Education Dr. Miswar Fattah, MSi Makassar, 6th June 1978 1997 : SMAK Depkes Makassar 2002 : Chemistry - UNHAS 2006 : Master of Science in Clinical Chemistry, Biomedicine- UNHAS 2012 : Doctor of Medicine - UNHAS Current position 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 LABORATORY TESTING RELATED COVID-19 DPP PATELKI Webinar Jakarta, 03th April 2020 Dr. Miswar Fattah, MSi Vice Presdient PATELKI Specialty & Research Laboratory Prodia Clinical Laboratory [email protected] Outline 01 Introduction: COVID-19, Structure of 2019-NCOV & Diagnosis 02 Nucleic Acid Testing related COVID-19 test 03 Antibody Testing & Other testing related COVID-19 04 Potential preanalytical & analytical vulnerabilities in the laboratory diagnosis of COVID-19 KEY EVENTS IN THE 2019-NCOV OUTBREAK 3th march 2020 1st Case report in Indonesia Modiefied from Seah I et al. 2020. Eye, pp. 1–3 SEVEN COVS THAT CAN INFECT HUMAN AND CAUSE RESPIRATORY DISEASES HCoV229E HCoVOC43 HCoVNL63 HKU1 SARSCoV MERSCoV SARSCoV2 Rabi, F.A., Al Zoubi, M.S., Kasasbeh, G.A., Salameh, D.M., Al-Nasser, A.D., 2020. Pathogens 9, 231. NAMING VIRUS AND DISEASE Gorbalenya AE et al. 2020. Nature Microbiology. 5(4):536–44 9,5% 6,26% Coronavirus COVID-19 (2019-nCoV) [WWW Document], n.d. URL https://gisanddata.maps.arcgis.com/apps/opsdashboard/index.html#/bda7594740fd40299423467b48e9ecf6 (accessed 4.3.20). MAIN STRUCTURE OF CORONAVIRUSES LABORATORY TEST FOR COVID-19 LABORATORY TESTING RELATED OUTBREAK Patient management Perspective Epidemiology & Control outbreak 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 PRIORITIZATION FOR TESTING SHOULD BE GIVEN TO: symptomatic health workers (including emergency services and non-clinical staff) 3 2 1 Laboratory testing strategy recommendations for COVID-19: interim guidance, 22 March 2020. https://apps.who.int the first symptomatic individuals in a closed setting (e.g. schools, long-term living facilities, prisons, hospitals) people who are at risk of developing severe disease and vulnerable populations, who will require hospitalization and advanced care for COVID-19 CORRESPONDENCE BETWEEN DEVELOPMENT OF VIRAL LOAD DURING SARSCOV-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. Unclear Mechanism of Disease Different target of Gene High potencial False Negative High Potencial False Positive Diagnostic tool under develop Common problem in diagnostics new disease outbreak Lack of standardization of sampel type Not yet standardize method Unclear Sampling, storage, handling sample Protocol RACING TO DEVELOP COVID-19 TESTS HE tissue Electron microscope Viral Culture NGS RT PCR, LAMP Chemiluminoscence Autoanalyzer Rapid Lateral Flow ELISA Spesific Antibody Protein Isolation “If you have a sequence today, you have a PCR tomorrow” GLOBAL DATA SEQUENCE OF 2019-NCOV Released Genome Sequences - 2019 Novel Coronavirus Resource. https://bigd.big.ac.cn 3th April 2020 Type of Biomarkers Predictive, Risk, susceptibility Early detection & Screening Diagnosis, Confirmatory & Staging Pharmacogenetics & Targeted Therapy Monitoring Therapy & Prognostic DIFFERENT TYPE OF ANALYTE LABORATORY TESTING RELATED COVID-19 RNA ORFla/b Gene N Gene E Gene Host Respons Antigen Antibody N Protein IgM S Protein IgG D Dimer ACE2 Gene IgA SGOT HLA Gene S Gene CBC CRP Potencial Succebility Albumin LDH, etc rRTPCR, LAMP, NGS ELISA, Immunochromatography ELISA, Immunochromatography, Chemiluminoscence immnoassay Enzymatic, colorimetry, flowcytometry, impedance Genotyping microarray, RTPCR, Sanger Seq, NGS NUCLEIC ACID TESTING (RNA SARS CORONA 2) Upper airway specimens Lower airway specimens Other Nasopharyngeal swabs Sputum Blood Feces Nasal swabs Airway secretion Urine Nasopharyngeal secretions Bronchoalveolar lavage fluid Conjunctival secretions collected by a healthcare professional, If both NP and OP swabs both are collected, they should be combined in a single tube to maximize test sensitivity and limit testing resources SAMPLE TYPE: CDC RECOMENDATION FOR INITIAL DIAGNOSTIC TESTING FOR COVID-19 Nasopharyngeal specimen (NP) Oropharyngeal (OP) specimen nasal mid-turbinate (NMT) swab collected by a healthcare professional anterior nares specimen lower respiratory tract specimens (sputum or BAL) If both NP and OP swabs both are collected, they should be combined in a single tube to maximize test sensitivity and limit testing resources SAMPLE TYPE INDONSIAN GUIDELINE Kementerian Kesehatan Republik Indonesia. www.kemkes.go.id Kementerian Kesehatan Republik Indonesia. www.kemkes.go.id THE ETIOLOGICAL DIAGNOSIS OF SARS-COV-2 INFECTION IS CURRENTLY BASED ON: Collection of an upper respiratory specimen (i.e.,nasopharyngeal AND oropharyngeal swabs) Analysis of the sample by (real-time) reverse transcription polymerase chain reaction (rRT-PCR) https://www.cdc.gov/coronavirus/2019-nCoV/lab/guidelinesclinical-specimens.html Gold standard for Diagnosis COVID-19 Real-time reverse transcription polymerase chain reaction (rRT-PCR) is the current gold Additional confirmatory standard for diagnosing suspected cases of screening: N COVID-19 Confirmatory screening: RdRP gene Gene target for Diagnosis COVID-19 First line screening: E gene gene 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 Infectious Diseases, 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 COMPARISON WHO VS. CDC METHOD FOR RTPCR SARC CORONA 2 Jung YJ et al. 2020. Comparative analysis of primer-probe sets for the laboratory confirmation of SARS-CoV-2. Microbiology RELATIVE POSITIONS OF AMPLICON TARGETS ON THE SARS CORONAVIRUS AND THE 2019 NOVEL CORONAVIRUS GENOME Corman VM et al. 2020. Euro Surveill. 25(3): DIFFERENT TARGET GENE Corman VM et al. 2020. Euro Surveill. 25(3): VARIATION DYNAMICS FOR SARS COV-2 GENE 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 CRITERIA FOR A CASE TO BE CONSIDERED AS LABORATORYCONFIRMED BY VALIDATED NAAT ASSAYS ACCORDING TO THE WHO: IFCC Information Guide on COVID-19 - IFCC. www.ifcc.org FAVORABLE TECHNOLOGY DETECTION RELATED SARS COV-2 Molecular Based testing • Closed System • More Safety & Standardize • Open system • Easy & Faster to develop new test Immunoassay Based testing • Rapid immunochromatigraphy • Faster results, Easy to use • ELISA or Chemiluminoscence • More standardize & possible to Quantify DIFFERENT TYPE OF ANALYTE LABORATORY TESTING RELATED COVID-19 RNA ORFla/b Gene N Gene E Gene Host Respons Antigen Antibody N Protein IgM S Protein IgG D Dimer ACE2 Gene IgA SGOT HLA Gene S Gene CBC CRP Potencial Succebility Albumin LDH, etc rRTPCR, LAMP, NGS ELISA, Immunochromatography ELISA, Immunochromatography, Chemiluminoscence immnoassay Enzymatic, colorimetry, flowcytometry, impedance Genotyping microarray, RTPCR, Sanger Seq, NGS LEVELS OF IGM, IGA, AND IGG ANTIBODIES AGAINST SARS-COV-2 IN PLASMA SAMPLES AFTER SYMPTOM ONSET L. Guo et al., Clin. Infect. Dis. (2020), doi:10.1093/cid/ciaa310. L. Guo et al., Clin. Infect. Dis. (2020), doi:10.1093/cid/ciaa310. ANTIBODY IGG & IGM IN SEVERE COVID-19 PATIENTS J. Zhao et al., Clin Infect Dis, doi:10.1093/cid/ciaa344. SEROLOGICAL TESTING FOR SARS CORONA 2 • There has been much debate regarding the current value of serological testing in COVID-19 diagnosis and monitoring. • Serologic based tests are not currently recommended by the CDC, NHS or other health organizations. • There is general concern regarding their use in the acute phase of infection as they detect infection too late in the course of illness (usually more than 7-10 days) and they also may cross-react with serologic responses to seasonal coronaviruses. • However, there is anticipated value in using improved serological testing in the future for public and occupational health monitoring and assessment IFCC Information Guide on COVID-19 - IFCC. www.ifcc.org SEROLOGICAL TESTING FOR SARS CORONA 2 • For serum antibody monitoring, according to the “Handbook of Covid-19 Prevention and Treatment” (Zhejiang University School of Medicine) • Serum lgM is detectable 10 days after symptom onset • Serum lgG is detectable 12 days after symptom onset. • A positive interpretation has been defined as a positive lgM, or an increased lgG titer > 4 times than that in the acute phase. IFCC Information Guide on COVID-19 - IFCC. www.ifcc.org J. Zhao et al., Clin Infect Dis, doi:10.1093/cid/ciaa344. THE POSITIVE DETECTION RATE IS SIGNIFICANTLY INCREASED WHEN COMBINED IGM ELISA ASSAY WITH PCR L. Guo et al., Clin. Infect. Dis. (2020), doi:10.1093/cid/ciaa310. pdspatklin. Alur Px Rapid Test Covid-19 PDS PatKLIn. PDSPATKLIN. www.pdspatklin.or.id RAPID TEST ANTIBODY GUIDELINE Kementerian Kesehatan Republik Indonesia. www.kemkes.go.id RAPID TEST ANTIGENE GUIDELINE Kementerian Kesehatan Republik Indonesia. www.kemkes.go.id TESTS FOR SARS–COV-2/COVID-19 AND POTENTIAL USES Report from the American Society for Microbiology COVID-19 International Summit, 23 March 2020: Value of Diagnostic Testing for SARS–CoV-2/COVID-19 R. Patel et al., mBio. 11 (2020), doi:10.1128/mBio.00722-20. IFCC Information Guide on COVID-19 - IFCC. www.ifcc.org RECOMMENDED TEST LIST: FREQUENT LABORATORY ABNORMALITIES IN PATIENTS WITH COVID-19 INCLUDE: • • • • • Lymphopenia (35-75%) C reactive protein (CRP 75-93%) Lactate dehydrogenase (LDH; 27-92%) Erythrocyte sedimentation rate (ESR; up to 85%) D-dimer (36-43%) • Albumin (50-98%) • Hemoglobin (41-50%) MAJOR PREDICTORS OF COVID-19 SEVERITY ARE: • Lymphocyte count • Albumin • • • • • • • Neutrophil count Lactate dehydrogenase (LDH) Aminotransferases Cardiac biomarkers (e.g., cardiac troponins) D-dimer Procalcitonin C reactive protein (CRP) Lippi G, Plebani M. Laboratory abnormalities in patients with COVID-2019 infection. Clin Chem Lab Med. 2020 Mar 3. PREANALYTICAL General • Lack of identification/misidentification • Inadequate procedures for specimen (e.g. swab) collection, handling, transport Storage • Collection of inappropriate or inadequate material for quality or volume • Presence of interfering substances • Manual (pipetting) errors Specific • Sample contamination • Testing in patients receiving antiretroviral therapy G. Lippi, A.-M. Simundic, M. Plebani, Clinical Chemistry and Laboratory Medicine (CCLM). 1 (2020), doi:10.1515/cclm-2020-0285. POTENTIAL PREANALYTICAL AND ANALYTICAL VULNERABILITIES IN THE LABORATORY DIAGNOSIS OF CORONAVIRUS DISEASE 2019 (COVID-19) USING (REAL TIME) RT-PCR. Testing carried out outside of the diagnostic window Active viral recombination Use of nonadequately validated assays Lack of harmonization of primers and probes Instrument malfunctioning Insufficient or inadequate material Non-specific PCR annealing Misinterpretation of expression profiles G. Lippi, A.-M. Simundic, M. Plebani, Clinical Chemistry and Laboratory Medicine (CCLM). 1 (2020), doi:10.1515/cclm-2020-0285. WHY MIGHT COVID-19 TESTS FAIL? They may be in the early stage of the disease with a viral load that is too low to be detected. They may have no major respiratory symptoms, so there could be little detectable virus in the patient’s throat and nose. There may have been poor handling and shipping of samples and test materials. There may have been a problem with sample collection, meaning there was very little sample to test. There may have been technical issues inherent in the test, e.g. virus mutation. CURRENT SARS-COV-2 NOMENCLATURE FOR LABORATORY STANDARIZATION (POST ANALYTIC) • Virus: Severe acute respiratory syndrome coronavirus 2 (SARS coronavirus 2; SARS-CoV-2) • Species: SARS-related coronavirus • Disease: COVID-19 (based on Coronavirus disease 2019) the Logical Observation Identifiers Names and Codes (LOINC) terms for SARS-CoV-2 lab tests have SARS coronavirus 2 in the Component because the tests are looking for the virus, not the disease LOINC MULTIAXIAL HIERARCHY SARS-CoV-2/COVID-19 LOINC Meeting| March 2020 Coronavirus COVID-19 HOW TO PREVENT 2m ` If you have fever or cough you should stay at home regardless of your travel or contact history. WASH COVER AVOID CLEAN STOP DISTANCE your hands well and often to avoid contamination your mouth and nose with a tissue or sleeve when coughing or sneezing and discard tissue touching eyes, nose, or mouth with unwashed hands and disinfect frequently touched objects and surfaces shaking hands or hugging when saying hello or greeting other people. yourself at least 2 meters (6 feet) away from other people, especially those who might be unwell. COUGH SHORTNESS OF BREATH BREATHING DIFFICULTIES SYMPTOMS FEVER (High Temperature) EVALUASI PEMBICARA PATELKI www.bit.ly/kuisionerPNTP Nama: Miswar Fattah Judul: Laboratory testing related covid-19 Nama Acara: WEBINAR COVID-19 Penyelenggara: DPP PATELKI