microbiological investigation of osteo-articular infections

Microbiological Investigations of the joint and spine infections

Dr. Mahen Kothalawala MBBS, Dip in Microbiology, MD, MPH(NZ)Consultant Clinical Microbiologist

Teaching Hospital Kandy

• Diagnostic aspect of of SA

• TB arthritis and TB spine

• PCR based molecular assays in finding aetiology of osteo- artricular infections

A. Diagnosis of SA

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

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

Diagnosis of SA – challenging….

• Made on Clinical Suspicion and confirmed with laboratory tests

• Two types of tests are employed1. Synovial Fluid Analysis

2. Routine blood tests/ tests for Inflammatory markers

1. Synovial Fluid Analysis

• If properly performed provide valuable information to evaluate for Joint Disorders and guide treatment

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

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

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 glucose1 to 3g/dL

> 3g/dL in SAHigh 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

WBC BreakpointsCut 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

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

2. Blood analysis

• Blood culture (Sensitivity 50%)

• Inflammatory markers (Supports ∆ of SA)– CRP, – Pro-calcitonin

• ESR and Full count

04/12/2023

Detection of Septic insults to human body –Which inflammatory marker is better?

False Positives

Receiver observer characteristicsTrue positives

04/12/2023

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)

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

Organism RemarksStaphylococcus aureus most frequent organism in all ages and risk groups except > 2years

Incidence of MRSA on the rise - >25% of cases are due to MRSA37%-56% of All

Streptooccus sp Streptococcus pyogenes most common, usually after a trauma, procedure or Secondary to chronic skin conditionGroup B – specially in elderly with chronic diseases such as DM,Cirrhosis, CRF etcGroup 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

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 entericbacteria.

Synovial Fluid

• Request for - Three “C”s– Cell count– Crystals &– Culture

• Gram Stain

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

B. Diagnosis of TB arthritis and TB spine

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

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.

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

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

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

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.

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%.

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

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

Synovial fluid ADA level

• High in all inflammatory arthritis

• But not as high as in TB arthritis (Zamani et al, 2010)

A synovial fluid ADA level of 31 U/l gave a sensitivity of 83.3% (95%CI 35.9-99.6) and a specificity of 96.7%

(95%CI 82.8-99.9) with a Kappa of agreement of 0.8 (p<0.001).

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

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

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

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

RT-PCR

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

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

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

• Thank you