Covid 19 sample collection and diagnosis

Covid 19 sample collection and diagnosis

1. Morphology, Specimen collection, Processing of samples Covid 19 Sample Collection and Laboratory Diagnosis Dr. Moumita Adhikary Associate Professor Dept. of Microbiology Nodal Officer : RTPCR laboratory Rampurhat Govt. Medical College and Hospital

2. Objectives Sample Collection: Procedure, Formalities and Packaging Diagnosis: Real Time RTPCR, TRUENAT, CBNAAT, RAT, Ab test

3. Specimen labelling and processing: PPEs: Personal protective equipment’s ( APRON, HAND LATEX GLOVES (DOUBLE LAYERED), FACE SHIELD, N95 MASKS, GOGGLES, PROTECTING CLOTHES, WATERPROOF BOOTS etc.): to be used Proper labelling (NAME/AGE/GENDER/SPECIMEN ID) on specimen container Filling up the ICMR form To fill the ICMR app for generation of SRF ID at the time of sample collection

4. Requirements for Clinical Samples Collection, Packaging and Transport

5. 1. Sample vials and Virus Transport Medium (VTM ) 2. Adsorbent material (cotton, tissue paper), paraffin, scissors, cello tape 3. A leak-proof secondary container (e.g., ziplock pouch, cryobox , 50 mL centrifuge tube, plastic container) 4. Hard-frozen Gel Packs 5. A suitable outer container (e.g., thermocol box, ice-box, hard-board box) (minimum dimensions: 10 x 10 x 10 cm)

6. Collecting the Specimen Appropriate infection control precautions Interim Guidelines for Collecting, Handling, and Testing Clinical Specimens from Patients Under Investigation (PUIs) for 2019 Novel Coronavirus (2019-nCoV https://www.cdc.gov/coronavirus/2019-nCoV/guidelines-clinical-specimens.html ) - To follow specimen collection device manufacturer instructions for proper collection methods - Swab specimens: Collected using only swabs with a synthetic tip , such as Nylon or Dacron ®, and an Aluminum or Plastic Shaft Not Recommended: Calcium alginate swabs/cotton swabs with wooden shafts Place swabs immediately into sterile tubes containing 1-3 ml of viral transport media

7. Transporting Specimens Package, Shipped and Transport: According to the current edition of the International Air Transport Association (IATA) Dangerous Goods Regulation Follow shipping regulations for UN 3373 Biological Substance, Category B when sending potential 2019-nCoV specimens Storage: Store specimens at 2-8°C upto 72 hrs and ship on ice pack If a specimen is frozen at -70°C, ship on dry ice Extracted nucleic acid should be stored at -70°C or lower

8. Types of Specimens 1. Upper respiratory tract specimens : Nasopharyngeal and oropharyngeal swab 2. Lower respiratory tract specimens: D eep cough sputum, BAL, pulmonary tissue biopsy 3. Blood samples: A cute stage fasting blood within 7 days of onset, 5 ml in vacutainer with anticoagulants 4. Serum specimens: C ombine acute and recovery period double serum. 1 st serum to be collected as early as possible (preferably within 7 days of onset) and the 2 nd serum to be collected 3 to 4 weeks after onset. Amount: 5 ml, fasting blood, it is recommended to use vacutainer

9. Sample collection done at our college: RPHGMCH

10. Procedure for Specimen Packaging and Transport

11. 1. Use PPE while handling specimen 2. Seal the neck of the sample vials using parafilm 3. Cover the sample vials using absorbent material 4 . Arrange primary container (vial) in secondary container 5. Placing the centrifuge tube inside a zip-lock pouch 6. Placing the zip-lock pouch inside a sturdy plastic container and seal the neck of the container Note: Sample vials can also be placed inside a zip-lock pouch, covered in absorbent material and secured by heat- sealing or rubber bands. Then, the zip-lock pouch should be placed inside another plastic pouch and secured 7. Using a thermocol box as an outer container and placing the secondary container within it, surrounded by hard- frozen gel packs

12. 7 . Using a hard card-board box as an outer container and placing the secondary container and the gel packs 8. Placing the completed Specimen Referral Form (ICMR) and request letter inside a leak-proof, zip-lock pouch 9. Securing the zip-lock pouch with the Specimen Referral Form on the outer container 10. Attaching the labels: Senders’ address, contact number; Consignee’s address /contact number; Biological substance- Category B; ‘UN 3373’; Orientation label, Handle with care

13. Documents to accompany Packaging list/proforma Invoice Airway bill (for air transport) (to be prepared by sender or shipper) Value equivalence document (for road/rail/sea transport) [ Note: 1. A vaccine-carrier/ice-box can also be used as an outer container 2. The minimum dimensions of the outer container should be 10 x 10 x 10 cm (length x width x height)]

14. Laboratory & POC Diagnosis

15. Testing Methods Real Time RTPCR TRUENAT CBNAAT POC RAT Ab test

16. Real Time Reverse Transcriptase Polymerase Chain Reaction Gold standard frontline test for diagnosis of COVID19 Epidemiological and surveillance information Quick, Super Sensitive and Specific Qualitative detection of nucleic acid from the SARS-CoV-2 in respiratory specimens ( nasopharyngeal or oropharyngeal swabs, sputum, tracheal aspirates and bronchoalveolar lavage) : Acute Phase of Infection Identification of SARS-CoV-2 RNA Principle: The assay is composed of 2 principle steps: (1) Extraction of RNA from patient specimens (2) One-step reverse transcription and PCR amplification with SARS-CoV-2 specific primers and real-time detection with 2019-nCoV specific probes

17. PCR Laboratory at RPHGMCH

18. The assay targets regions of the virus nucleocapsid N gene (N1 & N3) Specimens are identified from populations based on positive rate (by zip code or by client) Result: after 35 cycles. Around 35 billion new copies. 2-5 hours: TAT NEGATIVE: Negative results must be combined with clinical observations, patient history and epidemiological information POSITIVE: Do not rule out bacterial infection or co-infection with other viruses POOLING: 5 patient specimens are combined Pool - Positive/Inconclusive/Invalid. Each of the samples are re-tested as a separate individual specimen - Negative: Each constituent sample is reported as negative - Specimens with low viral loads may not be detected in sample pools due to the decreased sensitivity of pooled testing Interpretation

19. Precautions To f ollow standard precautions All patient specimens and positive controls should be considered potentially infectious and handled accordingly No: Eat, Drink, Smoke, apply cosmetics or handle contact lenses in areas where reagents and human specimens are handled To Handle all specimens as if infectious using safe laboratory procedures Refer to Interim Laboratory Biosafety Guidelines for Handling and Processing Specimens Associated with 2019-nCoV https://www.cdc.gov/coronavirus/2019-nCoV/labbiosafety-guidelines.html Specimen processing should be performed in accordance with national biological safety regulations

20. Limitations Trained Personal: Perform the assay independently. Should Use, analyse and report diagnostic results Performance: Only established for upper and lower respiratory specimens Interpretation: Negative: Should not be used as the sole basis in patient management False Negative/Inconclusive - Specimen is improperly collected, transported or handled - If amplification inhibitors are present in the specimen - Inadequate number of organisms are present in the specimen - Prevalence of disease is high False positive: Prevalence is moderate to low Cannot be used to detect a past infection Require specialized laboratory facilities in terms of equipments , biosafety and biosecurity

21. TRUENAT

22. TRUENAT Chip-based, portable RT-PCR machine Diagnosis of Tuberculosis by Goa-based startup Molbio Diagnostics Main diagnostic tool for COVID-19 as India has expanded its testing capacity Fastest available PCR-based technique recommended by ICMR till date The latest versions of the TrueNat machine can detect an enzyme (called RdRp ) found in the RNA of the coronavirus SARS-CoV-2: ICMR advised confirmation TAT: Faster than the standard RT-PCR tests ICMR has advised private laboratories in States and Union Territories that intend to use TrueNat for COVID-19 testing to apply for NABL accreditation immediately

23. TRUENAT: Approval from ICMR via AIIMS Bhuvaneshwar

24. CBNAAT

25. CBNAAT Cartridge-based nucleic acid amplification test (CBNAAT) using Cepheid's Xpert Xpress SARS-CoV-2 test Done under the biosafety 2 level (BSL-2) conditions and with appropriate precautions ICMR: Any new govt. laboratory seeking to initiate the CBNAAT must meet the minimum ICMR requirements such as:- 1. Availability of a BSL-2 level laboratory facility 2. Molecular biology setup for virological diagnosis and a functioning and calibrated biosafety cabinet type 2A/2B in the laboratory 3. Staff having experience in molecular virology 4. Robust institutional policy on biomedical waste management Private laboratories should have National Accreditation Board for Testing & Calibration Laboratories (NABL) accreditation for molecular detection of RNA viruses either by the real-time PCR/CBNAAT

26. POC RAT - Urgent need - Reliable point-of-care rapid antigen detection test with good sensitivity and specificity for early detection - Standard Q COVID-19 Ag detection kit point-of-care test for SARS-CoV-2: (ICMR)

27. Antigens (visible protrusions on its surface): Nucleocapsid Phosphoprotein Spike glycoprotein Reveals: Current Infection Once the infection has gone, the antigen disappears Accuracy: lower sensitivity than PCR Relatively cheap more amenable to point-of-care use, which could make them more suitable for testing in the community and in remote regions

28. Procedure: Nasopharyngeal Swab to be collected by a trained healthcare worker following full infection control practices including use of proper PPE Time: After 15 minutes of putting the sample into the well. Maximum duration for interpreting a positive or negative test is 30 minutes Observation: Appearance of test and control lines which can be read with naked eye, requiring no specialized equipment. Test strip should be discarded Storage : Test kit should be stored between 2° to 30° C Not as sensitive as RTPCR Interpretation: Positive results from antigen tests are highly accurate but there is a higher chance of false negatives (so negative results do not rule out infection and need a confirmation via the RT-PCR test) Advantage : Gives results much faster within 30 minutes

29. Low sensitivity: Higher likelihood of false negatives Eg , 1st antigen test kit approved by the ICMR was made by SD Biosensor, a South Korean company manufacturing in Gurugram, India. High specificity (ability to detect true negatives), relatively low sensitivity (ability to detect true positives) Specificity : 99.3% to 100%, Sensitivity : 50.6% to 84% Means, positive result: 99.3% to 100% accurate, negative result: 50.6% to 84% accurate Limitations:

30. Government’s stand: Those with symptoms of Covid-19 can get tested with the antigen test. If their result comes out negative, a re-test using the RT-PCR must be undertaken Field level test for early detection of infection and quick containment & quick diagnosis Use: Field settings (outside of the laboratory) Hospitals along with the RT-PCR test technology So far, antigen tests manufactured by 3 companies approved by the ICMR for use in India

31. Containment zones or hotspots (to be performed onsite under strict medical supervision and maintaining kit temperature between 2°to 30°C i ) All symptomatic Influenza Like Illness (ILI) ii) Asymptomatic direct and high-risk contacts with co-morbidities (lung disease, heart disease, liver disease, kidney disease, diabetes, neurological disorders, blood disorders) of a confirmed case to be tested once between day 5 and day 10 of coming into contact B. Healthcare settings to be performed onsite under strict medical supervision and maintaining kit temperature between 2° to 30° When will the test be done?

32. All symptomatic ILI patients presenting in a healthcare setting and are suspected of having COVID-19 infection ii) Asymptomatic patients who are hospitalized or seeking hospitalization , in the following high risk groups : Patients undergoing chemotherapy, Immunosuppressed patients including those who are HIV+; Patients diagnosed with malignant disease; Transplant patients; Elderly patients (>65 yrs of age) with co-morbidities (lung disease, heart disease, liver disease, kidney disease, diabetes, neurological disorders, blood disorders) iii) Asymptomatic patients undergoing aerosol-generating surgical / non-surgical interventions : Elective/emergency surgical procedures like neurosurgery, ENT surgery, dental procedures; Non-surgical interventions like bronchoscopy, upper GI endoscopy and dialysis

33. ANTIBODY TESTING

34. Antibody test Reveals if a person has already been exposed to an infection Blood/Serum ELISA (enzyme-linked immunosorbent assay)/ CIA (chemiluminescent immunoassay)/Point-of-care test based on lateral flow technology IgM antibody response peaks around 2 weeks after infection, followed by the IgG antibody peak at 3 weeks Advantage: 1. Track the spread of disease. Estimates how many people have already been infected with SARS-CoV-2, which can be important in assessing herd immunity 2. Distinguish between IgM and IgG: Identify phase of infection indicating how long ago the person was infected with SARS-CoV-2 3. Identify those who should be prioritized for vaccinations when they become available 4. Identify potential donors for convalescent plasma therapy

35. Interpretation: Negative test result may occur if the test is taken too soon after infection before antibodies have been produced by the body False positive : Results could also occur due to cross-reactivity with antibodies present as a result of previous infection with other coronaviruses Limitations Data from a recent study showed that antibody levels begin to wane after two months . This could mean that the window for which antibody testing can identify people who have been infected is relatively short, and therefore antibody test results will need to be interpreted with caution Availability: developed by Roche, Beckman-Coulter, EUROIMMUN and Abbott

36. Take Home Message Go for Antigen testing for all the patients in our hospital If negative with symptoms advice Real time RTPCR Herd Immunity is still a long way to reach therefore, Follow all precautions, maintain hygiene, protect yourself by wearing mask and face shield and maintain social distancing THANK YOU !