Seropositive test,When to treat?

Prof. Khaled Al-JarallahConsultant Internist, RheumatologistFRCPC, FACR, FACP, FRCP

Disclosures

I have no conflict of interest to declare

References

• Clinical Reasoning Series , Rahul patwari, 2017.

• Medow MA, Lucey CR.BMJ Evidence-Based Medicine. A qualitative approach to Bayes theorem. 2011;16:163-167.

• Schur Peter, Laboratory testing for Diagnosis, Management of Patients with Rheumatic Disease. The Rheumatologist, Dec 1,2014.

Referral Scenarios

• 48-year-old female referred from polyclinic

with 2 weeks history of generalized body ache & +RF 1:80

R/O Rheumatoid arthritis?

• Asymptomatic 25-year-old female, referred from polyclinic after employment medical check-up with lab tests showing ANA 1: 640

• R/O SLE ?

Lab test

❑ For informing us of an emerging disease

❑ Diagnose a specific disease

❑ Predict prognosis

❑ Biomarker of disease

❑Monitor of disease activity

!!

Why was the test requested?

Good referral

❑ 34-year-old female

❑ 12 week history of fatigue, joints pain&swelling in both hands.

Sensitivity, Specificity

Autoantibody positivity alone does not make a diagnosis

Similarly, antibody negativity does not exclude a diagnosis

❑If highly sensitive, diagnosis can be excluded in case ofnegativity

❑If test is highly specific, diagnosis confirmed in case of positivity

Pre and Post test probability Birtane M. et al 2017

The likelihood nomogram used in SLE with an antinuclear antibody test

Categorical probabilitiesA qualitative approach to Bayes theorem

Categorical probability Numerical probability

Very unlikely Less likely than 10%

Unlikely Between 10% and 33%

Uncertain Between 34% and 66%

Likely Between 67% and 90%

Very likely More likely than 90%

Catherine R Lucey,

Medow MA, Lucey CR.BMJ Evidence-Based Medicine 2011;16:163-167

Clinical reasoning approach

• Construct patient script (data gathering)

• Construct disease script (disease population studies)

• Match= treat

• Mismatch= rule-out

• Uncertain= test

Clinical Reasoning , Rahul patwari, 2017

Clinical reasoning approach

Catherine R Lucey,

Medow MA, Lucey CR.BMJ Evidence-Based Medicine 2011;16:163-167

Clinical Reasoning , Rahul patwari, 2017

Clinical Reasoning , Rahul patwari, 2017

Clinical Reasoning , Rahul patwari, 2017

Lupus in Arab world - disease script

Adwan M, Arch Rheumatol 2018;33(4);455-463

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Frequency of autoantibodies in Arab patients with systemic lupus erythematosus

Adwan M, Arch Rheumatol 2018;33(4);455-463

Lupus in Arab world – disease script

Frequency of systemic lupus erythematosus manifestations in Arab world.

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Adwan M, Arch Rheumatol 2018;33(4);455-463

Lupus in Arab world – disease script

Traditional approach to lab study

❑Why was the test requested? Diagnostic

❑What was the lab test method? Methodology

❑What was the lab results? Error in interpretation

Critical interpretation of serology test

❑ Sensitivity (proportion of patients with the targetdisorder who have positive test)

❑ Specificity (proportion of patients who are free of thetarget disorder who have negative or normal test)

❑ Positive and negative predictive value (based onpretest probability)

❑ Look for high Sensitive & high Specific diagnostic test!

History- Progress in Rheumatology Serology

❑ 1940s (RF and LE cell perp)

❑ 1950s (ANA and anti- DNA)

❑ 1960s (Sm, RNP, Ro, La)

❑ 1982 (ANCA)

❑ 1990s (Anti-CCP)

Hospitals covered by FOM immunology lab

• Mubarak Hospital

• Amiri Hospital

• Jaber Hospital

• Adan Hospital

• Ahmadi Hospital

• Military Hospital

Current methods used in FOM immunology Lab

❑ CRP : Nephelometry

❑ RF : Nephelometry

❑ Anti CCP 1, 2 : Enzyme-linked immune sorbent assay (ELISA)

❑ ANA : Immunofluorescence (IF)

❑ Anti dsDNA : ELISA

❑ ENA: Immunoblot Assays

❑ ANCA : Immunofluorescence (IF) ; MPO: ELISA, PR3: ELISA

❑ Complements(C3,C4) : Nephelometry

Rheumatoid Factor (RF)

❑What is it? RA and related diseases causes the production of globulin known as RF which is an autoantibody directed against the Fc portion of IgG, can be IgM , IgA, IgG, IgE , IgD

❑ That antibody binds to normal circulating IgG, forming immune complexes that are deposited in the joints which leads to inflammation of the joints

Rheumatoid Factor (RF)

❑ Positive in ~ 70% RA patients

❑ High RF indicator of worse prognosis. Also show aggressive,erosive joint disease, rheumatoid nodules, extra articularinvolvement

❑ Positive also in Sjogren’s syndrome, SLE, cryoglobulinemia,interstitial fibrosis, malignancy, various infectious disease

❑ Low titrations seen in 5% healthy population

❑ RF not used for monitoring treatment response and disease

Principle of the RF test

❑ RF anti-antibody can be detected in thelaboratory by its ability to bind and formclumps with latex particles or red blood cells(Rose-Waaler test) that contain humanImmunoglobulin G (IgG).

❑ If the RF is present in the patient’s blood it attaches to the IgG coating the latex particles causing clumps.

❑ Agglutination is considered a positive reaction that indicates the presence of rheumatoid factor at a detectable level. Shows if test is Positive or negative

Principle of the RF test

❑ Nephelometry technique is used in clinical laboratories forqualitative assessment of RF. It is relatively easily automated.

❑ In nephelometry, levels of several blood plasma proteins is madeby measuring the light passed through the sample.

IMMage Machine, used in FOM immunology lab for Nephylometry.

Anti-Cyclic citrullinated peptide antibodies(anti-CCP)

❑ Anti-CCP are autoantibodies produced by the immune system that are directed against cyclic citrullinated peptides (CCP).Changes happen in structure of CCP which make them a target for IgG antibodies in RA

❑ Detected using ELISA

❑ Anti CCP has higher specificity than RF

❑ Anti-CCP1(96% specificity, 53% sensitivity for RA); Anti-CCP2(99%specificity, 61.6% sensitivity for RA)

❑ Occurs years before development of clinical symptoms of RA;Associated with aggressive and erosive disease

Anti-CCP

• Early assays (ELISA) had sensitivity of 67% and specificity of 95% (compared to 69% and 85% for RF). Later generation assays are even better.

• However no test is perfect

• False positives seen in active TB, Sjogren’s, SLE, scleroderma, and poly-and dermatomyositis.

• In these cases, however, titers are lower than those seen in RA

True Positive or False Positive

RF & Anti-CCP in Rheumatic & Other Diseases

Schur P H, The Rheumatologists, 2014

Autoantibodies to nuclear antigens

❑ Antibodies developing against DNA, RNA, histones,centromeres, nucleolus and other nucleoproteins incell nucleus

❑ High sensitivity, low specificity

❑ High titration does not correlate with disease activityor severity, so not used for monitoring disease activity

Antinuclear Antibodies (ANA)

Sensitivity of ANA in Autoimmune and Non Rheumatic Disease

Schur P H, The Rheumatologist,2014

Autoantibodies to nuclear antigens

❑ ANA measured in 2 ways

❑ Generic ANA measurement completed with Immunofluorescence (IF) and Enzyme-linked immune sorbentassay (ELISA)

❑ If ANA positive, specific antibodies detected with automatedmethods

❑ ANA staining patterns has been recognized to have a lowsensitivity and specificity for different autoimmune disorders.

Common immunofluorescence antinuclear antibodies associated with

specific diseases

ANA Disease AssociationsSensitivity Specificity of Antinuclear antibodies

Schur P H, The Rheumatologist, 2014

Anti-dsDNA antibodies

• Anti-dsDNA antibodies are used in the evaluating and managing patients with SLE.

• Anti-dsDNA antibodies are of primary importance in the pathogenesis and disease activity i.e. lupus nephritis

Anti-dsDNA antibodies Disease associations

• Anti-dsDNA antibodies was reported in patients with otherdisorders, including rheumatoid arthritis, Sjögren's syndrome,scleroderma, overlap connective tissue disease, myositis,uveitis, juvenile arthritis, antiphospholipid syndrome, Grave'sdisease, autoimmune hepatitis, infections and lymphoma.

• Anti-dsDNA antibodies have been reported in patients treated with minocycline, etanercept, and infliximab.

Extractable Nuclear Antigens (ENA)

❑ Over 100 different soluble cytoplasmic and nuclearantigens

❑ Example: Ro, La, Sm, RNP, Scl-70 and Jo1

❑ Detected by immunoblotting techniques

Machine used in clinical lab for Immunoblotting.

Anti-neutrophil cytoplasmic antibodies (ANCA)

❑ ANCA supportive in diagnosis ofvasculitic conditions

❑ 2 forms of Immunofluorescence (IF)patterns

❑Measured using ELISA

cANCA (PR3-ANCA), proteinase 3-ANCA,sensitivity 90%, specificity 50% seen inWegner’s granulomatosis(WG)

pANCA (MPO-ANCA), myeloperoxidaseANCA seen in immuneglomerulonephritis, microscopicpolyangitis, Churg-Strauss syndrome,sometimes in WG

• A58-year-old woman with a history of cervical spondylosis andhypothyroidism presented with a 2-week history of joint pain.• She noted pain and swelling in her right knee, which migrated tothe right ankle and then affected the MCP and PIP joints of herfingers.• About 2weeks before her symptoms began, her 5-year-oldgrandson had a febrile illness characterized by headaches, bodyaches, malaise, and rash on the cheeks, trunk, and extremities butno arthritis• Manifestations in all of the family members resolvedspontaneously within 1week

Physical Examination

• Patient was afebrile with normal vital signs.

• A malar rash was present

• Had swelling and tenderness of the MCP and PIP joints and had difficulty making a fist and fully extending her fingers.

JAMA Clinical Challenge case

Laboratory tests

• Mild normochromic normocytic anemia with ahemoglobin level of 11.5 g/dL , normal white blood celland platelet counts, and normal biochemistry

• She had a positive ANA

• (1:160[homogeneous pattern; negative <1:80 serumdilution]).

• Testing for rheumatoid factor was negative.

• Levels of inflammatory markers were normal.

What would you do next?

A . Check anti-dsDNA antibodies

B . Order anti CCP antibody

C . Perform bilateral hand ultrasound

D . Check anti-Smith antibodies

E . Order serologic testing for parvovirus

Serology positive for:• Parvovirus B19–specific IgM antibodies

Final diagnosis:• Parvovirus-associated arthritis

Diagnostic test

• 25-year-old woman was admitted with history of a fever and polyarthritis for 2 months.

• H/o tonsillectomy of recurrent tonsillitis at age of 9 years, for positive throat cultures for beta-haemolytic group A streptococci

• At the age of 16 years a heart murmur was found and at the time considered innocent

• H/o irritability, malaise and fatigue, and AM stiffness for 30 min

Physical examination

• Look ill , sweaty, pale, no clubbing.

• Right ankle, both knees and DIP joints were tender on pressure ,Swelling , and redness.

• Heart examination revealed sinus tachycardia, a third heart sound, a grade 2/6 holodiastolic murmur over the aortic area and a grade 3/6 systolic murmur apex which radiated to the axilla.

What would you do next?

A. Order Rheumatoid factor test

B. Order ANA test

C. Order Blood Culture & Echocardiogram

D. Check ESR

E. Give NSAIDs

Diagnostic tests

• Urinalysis: slight proteinuria and microscopic hematuria (5-15 erythrocytes)

• ESR 70 mm in the first hour

• Antistreptolysin O (ASO) titer of 333 Todd units

• Positive latex test for rheumatoid factor(+160)

• ANA + (1:80) ELISA

• Mild normocytic anemia

• Blood cultures Enterococcus faecalis.

• Echocardiography showed valve vegetation's

MCQ

A 55 years old housewife referred from her GP with pain in her hands, shoulders and knees for the last one year. She felt stiff when she tried to get up for the first half an hour. Systemic review were negative. No skin rash or alopecia. Examination revealed swelling in the PIP and DIP joints as well as tenderness of both shoulders and knees. Lab. test ordered by her GP showed Rheumatoid factor + 1:20 , ESR of 35mm/h, ANA+ 1:80 fine speckled pattern.

What would be your diagnosis?

A. Rheumatoid arthritis

B. Systemic lupus erythematosus

C. Osteoarthritis

D. Polymyalgia Rheumatica

E. Not certain

MCQ

A 45 years old women presented in the OPD with fatigue& intermittent headaches for the last two years. She experienced generalized body aches and sleep disturbance in the last one year. No history of morning stiffness, alopecia, skin rash. Systemic review were unremarkable. Examination revealed generalized body tenderness but no arthritis or muscle weakness. Her GP told the patient that her blood tested positive for autoimmune disease. RF+ 1:40 , ANA + 1:160 homogenous & rest of the blood tests were normal.

What would be your likely diagnosis?

A. SLE

B. Fibromyalgia

C. Rheumatoid arthritis

D. Myopathy

E. Myasthenia gravis

Referral Scenarios

• 48-year-old female referred from polyclinic

with 2 weeks of generalized body ache & + RF 1:80

Post viral infection

• Asymptomatic 25-year-old female, referred from polyclinic after employment medical check-up with lab tests showing ANA 1: 640

Healthy asymptomatic

Low pretest probability ( very unlikely ).

Take Home Massage

• Physician must first evaluate the patient clinically and then request appropriate diagnostic tests.

• Test selection should be guided by clinical impression.

• Test interpretation require knowledge of the diagnostic power of each test.

• Good diagnostic test needs to discriminate between the diseased and the healthy state; a screening tool needs high sensitivity, while testing for a very rare condition requires high specificity.

• Reaching treatment threshold decision is the result of disease script matching illness script with positive diagnostic test.