Education. How... · 2020. 5. 9. · Education Current position Dr. Miswar Fattah, MSi Makassar,...
Transcript of Education. How... · 2020. 5. 9. · Education Current position Dr. Miswar Fattah, MSi Makassar,...
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 [email protected]
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