Education

Current position

Dr. Miswar Fattah, MSiMakassar, 6th June 1978

1997 : SMAK Depkes Makassar 2002 : Chemistry - UNHAS

2006 : Master of Science in Clinical Chemistry, Biomedicine- UNHAS

2012 : Doctor of Medicine - UNHAS

1. Specialty & Research Laboratory Manager, Prodia Clinical Laboratory 2018- Now

2. PATELKI : Vice President 2017-Now & Member of Collegium PATELKI 2015 - Now

3. IACC: Member scientific committe, Indonesian Association for Clinical Chemistry 2013- Now

4. President of ASEAN Association of Clinical Laboratory Scientist (AACLS) 2018-2020

5. Corresponding Member Scientific Committee Asia Pacific Federation for Clinical Chemistry (APFCB) 2010 – Now

HOW TO CHOOSE & VERIFY KITS RELATED COVID-19

AACLS Webinar

Jakarta, 08th May 2020

Dr. Miswar Fattah, MSiAACLS PresidentVice Presdient PATELKISpecialty & Research LaboratoryProdia Clinical Laboratorymiswarfattah@gmail.com

COMBAT COVID-19

Introduction: COVID-19, Structure of 2019-NCOV & Diagnosis 01

Test related COVID-19 & Problems02

Types of IVD reagents & How to choose it03

Verification kits Molecular, Antigen & Antibody for

COVID-1904

Outline

KEY EVENTS IN THE 2019-NCOV OUTBREAK

3th march 20201st Case report in Indonesia

Modiefied from Seah I et al. 2020. Eye, pp. 1–3

AACLS members Country data

SEVEN COVS THAT CAN INFECT HUMAN AND CAUSE RESPIRATORY DISEASES

HCoV-229E

HCoV-OC43

HCoV-NL63

HKU1SARS-CoV

MERS-CoV

SARS-CoV2

NAMING VIRUS AND DISEASE

Gorbalenya AE et al. 2020. Nature Microbiology. 5(4):536–44

MAIN STRUCTURE OF CORONAVIRUSES

THE ETIOLOGICAL DIAGNOSIS OF SARS-COV-2 INFECTION ISCURRENTLY BASED ON:

Collection of an upper respiratory specimen

(i.e.,nasopharyngeal AND oropharyngeal swabs)

Analysis of the sample by (real-time) reverse

transcription polymerase chainreaction (rRT-PCR)

https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelinesclinical-specimens.html

AACLS members Country data

LABORATORY TEST FOR COVID-19

LABORATORY TESTING RELATED PANDEMIC

Patient management Perspective

Epidemiology & Control outbreak

DIFFERENT TYPE OF ANALYTE LABORATORY TESTING RELATED COVID-19

RNA

RdRp

ORFla/b Gene

N Gene

E Gene

S Gene

Antigen

N Protein

S Protein

Antibody

IgM

IgG

IgA

Host Respons

CBC

CRP

D Dimer

SGOT

Albumin

LDH, etc

Potential susceptibility

ACE2 Gene

TMPRSS2 Gene

HLA Gene

rRTPCR, LAMP, NGS

ELISA, Immuno-chromatography

ELISA, Immuno-chromatography,

Chemiluminoscence immnoassay

Enzymatic, colorimetry,

flowcytometry, impedance

Genotyping microarray, RTPCR,

Sanger Seq, NGS

THE DRAMATIC IMPACT OF THE RAPID DETECTION OF INFECTIOUS DISEASES IN CONTROLLING AND PREVENTING AN OUTBREAK

Nguyen T et al. 2020. Micromachines. 11(3):306

SHORTAGE OF REAGENTSInclude high skills ofCLINICAL LABORATORY SCIENTIST

High potencial False Negative

High Potencial False Positive

Common problem in diagnostics new disease outbreak

Unclear Mechanism of

Disease

Diagnostic tool under develop

Lack of standardization of sampel type

Unclear Sampling, storage,

handling sample Protocol

Not yet standardize

method

Donation kit without kit

review

RACING TO DEVELOP COVID-19 TESTS

HE tissueElectron

microscopeViral Culture NGS

RT PCR, LAMP

Protein Isolation

Spesific Antibody

ELISA

Rapid Lateral Flow

Chemiluminoscence

Autoanalyzer

“If you have a sequence today, you have a PCR tomorrow”

some reagent producers are not very well known by clinical laboratory scientists

in just 3 months the production of new reagents is very muchnot all reagents are listed on this website

https://www.finddx.org/covid-19/pipeline/

HOW TO CHOOSE KIT IN PANDEMIC

Avaibility CostPositioning of test / hospital base or

survailance

Goverment recomendation

Visible with current fasility

Best literature / Number

publicationCompare & verify

Sethuraman N, Jeremiah SS, Ryo A. 2020. JAMA

CORRESPONDENCE BETWEEN DEVELOPMENT OF VIRAL LOAD DURING SARS-COV-2 INFECTION, CLINICAL COURSE AND POSITIVITY OF RRT-PCR ASSAYS

G. Lippi, A.-M. Simundic, M. Plebani, Clinical Chemistry and Laboratory Medicine (CCLM). 1 (2020), doi:10.1515/cclm-2020-0285.

IFCC Information Guide on COVID-19 - IFCC. www.ifcc.org

TESTS FOR SARS–COV-2/COVID-19 AND POTENTIAL USES

R. Patel et al., mBio. 11 (2020), doi:10.1128/mBio.00722-20.

Report from the American Society for Microbiology COVID-19 International Summit, 23 March 2020: Value of Diagnostic Testing for SARS–CoV-2/COVID-19

FAVORABLE TECHNOLOGY DETECTION RELATED SARS COV-2

• Closed System

• More Safety & Standardize

• Open system

• Easy & Faster to develop new test

Molecular Based testing

• Rapid immunochromatigraphy

• Faster results, Easy to use

• ELISA or Chemiluminoscence

• More standardize & possible to Quantify

Immunoassay Based testing

SENSITIVITY & SPECIFICITY ANTIBODY VS. MOLECULAR

Antibody IgG/IgMSARS CoV-2

MolecularSARS CoV-2

https://finddx.shinyapps.io/COVID19DxData/

COMPARISON RTPCR KIT AVAILABLE IN INDONESIASpecification

Country

Producent

Gene target

LOD

Ct Cut off

PCR platform

Match current facility

Open/Close/POCT

Number of tube/test

Troughput per 2 h

Number of manual step

Regulatory status

Number of Publish

Seegen

South of Korea

Koogen

South of Korea

A Star

Singapore

Sansure

China

IDT CDC

Singapore

Genexpert

USA

Cobas c6800

Germany

Tibmolbiol

Germany

Country Producent Gene target LODAvaibility Ct Cut

off

Inter control PCR platformMatch current

facilityMatch wide range

VTMOpen/Close/POCT

Number of tube/test

Troughput per 2 hNumber of manual

stepRegulatory status Number of Publish

Institute Gene targets

China CDC, China ORF1ab and N

Institut Pasteur, Paris, France Two targets in RdRP

US CDC, USA Two targets in N gene (previusly 3)

National Institute of InfectiousDiseases, Japan

Pancorona and multiple targets, Spike protein

Charité, Germany RdRP, E, N

HKU, Hong Kong SAR ORF1b-nsp14, N

National Institute of Health, Thailand N

DIFFERENT TARGET GENE

Corman VM et al. 2020. Euro Surveill. 25(3):

COMPARATIVE ANALYSIS OF PRIMER-PROBE SETS FOR THE LABORATORY CONFIRMATION OF SARS CORONA 2

Jung YJ et al. 2020. Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2. Microbiology

First line screening:

E gene

Confirmatory screening:

RdRP gene

Additional confirmatory

screening: N gene

Gene target

for Diagnosis COVID-19

Real-time reverse transcription polymerase

chain reaction (rRT-PCR) is the current gold

standard for diagnosing suspected cases of

COVID-19

Gold standard for Diagnosis COVID-19

CHOOSE SAMPLE TYPE BASED ON RT-PCR POSITIVITY

Bronchoalveolar lavage specimens (93%)

Sputum (72%)

Nasal swab (63%)

Pharyngeal swab (32%)

Sethuraman N, Jeremiah SS, Ryo A. 2020. JAMA

Sample availability

Positivity

FALSE POSITIVE & FALSE NEGATIVE IN MOLECULAR TEST

False-negative results

Inappropriate timing of sample collection in relation to illness onset

Deficiency in sampling technique, especially of nasopharyngeal swabs

False Positive results

Occasional false-positive results may occur due to technical errors

reagent contamination

VALIDATION : Normal condition

1st: Selection

Application characteristics Methodology characteristics Performance characteristics

Factors that determine whether a method can be implemented in a Lab.

Factors that in practice, demonstrate how well a method performs

Factors that in principle contribute to best performance

Cost per test, type of specimen, turn around time, workload, operator

skills, etc

Reportable range, precision, recovery, interference, accuracy, etc.

Traceability of standards, chemical principle, measurement principle,

etc.

Westgard JO. Basic Method Validation, 3rd Ed. 2008

Validation/Verification

VALIDATION : Normal condition

Imprecision

(random error)

Performance characteristic :

Inaccuraccy

(systematic error)

Sensitivity

Reportable range

Reference intervals

Validated by :

Replication study --> controls, samples

- Comparison of methods

- Interference (constant systematic error)

- Recovery (proportional systematic error)

LoB, LoD, LoQ experiment

Linearity experiment

Verified by testing samples from healthy people

Non-FDA approved/LDT FDA-approved/cleared LDT

CLIA CAP CLIA CAP

Accuracy method comparison

+ + + +

Precisionreplication experiment

+ + + +

Reportable range linearity experiment

+ + + +

Establish reference range + + + +

Analytical sensitivity Limit of detection study

Not required Not required + +

Analytical specificityInterference study

Not required Not required + +

Recovery to determine proportional interferences Not required Not required + Not required

Westgard JO. Basic Method Validation, 3rd Ed. 2008

TYPES OF IN VITRO DIAGNOSTICS

RUOs (Research Use Only)

IUOs (Investigational Use Only)

EUA (Emergency Use Authorization) IVD

ASRs (Analyte Specific Reagents)

LDTs (Lab Developed Tests)

adequate IVD (In vitro diagnostics)

Companion Diagnostic IVDs

Higher utility in pandemic

Higher utility in pandemic

Higher utility in pandemic

EMERGENCY USE AUTHORIZATION

EUA: Emergency Use Authorization

HSA: Health & Safety/Sciences

Authority

MFDS: Ministry of Food & Drug Safety

MHRA: Medicinces& Health Care

Products Regulatory Agency

NRA: National Regulatory Authority

RUO: Research Use Only

TGA: Therapeutic Goods

Administration

WHO EUL: World Health

Organization Emergency Use

this is not a complete guide and the FDA guidance should be read closely for all compliance details. Information is changing quickly and is likely to change more if the number of COVID-19 cases in the each country increases

Version 4:27th March 2020

Version 3:Version 1-2

Rapidly changes of Guidellines in Pandemic

DEVELOP NEW TEST VERY HARD FOR CLINICAL LABORATORY SCIENTIST

Pressure in pandemic significantly increase

PRO & CONTRA OF “EMERGENCY USE ONLY” VALIDATION

Simplification of the validation methodology

Low cost

Fast process of designing and

developing a new kit

Rapid implementation of

new kits on the market

Rapid reporting of laboratory results

Clinical decisions based on

laboratory results

Less quality & regulation (poor testing)

Recognized manufacturers are developing new tests in a stressed production scenario

Manufacturers of unknown quality are producing reagent

kits

Supply chains are strained, presenting a high risk of

shortage at several points, such as the availability of raw materials and reagent kits on

the market

The effect of false results in an epidemic outbreak has

been misestimated

PRO CONTRA

Paulo Pereira, westgrad webinar 2020

QUALITY VS. AVAILIBILITY OF TEST

IDEAL PANDEMIC

Reliable & consistent QC

practices

Fast Avaibility of New Test

VALIDATION STUDY RECOMMENDATIONS BASED ON THETECHNOLOGICAL PRINCIPLES OF TESTS

Molecular Diagnostic

Tests

Antigen Detection

Tests

Serological Tests

Limit of Detection

Clinical Evaluation

InclusivityCross-

reactivity

Clinical Testing

Limit of Detection/Analytical

Sensitivity

Cross-reactivity/Analytical

Specificity

Microbial Interference

Clinical Agreement Study

Cross-reactivity/Analytical

SpecificityClass Specificity

Clinical Agreement Study

FDA EUA: VALIDATION OF MOLECULAR DIAGNOSTIC TESTSLOD: dilution series of three replicates per concentration with inactivated virus on actual patient specimen, then confirm the final concentration with 20replicates

Clinical Evaluation: minimum of 30 positive specimens and 30 negative specimens as determined by an authorized assay. Alternative contrived clinical specimens be spiked at a concentration of 1x2x LoD, 95% aggrement Positive and 100% for negatif spesimens

Inclusivity: in silico analysis indicating the percent identity matches against publicly available SARS-CoV-2 sequences

Cross-reactivity: common respiratory flora &d other viral pathogens at concentrations of >=106 CFU/ml (bacteria) & >=105 pfu/ml (viruses), except for SARS-Coronavirus and MERS-Coronavirus, which can be accomplished byin silico analysis

Clinical Testing: confirmation of the first five positive and the first five negative clinical specimens using an EUA-authorized assayFDA Document issued on

the web on May 4, 2020

HIGH VARIABILITY OF GENE TARGET

INSILICO VERIFICATION

INSILICO VERIFICATION

Lablogatory A. 2020. How to Validate a COVID-19 Assay

Step 1. Perform one positive and one negative QC

Step 2. Option 1: Verification panel with commercial synthetic material, dilutions of positive material should be made to test 5 high and 5 moderate viral loads

Step 2. Option 2: Verification panel with residual patient samples, 10 (+) and 10 (-)residual samples should be tested. Negative samples should be 10 independent patient samples (i.e. not pooled). Positive samples can be either 10 independent patient samples or dilutions of a strongly positive patient sample

Determination of limit of detection is not required for on-label use

https://www.asm.org/ASM/media/Protocol-Images/ASM_EUA_verification_040220_FINAL.pdf

FREE ACCESS CLSI GUIDELINE

www.clsi-covid-19.org

FREE

1. CLSI EP19 ED2:2015; A Framework for Using CLSI Documents to Evaluate Clinical Laboratory Measurement Procedures, 2nd Edition

2. CLSI GP36 A:2014; Planning for Laboratory Operations During a Disaster; Approved Guideline

3. CLSI QSRLDT-2015; Quality System Regulation for Laboratory-Developed Tests : A Practical Guide for the Laboratory

4. CLSI POCT07 A:2010; Quality Management: Approaches to Reducing Errors at the Point of Care; Approved Guideline

5. CLSI MM22 A:2014; Microarrays for Diagnosis and Monitoring of Infectious Diseases; Approved Guideline

TERIMA KASIH