ROLE OF CRP FOR SCREENING RESPIRATORY TRACT …ROLE OF CRP FOR SCREENING RESPIRATORY TRACT INFECTION Prof. Dr. Aryati, dr., MS, SpPK(K) Ketua Umum Pengurus Pusat Perhimpunan Dokter

Rabu, 20 Mei 2020

ROLE OF CRP FOR SCREENING RESPIRATORY TRACT INFECTION

Prof. Dr. Aryati, dr., MS, SpPK(K)Ketua Umum Pengurus Pusat Perhimpunan Dokter Spesialis Patologi Klinik (PDS PatKLIn)

RESPIRATORY TRACT INFECTION

Respiratory tract infection (RTI) is defined as anyinfectious disease of the upper or lowerrespiratory tract.ü Upper respiratory tract infections (URTIs)

include the common cold, laryngitis,pharyngitis/tonsilitis, acute rhinitis, acuterhinosinusitis and acute otitis media.

ü Lower respiratory tract infections (LRTIs) includeacute bronchitis, bronchiolitis, pneumonia andtracheitis.

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COMMON AGENTS OF RESPIRATORY TRACT INFECTIONS (Dasaraju & Liu, 1996)CLINICAL ILLNESS BACTERIA VIRUSES FUNGI

Common cold (rhinitis, coryza)

Rare RhinovirusesCoronavirusesParainfluenza Viruses

AdenovirusesRSVInfluenza

Rare

Pharyngitis and tonsilitis

Group A β hemolytic streptococcusCorynebacterium diphteriaeNeisseria gonorrhoea

Mycoplasma pneumoniaeMycoplasma hominis (type 1)Mixed anaerobes

AdenovirusCoxsackievirus AInfluenza virusRhinovirusesCoronaviruses

Parainfluenza VirusesEipstein Barr Virus; cytomegalovirusHerpes simplex virus

Candida albicans

Epiglottitis and laryngotracheitis

(croup);

Haemophilus influenzae type BCorynebacterium diphteriae

RSVParainfluenza Viruses

Rare

Bronchitis and bronchiolitis

Haemophilus influenzaeStreptococcus pneumoniaeMycoplasma pneumoniae

RSVParainfluenza VirusesAdenovirusHerpes simplex virus

Rare

Pneumonia Streptococcus pneumoniaeStaphylococcus aureusStreptococcus pyogenesHaemophilus influenzaeKlebsiella pneumoniaeEscherichia coliPseudomonas aeruginosaMycoplasma pneumoniae

Legionella sppAnaerobic bacteriaMycobacterium tuberculosisCoxiella burnettiChlamidya psittaciChlamidya trachomatisChlamidya pneumoniae

AdenovirusParainfluenza VirusesRSVInfluenza virusVaricella-zoster virusMeasles virus

CytomegalovirusHerpes simplex virusHantavirus

Histoplasma capsulatumBlastomyces dermatitidisParacoccidioidesCoccidioides immitisCandida albicansFilobasidiellaCryptococcus neoformansAspergillusPneumocystis carinii 3

Respiratory tract infection

Rhodes et al, 2011. DOI: 10.1038/nrrheum.2011.37 4

5Gambar 1. Respon terhadap inflamasi (Reinhart et al. Clinical Microbiology Review. 2012; 25 : 609-634)

PATOGENESIS

COMPLETE BLOOD COUNT (CBC)

DR_ARYATI@YAHOO.COM 6

BLOOD SMEAR EVALUATION (BSE)

Toxic granule Vacuolization

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BIOMARKER OF INFLAMMATION

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ACUTE PHASE PROTEIN (APP)

They are produced from the liver as apart of immediate response to

infection or tissue injury.

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alpha 1 antitrypsin

CRP (C-reactive Protein)

Fibronectin

Haptoglobin

Lactoferrin

Procalcitonin (PCT)

INFLAMMATORY MARKERS

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C-REACTIVE PROTEIN

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STRUKTUR CRP

ü Produced as homopentameric protein, termedas native CRP (nCRP)

ü Will irreversibly dissociate at sites ofinflammation/ infection into five separatemonomers, termed monomeric CRP (mCRP)

ü Synthesized (primarily) in liver hepatocytes,but alse by:• Smooth muscle cells• Macrophages• Endothelial cells• Lymphocytes• Adipocytes

Sproston & Ashworth, 2018. doi: 10.3389/fimmu.2018.00754

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C-REACTIVE PROTEIN

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• Synthesized within 6-8 hours of exposure to an infective process or tissuedamage

• Half life 19hrs and may reach to 1000 fold during an acute phaseresponse

• Peaks at 36–50 hours. It decreased when there was no stimuli

• It has higher sensitivity and specificity than total neutrophils and I/T ratio (immature granulocyte to total).

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CRP mempunyai kelebihan dibandingkan LED, kenaikan suhu tubuh atau protein fase akut yang lain (a1-anti-trypsin, a1-acid glukoprotein, haptoglobin)

CRP meningkat dalam waktu 6-8 jam setelah adanya stimulus, mencapai puncak setelah 36-50 jam, dipertahankan selama masihada proses radang, inflamasi atau nekrosis jaringan dan menurundengan curam segera setelah proses radang, inflamasi atau nekrosismenghilang.

Keuntungan penentuan CRP, tidak dipengaruhi oleh obat-obatan kortikosteroid atau obat anti radang lain.

HOW TO EVALUATE THE CRP RESULT

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< 10 mg/L • Normal concentration10 - 25 mg/L • Increased but diagnostically of less

importance• During antibiotic treatment CRP should be reduced below this level• Does not exclude bacterial infection if the disease has lasted short• Take a new sample some hours later

25 - 50 mg/L • There is “something”. Bacterial or viral?

50 - 100 mg/L • Common in bacterial infections• Seldom seen with viral infections

> 100 mg/L • Fairly common in bacterial infections

CRP PADA RESPIRATORY INFECTION

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Ruiz-Gonzales, 2016. http://dx.doi.org/10.1155/2016/2198745 17

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20Song et al, 2011. DOI:10.1111/j.1750-2659.2011.00269.x

CRP: a cutoff of 86,5 mg/L best differentiated concomitant⁄secondary bacterial pneumonia from primary influenzapneumonia (sensitivity 81,8% and specificity 59,3%)

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CRP PADA COVID-19

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Tan et al, 2020. DOI: 10.1002/jmv.25871 23

Stage 1 : Initial stageStage 2 : Progression stageStage 3 : Peak stageStage 4 : Recovery stage

“CRP increased significantly at the initial stage in severe COVID-19 patients; while still no significant difference in the CT scores were found between the severe and mild groups”

CRP meningkat>10mg/L pada seluruhpasien dengan rata-rata CRP 23,5 mg/L pada kelompok pasienCOVID-19 kritis

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Liu et al, 2020

• Both CRP levels and diameter of the largestlung lesion increased with diseaseprogression.

• CRP levels were positively correlated withlung lesions and severe presentation(correlation coefficient = 0.873,0.734, P ˂ 0.001).

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METODE PEMERIKSAAN CRP

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METODE PEMERIKSAAN CRP

• Kualitatif• Semi

kuantitatif

Kuantitatif

Latex agglutination

• Enzyme immunoassay• Immunoturbidimetry (PETIA)• Nephelometry• Reflectometry Immunoassay

Aryati, 2018

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• Prinsip : reaksi aglutinasi antara antibodi anti human CRP dan CRP pada sampel

Sampel + goat IgG antihuman CRP berlapispartikel latex à komplek antigen-antibodi à

aglutinasi

Semikuantitatif : titrasi sampel dengan two fold dilution

Latex agglutination

Aryati, 2018

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• Prinsip : solid phase enzyme-linked immunosorbent assay Sampel + mouse monoclonal anti CRP antibody

(solid phase)

+ goat anti-CRP antibody berlabel HRP

Perubahan warna (biru) à ELISA reader

Enzyme Immunoassay

Inkubasi, washing

Substrat TMB

Aryati, 2018

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• Prinsip : reaksi antara antibodi spesifikmenghasilkan komplek imun tidak terlarut

Sampel + antibodi monoklonal anti-CRP àkomplek antigen-antibodià kekeruhan

Spectrophotometer

Konsentrasi dalam sampel

Immunoturbidimetry (PETIA)

Aryati, 2018PETIA = Particle Enhanced Turbidimetric Immunoassay

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• Prinsip : reaksi antara analit terlarut danantibodi anti CRP yang terikat dengan partikelpolystyrene

Sampel + partikel latex berlapis antibodimonoklonal anti-CRP àkomplek antigen-antibodi

dengan partikel latex

Light scaterring (nephelometric)

Konsentrasi dalam sampel

Nephelometry

Aryati, 2018

REFLECTOMETRY IMMUNOASSAY

Brand NycoCard™ Reader II Afinion 2TM

Made in Norway Norway

Sample typeWhole blood Whole bloodSerum, Plasma Serum, Plasma

Principle Immunometric assay – measurement reflecto meter Immunomatric assay – measurement reflecto meter

Time to results 3 minutes (all process) 3 minutes (all process)

Sample Volume 5µL 2.5µL

Touch Screen No Yes

Automatic Process Semi automatic Yes

Measuring RangeSerum/Plasma: 5-160 mg/L Serum/Plasma: 5-160 mg/LWhole blood : 5-200 mg/L Whole blood : 5-200 mg/L

Steps 5 3

Storage 2-8oC until expiry, 15-25oC 6 weeks unopened 2-8oC until expiry (12 months), 15-25oC 28 days unopened

Setting No. need set up instrument No. need set up instrumentCode number No Yes so the result will not false positive

Time to Room Temperature At least 10 minutes to reach 15-25oC At least 15 minutes to reach 18-30oC

Controls Black Calibration, White Calibration, 2 levels control Automatic calibration, 2 levels Afinion Controls

CRP POINT OF CARE TESTING - AFINION

§ 2.5 µL sample volume§ 3 minute test time§ Sample materials whole blood, serum or plasma§ Highly accurate results§ with automatic hematocrit correction

§ Measuring range whole blood; 5-200 mg/L§ No interference from common antibiotics and painkillers§ 4 week room temperature storage

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TAKE HOME MESSAGECRP merupakan acute phase reactant yang diproduksiterhadap infeksi/inflamasi, merupakan marker inflamasi(termasuk infeksi bakterial, nekrosis jaringan, autoimun)

CRP dapat digunakan sebagai skrining respiratory tract infection

Di era pandemi Covid-19, CRP dapat sebagai terjadinyaderajat penyakit yang berat

Diperlukan analisis menyeluruh dengan kondisi klinis, biomarker lain serta radiologis untuk tatalaksana diagnosis respiratory tract infection

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TANGGAL PANDUAN

7 Maret 2020 Manajemen Spesimen Dan Diagnosis Laboratorium KasusSuspek 2019-ncov

7 Maret 2020 Pencegahan Dan Pengendalian Infeksi Pada Suspek Infeksi2019-ncov

19 Maret 2020 Press Release Kewaspadaan Tes Cepat (Rapid Test) COVID-19 Igm/Igg Berbasis Serologi

21 Maret 2020 Alur Pemeriksaan Rapid Test Sars-Cov-2 (COVID-19) Usulan PDS Patklin

25 Maret 2020 Panduan Tatalaksana Pemeriksaan Rapid Test Antibody Sars-cov-2 Metode Imunokromatografi

20 April 2020 Daftar Rapid Test Serologi COVID-19 Yang SudahTerdaftar di FDA Negara

21 April 2020 Revisi Panduan Pemeriksaan Rapid Test Antibodi MetodeImunokromatografi

22 April 2020 Panduan Tatalaksana Pemeriksaan Tes Cepat Molekuler(TCM) dan Polymerase Chain Reaction (PCR) SARS-CoV-2 36

Berbagai Panduan COVID-19 oleh PDS PatKLIn

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