of TB arthritis and TB spine

23. B. TB arthritis and TB spine
• infect the musculoskeletal system(3% cases)
Commonest area – Spine
• Patho-physiology
– Hematogenous dissemination to long bones, spine
– Direct spread to bone → adjacent tuberculous
lymphadenitis
• Forms- caseating granulomatous inflammation
with bone necrosis - PATHLOGIC HALLMARK

24. Diagnosis of TB arthritis
• TB SA and spine are pauci-bacillary, Smears for
(AFB often negative) Typically, 104 – 106 organisms/ml
necessary for AFB positivity
• Classical constitutional symptoms rare finding
• Tuberculin skin testing (TST) and interferon-
gamma release assays (IGRA) are adjunctive
diagnostic tools.

25. Diagnosis
• Blood tests – ESR (usually >100mm/hr,
WBC/DC usually lymphocytes
• Sputum and urine for AFB to exclude
concurrent involvement of other sites (50% of
cases Positive)
• Synovial fluid culture for TB →gold standard
test

26. Synovial Biopsy for AFB and culture
• If performed correctly→ Higher yield for AFB
(TB prefers synovium)
• Culture – Gold standard- take up to 8/52
• Synovial Fluid PCR –Rapid results

27. TB arthritis
• 70% percent to 90% of patients may have a
positive TST.
• Similar numbers – Quantiferone gold +
• 50% have abnormalities on chest x-ray
consistent with TB

28. Establishment of Diagnosis of TB arthritis
• A high level of suspicion required with risk
factor identification
• Specimens for microbiology and histology.
• Gold Standard – by culture demonstration &
AST.
– Delay of up to 8 to 10 weeks
– Delays in diagnosis and initiation of therapy are
associated with increased mortality.

29. Role of Synovial Biopsy
• Specimen of choice to diagnose TB arthritis.
• Biopsy may yield culture positive in 90% to
95%-can be performed if the diagnosis of TB arthritis remains in
question
• Synovial fluid AFB smear is positive in <20%
but culture may be positive in up to 80%.

30. Diagnosis of TB arthritis using Adenosine Deaminase
levels in serum and synovial fluid
• ADA – Level reflects ↑ activity of monocyte
and lymphocyte stimulation
• Which occur in TB arthritis, Theoretically ADA
should increase in TB A

31. Serum ADA Levels
• ADA levels high → in inflammatory arthritis
• Can differentiate inflammatory from non
inflammatory arthritis
• High serum ADA reported in
– RA,
– crystal-induced arthritis
– septic arthritis

32. Synovial fluid ADA level
• High in all inflammatory arthritis
• But not as high as in TB arthritis (Zamani et al, 2010)

33. A synovial fluid ADA level of 31 U/l gave a sensitivity of 83.3% (95%CI 35
99.6) and a specificity of 96.7% (95%CI 82.8-99.9) with a Kappa of agreem
of 0.8 (p<0.001).

34. ADA in TB arthritis
• With reference to the gold standard test,
– Sensitivity> 95% &
– specificity > 80%,
• Synovial ADA might be an alternative tool for
the early diagnosis of TB arthritis

35. • C. PCR based Molecular assays in finding
aetiology of osteo- artricular diseases

36. PCR
• Multiplication of genetic material using series
of steps and detecting them subsequently
• Has very high sensitivity
• Chances of Contamination is high- need to
follow strict protocols
• Two method
– Conventional and Real time

37. PCR based tests
• IS useful in many fastidious, slow-growing or
uncultured bacteria,
• used when an empirical antibiotic treatment
has already been initiated.
• Two steps –
– Broad Base Primer to identify Bacterial etiology
– Specific primer- to identify specific pathogens

38. RT-PCR
• RT-PCR easier to interpret and allowed to
detect fours time more cases than
conventional PCR.

39. Specimens for PCR
• All specimens prior to commencement of
therapy – Can use
– Biopsy specimens
– Synovial biopsies
– Fine needle aspirates under guidance
Containers- Plain bottles, If anticoagulant necessary
use only heparin
Contact the service provider to obtain containers

40. Molecular diagnostic methods for TB
arthritis
• HSP 65 – 65 kDa gene which is highly
conserved and specific for Mycobacteriae
• Advantage of PCR based tests
– Prevent mis-identification as in phenotypic
analysis
– Early rapid diagnosis
– Not subject to false negative results
– Identification of unusual organisms

41. • Thank you