Kamis, 6 Agustus 2020

MULTIPLEX PCR SEBAGAI SALAH SATU PEMERIKSAAN YANG DIBUTUHKAN PADA

SAAT PANDEMI COVID-19

Prof. Dr. Aryati, dr., MS, SpPK(K)

SITUASI COVID-19 TERKINI

https://infeksiemerging.kemkes.go.id/ Update hingga 05 Agustus 2020 Pukul 08.00 WIB2

VIRUS SARS-COV-2

6 jenis Coronavirus yang menginfeksi manusia:

HKU1 (Beta Coronavirus)

OC43 (Beta Coronavirus)

229E (Alpha Coronavirus)

NL63 (Alpha Coronavirus)

SARS-CoV

MERS-CoV

SARS-CoV-2

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Ordo : Nidovirales

Family : Coronaviridae

Genus : Beta coronavirus

Subgenus: SarbecovirusSumber gambar: Peiris & Yuan, 2003. Severe acute respiratory syndrome. doi:10.1038/nm1143

Penyebab penyakit Coronavirus

Disease 2019 (COVID-19)

Penyebab Common

Cold

TARGET GEN RT-PCR SARS-COV-2

4

VIRUS SARS-CoV-2:

• Single-stranded positive sense RNA

• Genome length ∼30,000 nucleotides

• Genom mengkode 27 protein termasuk

RNA-dependent RNA polymerase (RdRP)

dan 4 protein struktural.

Similaritas dengan:

SARS-CoV : ~80%

MERS-CoV : ~50%

Bat Coronavirus RaTG13 : ~90%

Udugama et al, 2020. doi: 10.1021/acsnano.0c02624

DIAGNOSIS LABORATORIUM COVID-19

5Torres, 2008

Sampel Swab Naso/orofaring

Sampel Darah

GOLD STANDARD

6

Estimated Variation Over Time in Diagnostic Tests for Detection of

SARS-CoV-2 Infection Relative to Symptom Onset

aDetection only occurs if patients are

followed up proactively from the time of

exposure

bMore likely to register a negative than a

positive result by PCR of a nasopharyngeal

swab

Sethuraman et al, 2020. doi:10.1001/jama.2020.8259

RESPIRATORY TRACT INFECTION

Respiratory tract infection (RTI) is defined as any

infectious disease of the upper or lower

respiratory tract.

Upper respiratory tract infections (URTIs)

include the common cold, laryngitis,

pharyngitis/tonsilitis, acute rhinitis, acute

rhinosinusitis and acute otitis media.

Lower respiratory tract infections (LRTIs) include

acute bronchitis, bronchiolitis, pneumonia and

tracheitis.

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COMMON AGENTS OF RESPIRATORY TRACT INFECTIONS ( Dasaraju & Liu, 1996)

CLINICAL ILLNESS BACTERIA VIRUSES FUNGI

Common cold

(rhinitis, coryza)

Rare Rhinoviruses

Coronaviruses

Parainfluenza Viruses

Adenoviruses

RSV

Influenza

Rare

Pharyngitis and

tonsilitis

Group A β hemolytic

streptococcus

Corynebacterium

diphteriae

Neisseria gonorrhoea

Mycoplasma pneumoniae

Mycoplasma hominis (type

1)

Mixed anaerobes

Adenovirus

Coxsackievirus A

Influenza virus

Rhinoviruses

Coronaviruses

Parainfluenza Viruses

Eipstein Barr Virus;

cytomegalovirus

Herpes simplex virus

Candida albicans

Epiglottitis and

laryngotracheitis

(croup);

Haemophilus influenzae type B

Corynebacterium diphteriae

RSV

Parainfluenza Viruses

Rare

Bronchitis and

bronchiolitis

Haemophilus influenzae

Streptococcus pneumoniae

Mycoplasma pneumoniae

RSV

Parainfluenza Viruses

Adenovirus

Herpes simplex virus

Rare

Pneumonia Streptococcus pneumoniae

Staphylococcus aureus

Streptococcus pyogenes

Haemophilus influenzae

Klebsiella pneumoniae

Escherichia coli

Pseudomonas aeruginosa

Mycoplasma pneumoniae

Legionella spp

Anaerobic bacteria

Mycobacterium

tuberculosis

Coxiella burnetti

Chlamidya psittaci

Chlamidya trachomatis

Chlamidya pneumoniae

Adenovirus

Parainfluenza Viruses

RSV

Influenza virus

Varicella-zoster virus

Measles virus

Cytomegalovirus

Herpes simplex virus

Hantavirus

Histoplasma capsulatum

Blastomyces dermatitidis

Paracoccidioides

Coccidioides immitis

Candida albicans

Filobasidiella

Cryptococcus neoformans

Aspergillus

Pneumocystis carinii8

RESPIRATORY INFECTION

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POLYMERASE CHAIN REACTION

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Teknik Amplifikasi Regio Spesifik Dari Suatu Rantai DNA Secara Invitro

In order to use PCR, the exact sequence of nucleotides that flank (lay on eitherside of) the area of interest (the target area that needs to be amplified), mustbe known

Aplikasi : identifikasi penyakit genetik, infeksi virus, Genetic profiling inforensic, legal and bio-diversity applications, biologi evolusi, Site-directedmutagenesis of genes dan mRNA Quantitation di sel ataupun jaringan

Fatchiyah, 2005

POLYMERASE CHAIN REACTION (PCR)

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• EkstraksiDNA/RNA

• DNA / RNA preparasi

Pre-

• Denaturation

• Primer annealing

• Extension

PCR• Product

analysis

Post-

PROSEDUR PCR

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CELL DISRUPTION

MEMBRANE LIPID, PROTEIN &OTHERs REMOVAL

PURIFICATION

CONCENTRATION

Ali et al., 2017

Efisiensi PCR dipengaruhi oleh integritas molekul DNA/ RNA; konsentrasi enzym inhibitor perlu hati – hati terhadap kontaminasi RNAse

PRINSIP UTAMA EKSTRAKSI

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DNA template Primers DNA

polymerase

Deoxynucleotide triphosphate

(dNTPs)

Buffer, divalent cations & additives

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KOMPONEN DASAR PCR

Taq PCR Master Mix Kit :

-Sense dan antisense primer

-Template DNA

-RNAse Free water

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Template DNA : DNA yang mengandung sekuens target yang akan

diamplifikasi selama PCR

Primers : pasangan nukleotida sintetik (forward dan reverse) yang

komplementer terhadap ujung 3’ dari kedua untai DNA target

Enzim DNA polymerase misal Taq polymerase (yang diisolasi dari bakteri termofilik

Thermus aquaticus) atau tTH yang digunakan untuk mengkatalisis proses sintesis DNA

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deoxynucleoside triphosphates (dNTPs) : terdiri dari dATP, dCTP, dGTP, dTTP dalam

jumlah yang sama; digunakan untuk proses sintesis salinan baru menggunakan enzim DNA

polimerase

Divalent cations : umumnya Mg2+ dalam konsentrasi optimum untuk aktivitas DNA

polimerase dan beberapa tahapan dalam PCR

Buffer solution : menjaga suasana ionik untuk aktivitas dan stabilitas

optimum DNA polimerase

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TAHAPAN PCR DALAM ALAT PCR (THERMAL CYCLER)

3 tahap utama yang diulangi ~

30-40 siklus

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19

Produk PCR : 2n

Pada

umumnya:

30-40

siklus

PEMBACAAN PRODUK PCR

Gel electrophoresis dengan pewarnaan ethidium bromide (konvensional)

SYBR Green I fluoresence

Probe detection : Molecular beacon, Taqman probe, Scorpion dll

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Conventional PCR

Nested PCR

Multiplex PCR

Reverse transcription PCR

(RT-PCR)Real time PCR

(qPCR)

In situ PCR

Digital PCR

VARIASI TEKNIK PCR

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PCR Konvensional:

seminested,nested,

multiplex

OTHERS :

- NASBA

- RT-LAMP

JENIS PCR

Pre-PCR

Ekstraksi & DNA / RNA

template preparation

PCR Analysis

Gel electrophoresis

Thermalcycler 23

CONVENTIONAL PCR

3000

500

1000

100

M 1 2 3 4 M 5 6 7 8 9 10 11 12 13 14 M

M: 100 bp ladder

Lane 1: DEN-1 pos ctrl

Lane 2: DEN-2 pos ctrl

Lane 3: DEN-3 pos ctrl

Lane 4: DEN-4 pos ctrl

Lane 5: #023 = DEN-3

Lane 6: #024 = DEN-3

Lane 7: #025 NEG

Lane 8: #026 = DEN-1

Lane 9: #027 = DEN-3

Lane 10: #029 = DEN-4

Lane 11: #030 = DEN-3

Lane 12: #031 =DEN-1

Lane 13: #032 = DEN-4

Lane 14: Neg ctrl

Dengue PCR Typing results

D1/TS1: 482 bp

D1/TS2: 119 bp

D1/TS3: 290 bp

D1/DEN4: 389 bp

24(Aryati et al, 2010)

PCR KONVENSIONAL

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Real Time RT-PCR (qRT-PCR)

Multiplex PCR memungkinkan amplifikasi beberapa target berbeda dalam satu tube PSC secara bersamaan.

Menghemat waktu, reagen dan sampel & membandingkan beberapa amplikon secara simultan

thermofisher 26

MULTIPLEX PCR

Pengukuran reaksi PCR secara kinetik pada deteksi amplifikasiPCR selama fase awal reaksi

Monitor emisi florescence selama reaksi sebagai indikator amplifikasidari setiap cycle (real time)

Peningkatan sinyal floresens sebanding dengan produk PCR

Computer based, kualitatif/kuantitatif, tidak perlu elektroforesis

Methode deteksi fluorescence

DNA binding agents: Sybr Green

Sequence specific probes: Taqman™, Molecular beacons,Scorpions™

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REAL TIME PCR

MICROARRAY

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AFFYMETRIX EXPRESSION ARRAYS

http://www.affymetrix.com/technology/ge_analysis/index.affx29

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ANALYSIS OF MICROARRAYS

Microarrays allow for the simultaneous analysis of the expression of thousands of mRNAs.

Useful for determining changes in gene expression patterns from one sample tissue to another.

For example, microarrays have been used to study differences in gene expression in different tumor tissues.

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PENTINGNYA DETEKSI MULTIPEL PATOGEN

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The Challenge of Syndromic Disease (same symptoms, many causes)

Biomerioux; Aryati, 2019, Suramade Denpasar

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35

The prevalence of co-infection was variable it could be up to 50% among non-

survivors

Co-pathogens included bacteria, such as Streptococcus pneumoniae,

Staphylococcus aureus, Klebsiella pneumoniae, Mycoplasma pneumoniae,

Chlamydia pneumonia, Legionella pneumophila and Acinetobacter

baumannii; Candida species and Aspergillus flavus; and

Viruses such as: influenza, coronavirus, rhinovirus/enterovirus, parainfluenza,

metapneumovirus, influenza B virus, and human immunodeficiency virus

Clinicians can neither rule out other co-infections caused by respiratory

pathogens by diagnosing SARS-CoV-2 infection nor rule out COVID-19 by

detection of non-SARS-CoV-2 respiratory pathogens

Lai et al, 2020. https://doi.org/10.1016/j.jmii.2020.05.01336

The rates of viral co-infection (6, 35.3

%), fungal co-infection (5, 29.5 %) and

bacterial-fungal co-infection (5, 29.5 %)

were the highest in severe/critical

category (P > 0.05)

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KO-INFEKSI SARS-CoV-2 DENGAN VIRUS LAINNYA

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CONTOH MULTIPLEX MOLECULAR DIAGNOSTIC TEST DLM MICROARRAY-POCT PLATFORM

1. CLART Genomica

2. BIOFIRE Filmarray Biomerioux

3. QIAstat

4. Vivalytic Randox-Bosch

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CLART GENOMICA

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CLART menggunakan

teknologi platform secara

low-density microarray yang

berada pada dasar tabung.

Probe yang terimobilisasi di

dasar tabung akan

berhibridisasi dengan target

dan menghasilkan titik hitam.

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WAKTU PENGERJAAN: < 8 JAM

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BIOFIRE Filmarray Biomerioux

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ONE SYSTEM & ONE POUCH FOR ONE TEST

• Including : extraction reagents, control RNA & DNA, and mastermix

2nd

Stage

PCR

1st Stage

Multiplex

PCR

Sample

Extraction &

Preparation

+Conventional

BIOFIRE - FILMARRAY

WORKFLOW: SIMPLE, EASY, FAST

BIOFIRE - FILMARRAY

Parainfluenza 1

Parainfluenza 2

Parainfluenza 3

Parainfluenza 4

RSV

Bacterial

Bordetella pertussis

Bordetella parapertussis Chlamydia pneumoniae

Mycoplasma pneumoniae

Viral

Adenovirus

Coronavirus 229E

Coronavirus HKU1

Coronavirus OC43

Coronavirus NL63

SARS COV 2Human Metapneumovirus

Human Rhinovirus/ Enterovirus

Influenza A

Influenza A/H1

Influenza A/H1-2009

Influenza A/H3

Influenza B

Respiratory 2.1 EUA Panel Sample Type: Nasopharyngeal swabs (NPS) Volume: 300 μL

22 pathogens 18 viruses 4 bacteria

Processing Time + 45 minutes

Samples in VTM/UTM :

(18–30 ºC) up to 4 h,

2-8 ºC up to 3 days,

< -15 ºC up to 30 days

Sensitivity 97,1%; Specificity 99,3% overall panel

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PRIMER DESIGN

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49

PANEL LAINNYA:

Pneumonia Panel

Blood Culture Identification Panel

Gastrointestinal Panel

Meningitis Panel

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QIASTAT-Dx

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QIASTAT-DX RESPIRATORY SYNDROMIC PANEL

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Detection of 21 pathogens in about an hour

Can accept both direct swabs and liquid samples (300 μL)

Room temperature stable

Less than a minute hands-on time

True Sample to Insight solution

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Detection of 24 Pathogens (6 viruses, 14 bacteria, 4 parasites) in about an hour

Can accept liquid samples (200 μL)

Room temperature stable

Less than a minute hands-on time

True Sample to Insight solution

QIAstat-Dx Gastrointestinal Syndromic Panel

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Deliver diagnostic actionable information with

confidence

Facilitate fast care decision making

Lab delivers actionable information

Color coding and symbols used to provide

test results:

• All pathogens detected and identified in

sample preceded by

• Pathogens tested but not detected are

preceded by

Amplification curves can also be viewed:

• Ct values and amplification curves are

reported by QIAstat-Dx when a pathogen is

detected

• Physicians can use Ct values to identify the most

prevalent pathogen in co-infections and treat the

patient accordingly.

• Potentially aid in assessing the severity of an

infection – readings above Ct 35 are generally

not considered significant

QIASTAT-DX SYNDROMIC TESTING COMPREHENSIVE

VIVALYTIC RANDOX - BOSCH

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Chemiluminescent SignalThe Art of Detection

The biochip detection system is

based on a chemiluminescent signal.

This is the emission of light, without

heat, as a result a chemical reaction.

An enzyme is used to catalyze the

chemical reaction of the biochip

which generates the

chemiluminescent signal.

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Vivalytic Worfklow

Sample Type: Nasopharyngeal Swab, Sputum, BAL

Sample Volume: 200 μl

Detection Method: Biochip Technology

Result Time : 2,5 hours

Viral Respiratory Tract Infection Array (VRI)

Virus

SARS-CoV-2 Adenovirus A/B/C/D/E

Coronavirus 229E/NL63 Enterovirus A/B/C

Coronavirus OC43/HKUI Influenza A

Middle East Respiratory

Syndrome Coronavirus (MERS-

CoV)

Influenza B

Pan-Coronavirus

(SARS, SARS like, 2019-nCoV)

Rhinovirus A/B

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59https://www.captodayonline.com/vivalytic-test-for-detecting-sars-cov-2/

VIRAL

Adenovirus Respiratory syncytial virus B Influenza virus B

Metapneumovirus Rhinovirus A/B/C

Respiratory syncytial virus A Influenza virus A

BACTERIAL

Achromobacter xylosoxidans Maraxella catarrhalis Pseudomonas aeruginosa

Bordetella pertussis Mycoplasma pneumoniae Staphylococcus aureus

Burkholderia cepacia complex (21 spp) Non-tuberculous mycobacterium (15 spp) Stenotrophmonas maltophilia

Burkholderia cenocepacia Mycobacterium abscessus subgroyp (4

spp)

Streptococcus pneumoniae (21pp)

Burkholderia multivorans Mycobacterium avium complex (4pp) Streptococcus species (19 spp)

Chlamydia pneumoniae Pandoraea species (5 spp) Veillonella species (3 spp)

Haemophilus influenza Prevotella species (16 spp)

FUNGAL

Aspergillus fumigatus Candida albicans Exophialia dermatitidis

ANTBIOTIC RESISTANCE MARKERS

mecA (incl MRSA)

Chronic Lung Infection Array Sample Type: Sputum

Sample Volume: 200 μl

Detection Method: Biochip Technology Chronic Lung Disease Panel

TAKE HOME MESSAGE

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Multiplex PCR dapat mendeteksi berbagai patogen dari1 sampel dalam 1x pengerjaan

Dapat memberikan hasil yang cepat dalam waktu 1-8 jam

Dapat memberikan pertimbangan tatalaksana manajemenpasien yang cepat terutama di era pandemi Covid-19

TANGGAL PANDUAN

7 Maret 2020 Manajemen Spesimen Dan Diagnosis Laboratorium Kasus

Suspek 2019-ncov

7 Maret 2020 Pencegahan Dan Pengendalian Infeksi Pada Suspek Infeksi

2019-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 Sudah

Terdaftar di FDA Negara

21 April 2020 Revisi Panduan Pemeriksaan Rapid Test Antibodi Metode

Imunokromatografi

22 April 2020 Panduan Tatalaksana Pemeriksaan Tes Cepat Molekuler

(TCM) dan Polymerase Chain Reaction (PCR) SARS-CoV-2 62

Berbagai Panduan COVID-19 oleh PDS PatKLIn

63

1. Email : infokompdspatklin2019@gmail.com

2. Facebook : infokompdspatklin2019@gmail.com

3. Website : www.pdspatklin.or.id

4. Youtube : PDS PatKLIn Dokter Patologi Klinik

5. Instagram : DOKTER PATOLOGI KLINIK

6. Twitter : www.twitter.com/patologi_klinik

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TERIMA KASIH