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18 August 2021

A HANDBOOK ON

COVID-19 TESTING

To inform and guide laboratory personnel and stakeholders

involved in diagnostic testing for SARS-CoV-2

An initiative supported by PATH and the Rockefeller Foundation

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© FIND/Katie G. Nelson 2020

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Acknowledgements

This volume would not have been possible without the support of PATH and the Rockefeller

Foundation. We are grateful to the team at FIND and our partners - Translational Health Science

Technology Institute (THSTI), Molbio Diagnostics Pvt Ltd, Cepheid India Pvt Ltd and ROCHE

Diagnostics, for their gracious inputs and contributions. We owe individual contributors a

significant debt for providing insightful perspective, derived from their on-ground laboratory

experience.

CONTRIBUTORS

Academics (in alphabetical order)

1. Dr Ankur Kumar, Microbiologist VDRL Lab, Dept. of Microbiology, Baba Raghav Das

Medical College, Gorakhpur, Uttar Pradesh

2. Dr Vikas Mishra, Associate Professor, Dept. of Microbiology, Ganesh Shankar Vidyarthi

Memorial Medical College, Kanpur, Uttar Pradesh

3. Dr Amresh K. Singh, Head of Department, Dept. of Microbiology, BRD Medical College

Gorakhpur Uttar Pradesh

FIND, the global alliance for diagnostics (India)

1. Dr. Daniel Bausch, Director of Emerging Threats & Global Health Security

2. Dr. Sanjay Sarin, Director, Country Programmes

3. Dr. Sarabjit Chadha, Regional Technical Director, India & SEA

4. Dr. Archana Beri, Medical Officer

5. Dr. Kamlesh Bhatt, Microbiologist

6. Ms. Keertana Duppala, Project Manager

7. Dr. Jogeswar Gadi, Laboratory Specialist

8. Ms. Beatrice Gordis, Communications Officer

9. Ms. Natalie Jotikasthira, Communications Officer

10. Ms. Kritika Kamthan, Communications Lead

11. Dr. Mayank Mittal, Microbiologist

12. Ms. Rajashree Sen, Deputy Head-Programs and Partnership

13. Dr. Prashant Kumar Singh, Project Coordinator

14. Dr. Navneet Tewatia, Project Manager

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PATH

1. Dr. Nimmy Dominic, Technical Officer

2. Ms. Isha Jain, Senior Communications Coordinator

3. Mr. Praveen Kandasamy, Senior Program Officer (Laboratory Strengthening)

4. Ms. Preetishirin Katapur, State Programme Officer (Punjab)

5. Dr. Mayank Sharma, Senior Programme Officer

Translational Health Science Technology Institute

1. Prof. Guruprasad R. Medigeshi, Laboratory Director, Bioassay Lab

2. Dr. Madhu Pareek, Technical Officer, Bioassay Lab

3. Mr. Ananth Raj, Technical Officer, Bioassay Lab

4. Mr. Sharanabasava, Technical Manager, Bioassay Lab

Industry

1. Dr. Shivraj Bhoite, Manager, Technical Services, Molbio Diagnostics Pvt Ltd

2. Mr. Sanjay Singh, Project Specialist, ROCHE Diagnostics

3. Mr. Varun Suneja, Product Manager, Cepheid India Pvt Ltd

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Table of Contents

1.1 Facts about COVID-19 .................................................................................................... 7

1.2 Testing for COVID-19 .....................................................................................................10

1.3 COVID-19 prevention, control and treatment ..................................................................13

2.1 Setting up a COVID-19 testing laboratory for optimal workflow and safety .....................17

2.2 The layout of an RT-PCR laboratory ...............................................................................22

2.3 Equipment & consumables required for COVID-19 RT-PCR testing laboratory ..............23

2.4 Specimen collection and transport ..................................................................................23

2.5 RT-PCR testing and guidelines for COVID-19 ................................................................29

2.6 COVID-19 immunoassays ..............................................................................................34

2.7 Quality management mechanisms in the laboratory .......................................................37

2.8 Guidance on COVID-19 test results ................................................................................40

2.9 Laboratory safety measures ...........................................................................................44

2.10 Biosafety management ................................................................................................46

2.11 Laboratory waste management ....................................................................................47

3.1 Advisory on strategy for COVID-19 testing in India .........................................................48

3.2 Frequency of testing .......................................................................................................50

3.3 Information about COVID-19 test kit manufacturers........................................................53

3.4 Concluding note .............................................................................................................54

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Abbreviations

BSC biosafety cabinet

EQA external quality assessment

ICMR Indian Council of Medical Research

ILI influenza-like illness

MERS Middle East respiratory syndrome

LAF laminar air flow

MOHFW Ministry of Health and Family Welfare

NAAT nucleic acid amplification test

PPE personal protective equipment

QA quality assurance

QC quality control

RDT rapid diagnostic test

RT-PCR reverse transcription polymerase chain reaction

SARS severe acute respiratory syndrome

SARS-CoV-2 severe acute respiratory syndrome coronavirus-2

SRF specimen referral form

WHO World Health Organization

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Overview

With the sudden outbreak of the deadly COVID-19 pandemic that hit the world at the end of 2019,

and the problems arising ever since, there is an urgent need for reliable and efficient laboratory

tests to diagnose SARS-CoV-2, the virus that causes COVID-19. The crucial role played by

laboratories in helping people obtain an accurate diagnosis and effective care has never been

more evident than during the current pandemic.

This document aims to provide complete and accessible information about COVID-19 and its

diagnosis. Created especially for laboratory personnel and stakeholders involved in laboratory

work, this “handbook” on COVID-19 Testing on COVID-19 testing includes guidance on various

diagnostic techniques for COVID-19. Aside from providing basic information about COVID-19

testing techniques and biosafety measures, this report also covers information about the standard

procedures needed to set up a COVID-19 laboratory and how to carry out validation and approval

of test kits for COVID-19 using antigen rapid diagnostic tests (Ag-RDTs) and molecular

diagnostics, like polymerase chain reaction (PCR).

It is hoped that this document will act as a handy and useful guide for laboratory personnel

involved in COVID-19 diagnosis.

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UNDERSTANDING COVID-19 This section covers the basics about the SARS-CoV-2 virus, COVID-19 disease, its symptoms,

prevention, control, and treatment, before delving into deeper aspects related to COVID-19

diagnostics, i.e., testing procedures, mechanisms and practices. The first chapter comprises a

question/answer format to act as a quick reference source about some of the most pertinent queries

related to COVID-19.

1.1 Facts about COVID-19

What is a coronavirus?

©US Government department: Public Health Image Library, Centers for Disease Control and Prevention

Coronaviruses are a large family of viruses that can cause illness in humans and animals. In

humans, several coronaviruses are known to cause respiratory infections, ranging from the

common cold to more severe diseases, such as Middle East respiratory syndrome (MERS) and

severe acute respiratory syndrome (SARS). A coronavirus discovered in 2019, SARS-CoV-2 is

responsible for the ongoing COVID-19 pandemic1.

1https://www.hopkinsmedicine.org/health/conditions-and-diseases/coronavirus

CHAPTER 1

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What is COVID-19?

COVID-19 is an infectious disease caused by a newly discovered coronavirus, named

SARS-CoV-2. The World Health Organization (WHO) first learned of this new virus following a

report of a cluster of cases of viral pneumonia in Wuhan, China2.

What are the symptoms of COVID-19?

The most common symptoms of COVID-19 are fever,

dry cough, fatigue and others as listed in Table 1

below.

Individuals of any age who experience fever and

cough along with difficulty in breathing, shortness of

breath, chest pain or pressure, or loss of speech or

movement should seek medical care immediately. If possible, the individual’s healthcare provider

should be contacted; otherwise, the national helpline number, 1075, or the nearest health facility

should be contacted immediately, so that he/she can be directed to the correct clinic.

Table 1: COVID-19 symptoms

Most common symptoms

Other symptoms

Less common symptoms

Symptoms of severe COVID-19 disease

Fever, dry

cough, fatigue

Loss of taste or

smell, nasal

congestion,

conjunctivitis, sore

throat, headache,

muscle or joint pain,

skin rash, nausea or

vomiting, diarrhoea,

chills, or dizziness

Irritability, confusion,

reduced consciousness

(sometimes associated

with seizures), anxiety,

depression, sleep

disorders, more severe

and rare neurological

complications such as

stroke, brain inflammation,

delirium, and nerve

damage

Shortness of breath,

loss of appetite,

confusion, persistent

pain or pressure in

the chest,

temperature (37.8C

or higher)

2https://covid19.who.int/

In the name “COVID-19”, “CO”

stands for “corona”, “VI” for “virus”,

“D” for “disease” and “19” for

“2019”.

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What are the effects on people who are infected with COVID-19?

Among those who develop symptoms of COVID-19, most individuals (about 80%) recover from

the disease without requiring hospitalization. About 15% of individuals become seriously ill and

require oxygen, while 5% become critically ill and need intensive care. Complications leading to

mortality may include respiratory failure, acute respiratory distress syndrome, sepsis and septic

shock, thrombo-embolism, and/or multi-organ failure, including injury to the heart, liver and

kidneys. In rare situations, children can develop a severe inflammatory syndrome, a few weeks

following infection2.

Who is most at risk of severe illness with COVID-19?

People aged 60 years or more and those with underlying medical problems or comorbidities, such

as hypertension, heart and lung disease, diabetes mellitus, obesity and cancer, are at higher risk

of developing serious illness due to COVID-19. However, anyone can be infected and become

seriously ill at any age.

Are there long-term effects of COVID-19?

Some people who have had COVID-19, regardless of whether they required hospitalization,

continue to experience symptoms even after recovery from the acute phase of the infection, a

syndrome sometimes known as ‘long COVID-19’. These symptoms can include fatigue and

respiratory and neurological problems. Some patients may experience anxiety or depression and

have trouble sleeping.

How can we protect others and ourselves from COVID-19?

We can reduce the risk of being infected with COVID-19 by taking some simple precautions, as

outlined below.

Simple precautions to protect yourself and others from COVID-19 ✓ Strictly observe physical distancing (approximately 2 meters) ✓ Wear a good-quality mask that properly covers your nose and mouth ✓ Avoid crowded and poorly ventilated indoor spaces ✓ Thoroughly wash your hands regularly with soap and water; make frequent use of hand

sanitizer if soap and water are not available ✓ Get vaccinated against COVID-19 as per WHO and government guidelines ✓ Avoid coughing and sneezing in the proximity of others and make sure you cough into your

elbow or a tissue

✓ If you are infected with COVID-19 or have been exposed to a person who has COVID-19, you must quarantine or self-isolate for a 14 days to prevent further spread of the virus

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How is COVID-19 transmitted?

Recent evidence suggests that the SARS-CoV-2

virus is transmitted primarily via the airborne

route 3 , such as through coughing, sneezing,

shouting, singing etc. Although other routes of

transmission exist, the airborne route is thought

to be the main one. Further research is ongoing

to better understand the spread of the virus and

the settings that pose most risk for contracting the

disease. Research is also underway to study the

new variants of the virus that are emerging and why

some variants are more transmissible4.

1.2 Testing for COVID-19

When should someone get tested for COVID-19?

Any individual with COVID-19 symptoms should get tested immediately. People who do not have

symptoms but have come into contact with an infected person should be tested. Individuals

awaiting a test result should remain isolated to avoid spreading the infection to others. In situations

where testing capacity is limited, priority for testing should be given to those who are at higher

risk of infection and severe disease, such as health workers, elderly people, and those with

comorbidities. Local guidelines on testing should be followed.

3 Trisha Greenhalgh et al “Ten scientific reasons in support of airborne transmission of SARS-CoV- 2” The Lancet, vol 397, ISSUE

10285, P1603-1605, MAY 01, 2021; https://www.thelancet.com/article/S0140-6736(21)00869-2/fulltext 4 Coronavirus disease (COVID-19): How is it transmitted? (who.int)

© US Government department: The National Institute of Allergy and Infectious Diseases.

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What tests are used to diagnose COVID-19?

There are two types of diagnostic tests available to detect a current COVID-19 infection.

These are nucleic acid amplification tests (NAATs) and antigen rapid diagnostic tests (Ag

RDTs). To detect a past infection, an antibody test (serology test) can be performed. An

antibody test cannot be relied upon to diagnose a current infection.

What are NAATs?

Molecular diagnostic testing for COVID-19 is crucial for controlling and preventing the spread of

the disease. Reverse transcription polymerase chain reaction (RT-PCR), a type of NAAT, is the

most widely used molecular test for COVID-19. The test detects virus present in the sample by

amplifying the viral genetic material to detectable levels. Such a test can be used to confirm an

active infection, usually within a few days of exposure and around the time when symptoms

become apparent.

It is important that all areas/states affected by COVID-19 take steps to rapidly test symptomatic

individuals for COVID-19. Successful testing depends on the availability and efficiency of testing

facilities in the affected areas.

Useful FAQs about RT-PCR testing

➢ Should individuals who have already had a positive RT-PCR test for COVID-19 be tested again?

• An RT-PCR test should not be repeated for any individual who has previously tested positive, either by Ag-RDT or RT-PCR unless they show symptoms again.

➢ Is COVID-19 testing necessary for individuals who have recovered from the disease?

• No testing is required for individuals who have recovered from COVID-19. ➢ Is a test report necessary for domestic travel?

• The requirement for an RT-PCR test in healthy individuals undertaking interstate domestic travel may be removed to reduce the workload in laboratories.

➢ Are mobile testing facilities available?

• Mobile testing laboratories are now available on the GeM portal. States are encouraged to augment RT-PCR testing with mobile systems.

(Government e-Marketplace: Procurement Made Smart | National Portal of India)

The above information is derived from new guidelines (dated 4 May 2021) on RT-PCR testing5, released by ICMR during the second wave of COVID-19.

5ibid

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What are Ag-RDTs?

Antigen rapid diagnostic tests (Ag-RDTs), also referred to as rapid antigen tests, are used to

detect viral proteins, known as antigens. These are point-of-care tests that are cheaper and

provide more rapid results than RT-PCR, although they are generally less sensitive. Ag-RDTs are

very useful in situations where there is a high prevalence of COVID-19 and when samples are

taken from symptomatic individuals. If an Ag-RDT result is positive, it indicates that the individual

has COVID-19; however, if the result is negative but the individual is symptomatic, then an RT-

PCR test should be performed for confirmation.6

Useful FAQs on Ag-RDTs

➢ What are the recommended use-cases for Ag-RDTs?

• Ag-RDTs are allowed at all available government and private healthcare facilities as

well as dedicated Ag-RDTs booths in cities, towns and villages.

➢ What are the main guidelines for setting up testing booths?

• Testing booths may be set up in various locations, including healthcare facilities,

resident welfare associations, offices, schools, colleges, community centres and other

available vacant spaces. The booths should operate on a 24–7 basis to improve

access to testing. Drive-through Ag-RDT facilities may be established at convenient

locations, as identified by the local administration. Stringent measures must be

instituted to avoid overcrowding at testing facilities.

➢ Should vaccination status be mentioned in the sample referral form?

• The vaccination status of individuals tested for COVID-19 must be entered in the ICMR

sample referral form (SRF) for both individuals tested by RT-PCR as well as Ag-RDTs.

This information is very important.

As per the new guidelines (dated 4 May 2021) on Ag-RDTs, released by ICMR during the

second wave of COVID-19.

Is there a test I can take to find out whether I have had COVID-19 in the past?

Antibody tests can tell us whether someone has had an infection in the past, even if they did not

have any symptoms. Also known as serological tests, and usually performed on a blood sample,

these tests detect antibodies produced in response to an infection. In most people, antibodies

start to develop after a few days to weeks and can indicate if a person has had a past infection.

4Advisory_COVID_Testing_in_Second_Wave_04052021 (1).pdf

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Antibody tests cannot be used to diagnose COVID-19 during the early stages of infection.

Detecting an active infection requires an RT-PCR test or an Ag-RDT.

NB. More information on testing can be found on pp 29−37 of this document.

1.3 COVID-19 prevention, control and treatment

What is the difference between “‘isolation” and “quarantine”?

Isolation and quarantine are measures that can be used to help prevent the spread of COVID-19.

Quarantine means restricting the movement of anyone who has come into contact with someone

infected with COVID-19 to see if they become sick, irrespective of whether the infected person

had symptoms. Quarantine can take place in a designated facility or at home. For COVID-19,

quarantine should last anywhere from 7−14 days.

Isolation separates those who have tested positive for infection from the general population to

avoid spread of disease. In some cases, the patient may require hospitalization. If isolation in a

medical facility is not required/possible and one is not at high risk of developing severe disease,

isolation can take place at home. Patients with symptoms should remain in isolation for 10−14

days (from the date of the last day of symptoms). Infected individuals who are asymptomatic

should remain in isolation for 10 days from the time they test positive for COVID-19.

What should I do if I have been exposed to someone who has COVID-19?

If you have been exposed to someone who tested positive for COVID-19, you may have been

infected, even if you feel well and do not show any symptoms. In such a situation, it is important

that you take the following steps to help minimize the spread of the virus:

➢ Call your healthcare provider or COVID-19 hotline to find out where and when you can

have a test.

➢ Cooperate with contact-tracing procedures to help stop the spread of the virus.

➢ If testing is not available, stay home and away from others for 14 days.

➢ While you are in quarantine, do not go to work, school or public places. Ask someone to

bring you supplies.

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➢ Keep at least a 2-metre distance from others, even your family members.

➢ Wear a medical mask to protect others, including if/when you need to seek medical care.

➢ Wash your hands frequently with soap and water or use hand sanitizer (if soap is not

available).

➢ Stay in a separate room from other family members; if this is not possible, wear a medical

mask.

➢ Keep your room well ventilated.

➢ If you must share a room, place beds at least 2-metres apart.

➢ Monitor yourself for any symptoms for 7 to 14 days, taking your temperature using a

thermometer and, if possible, monitoring your oxygen levels using a pulse oximeter.

➢ Stay positive by keeping in touch with loved ones by phone or online; try to do some

exercise at home.

➢ If you live in an area with malaria or dengue fever, seek medical help if you have a fever.

➢ While travelling to and from a health facility and during medical care, wear a mask, keep

at least a 2-metre distance from other people and avoid touching surfaces with your hands.

This applies to both adults and children.

How long does it take to develop COVID-19 symptoms?

The time from exposure to COVID-19 to the time when symptoms begin, known as the incubation

period, is on average 5 to 6 days, although it can range from 1 to 14 days. Therefore, people who

may have been exposed to the virus are advised to remain at home and stay away from others,

ideally for 14 days, to prevent the spread of the virus. This is particularly important if testing

facilities are not easily available.

Is there a vaccine against COVID-19?

Yes. There are several COVID-19 vaccines authorized for use by different national regulatory

authorities. The vaccines approved for use in India are Covishield®, Covaxin®, Sputnik V, and

more recently one by Johnson & Johnson. The vaccines can be obtained by visiting the

government portal7.

7https://www.cowin.gov.in/

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Is there a treatment for COVID-19?

Scientists around the world are working to develop treatments for COVID-19. Optimal

supportive care for COVID-19 includes oxygen for severely ill patients and those who are at

risk of severe disease. Critically ill patients require more advanced respiratory care and may

need ventilator support. Corticosteroids can help reduce the length of time a patient requires

ventilation and can help to save the lives of patients with severe COVID-19 infections.

Results from the WHO solidarity therapeutics trial indicated that the drugs remdesivir,

hydroxychloroquine, lopinavir/ritonavir and interferon regimens appear to have little or no

effect on 28-day mortality or the in-hospital course of COVID-19 among hospitalized patients.

National guidelines on the same, published by the Ministry of Health and Family Welfare

(MOHFW), India, can also be referenced.

WHO does not recommend self-medication with any medicines, including antibiotics, as a

prevention or cure for COVID-19. WHO is coordinating efforts to develop treatments and will

continue to provide new information as it becomes available. The MOHFW has published

guidelines (dated 22 April 2021)8 on the clinical management of adults with COVID-19.

8https://www.mohfw.gov.in/pdf/COVID19ManagementAlgorithm22042021v1.pdf.

© US Government department: Public Health Image Library, Centers for Disease Control and

Prevention

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Are antibiotics effective in preventing or treating COVID-19?

Antibiotics only work against bacterial infections and thus should not be used to treat COVID-

19. However, in hospitals, doctors may sometimes prescribe antibiotics to prevent or treat

secondary bacterial infections, which can be a complication of COVID-19 in severely ill

patients. Antibiotics should only be used as directed by a physician.

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COVID-19 LABORATORY OVERVIEW

This section covers the essentials of COVID-19 diagnostics, i.e., lab setup, testing procedures,

mechanisms and practices, quality control mechanisms, biosafety and waste management

considerations.

2.1 Setting up a COVID-19 testing laboratory for optimal

workflow and safety

COVID-19 testing lab in Chennai, India © FIND/YRG Care 2020

Site readiness assessment and its importance

A site readiness assessment is a standard procedure used to determine if a laboratory is well

enough equipped to begin testing. It includes a checklist of questions that help to identify any

gaps and limitations that must be addressed when setting up a COVID-19 testing laboratory,

for example. It is mandatory for every COVID-19 laboratory to conduct a readiness

CHAPTER 2

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assessment, as even in an established laboratory, the safety, workflow, capacity and

equipment must be reviewed against the specific requirements for COVID-19 testing.

The readiness assessment reviews all areas relevant to the implementation of new tests. If

you are intending to use existing platforms, such as CBNAAT (GeneXpert) or Truenat™

(widely used for COVID-19 testing), or if laboratories do not have a BSC (e.g. they are

processing sputum for Xpert MTB/RIF testing on the bench), then this will be a limitation.

Hence, this aspect should be a key consideration when selecting a laboratory for COVID-19

testing9,10,11,12.

Key requirements for a COVID-19 testing laboratory

➢ Presence of trained laboratory staff who can cope with the workload.

➢ Adequate PPE that is available at all workstations.

➢ Implementation of appropriate spill and exposure procedures, waste

management procedures, stock management procedures, and security and

access control procedures.

➢ Availability of sample prioritization procedures, sample receipt procedures and

suitable sample storage facilities.

➢ Availability of a serviced and certified BSC for sample processing.

➢ Equipment that is correctly installed with adequate infrastructure, i.e.

uninterrupted power, sufficient workspace, a low dust environment, and

equipment has been serviced according to the manufacturer’s specifications and

is performing as expected.

➢ Appropriate results reporting procedures.

9https://apps.who.int/iris/handle/10665/331714&WHO-2019-nCoV-Lab_Assessment_Tool-2020.2-eng.pdf 10https://www.who.int/publications/i/item/assessment-tool-for-laboratories-implementing-covid-19-virus-testing 11https://ugc.futurelearn.com/uploads/files/fd/05/fd056db9-1817-4436-9481-e059c7295066/TA_MOOC_1.5_FIND_COVID-19_GUIDE-24.03.2020.pdf 12https://ugc.futurelearn.com/uploads/files/56/12/561292fe-ee46-4555-817d-61db15abb69e/1.9_RR_table_AT.pdf

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FAQs related to the site readiness assessment

What are the next steps once a site readiness assessment has been carried out?

➢ Have the readiness assessment reviewed by the facility staff and management.

➢ Highlight areas that have been accomplished and discuss any gaps along with improvements

that are needed.

➢ Identify the staff members responsible for addressing any identified gaps.

➢ Assign a timeline for completion of the tasks.

➢ Confirm that all gaps have been addressed before COVID-19 testing begins.

Which standard operating procedures, forms or reports must be created or updated?

During the site assessment, make a comprehensive list of documents that need to be developed

or modified to include COVID-19 procedural details. These documents may comprise SOPs,

bench aids, manuals or forms and include:

➢ Daily laboratory checklists

➢ Disinfectant preparation forms

➢ Sample reception registers

➢ Sample acceptance/rejection criteria

➢ BSC maintenance and servicing form

➢ Sample processing SOP

➢ SOPs and bench aids for all new equipment and consumables provided for COVID-19

testing, including maintenance instructions

➢ Results reporting form and SOP

➢ Temperature chart forms

How can you update SOPs, forms and reports for COVID-19 testing?

➢ Once you have identified the documents to be updated or written, assign the tasks to specific

people. Dividing the workload will ensure its timely completion.

➢ Set a timeline for the updating or writing of documents to be completed.

➢ Remove outdated documents from the laboratory and quality management system.

➢ Train staff in the changes, ensuring that they understand the content. Staff must sign a form

to confirm that they have read the revised or new document(s).

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How do I organize COVID-19 testing workflow in the laboratory?

It is crucial that the laboratory is systematically arranged so that patients and samples undergo a

streamlined process. This will also minimize the biohazard risk and improve efficiency in the

workflow. Some key laboratory areas and activities are described in Table 2 below13,14,15,16,17,18.

Table 2: Key laboratory areas and activities

Patient reception and sample collection area

This is the only area accessible to patients where

their samples are collected. Laboratories must

ensure that suitable SOPs are followed, and that

staff are trained in appropriate specimen

collection, storage, packaging, and transport.

Sample processing area

Multiple sample processing procedures are

performed in this area, including direct sampling on

cartridges, aliquoting of samples, and other sample

preparation techniques.

Instrumentation room

This is where the PCR instrument is installed.19 It

should be separated from the other activities to

protect the machine and ensure its cleanliness, as

well as to avoid cross-contamination while

performing tests.

Documentation or laboratory coordination room

This is where all non-diagnostic activities are

conducted, such as lab coordination and

administrative work.

A good laboratory workflow guarantees work optimization, adherence to infection control and

cross-contamination reduction measures, the safety of laboratory staff and the reliability of

diagnostic tests. Generally, activities assigned to one room should not crossover to another room

or area. This is to ensure strict infection and contamination control. An important aspect of

implementing laboratory workflow processes is good communication with laboratory workers (e.g.

in the form of SOPs, laboratory rules, bench aids), as well as providing advanced training on

essential infection control.

13https://www.icmr.gov.in/pdf/covid/labs/Advisory_on_sample_collection_strategies.pdf 14https://www.icmr.gov.in/pdf/covid/labs/Govt_labs_sample_retention_advisory_25062020.pdf 15https://www.icmr.gov.in/pdf/covid/labs/Revised_SRF_Form_16122020_1.pdf 16https://www.icmr.gov.in/pdf/covid/labs/SRF_ID_for_COVID_06052020.pdf 17Covid-19 Sample Collection Management System (covid19cc.nic.in) 18https://cdsco.gov.in/opencms/resources/UploadCDSCOWeb/2018/UploadPublic_NoticesFiles/approved%20PCR%20kits-17-04-200.pdf

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Training and competency assessment of laboratory staff

Adequate staff training and implementation of a competency assessment is essential prior to

starting any COVID-19 testing in a laboratory and when any new staff members are brought on

board to support testing. Training in COVID-19 testing should include both theoretical and

practical/hands-on training with trainer-led competency assessment. Training can also be

conducted by the test manufacturer or the authorized service provider. Some relevant

areas/topics for specific staff members are summarized in Table 3.

Table 3: Training topics for laboratory staff

Sample collection personnel

• Specimen collection,

identification, packaging

and storage of samples

• Appropriate sample

collection techniques

• Proper sample

identification, packaging

and storage

Sample collection

personnel and couriers

• Specimen packaging

and transport

Laboratory personnel/technicians

• Assay-specific training sessions

• COVID-19 testing techniques

and quality assurance, including

proficiency testing (PT), external

quality assessment (EQA) and

reporting of results

• Equipment management

• Operation and maintenance of

laboratory equipment

• Stock management

• Laboratory inventory

management cycle.

Data management personnel

• Data management for

COVID-19

• Analysis and interpretation

of COVID-19 testing data

Quality assurance officer

• Laboratory quality

assurance

• Implementation of

essential elements of

quality assurance in the

laboratory

Biosafety officer

• Biosafety

• Effective implementation of

safety guidelines in the

laboratory

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2.2 The layout of an RT-PCR laboratory

This section outlines the laboratory layout required for COVID-19 RT-PCR testing and the

equipment, supplies and consumables that such laboratories must have.

Suggested laboratory layout for COVID-19 RT-PCR testing highlighting the unidirectional

workflows.

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2.3 Equipment & consumables required for COVID-19 RT-PCR

testing laboratory

Equipment

1. Three Level II BSCs

2. Laminar air flow cabinet

3. Vortex mixer

4. Micropipettes – Four sets

5. Two Mini centrifuge -2 (6000 rpm)

6. High-speed cooling centrifuge

(15,000 rpm)

7. Freezers (-20 and -80C)

8. Two Refrigerators (2–8C)

9. 96-well plate rocker/shaker

10. RT-PCR machine with laptop

11. Two Desktop computers

12. Biowaste disposal dustbin

containers (yellow, red, blue)

13. Autoclave

Consumables

1. PCR tubes (0.2 ml), multi well strips,

multi well plates and sealers

2. Eppendorf tubes, 2 ml

3. Cryovials, 2 ml

4. Micropipette tips

5. Ethanol, 100%, molecular grade

6. Silver nitrate

7. Viral RNA extraction kit

8. COVID-19 PCR kits (Truenat, CBNAAT

and RT-PCR)

9. Biowaste disposal container

10. Poly-covers for bio-waste disposal

containers (yellow, red, blue)

11. Disposable Nitrile Gloves

12. Coveralls

13. Respirators (N-95/FFP2 NR)

14. Face shields

15. Goggles

16. Permanent markers

17. Registers for stock, sample, and

indicators

18. Viral transport medium

19. Ag-RDT kits

2.4 Specimen collection and transport

It is important that samples are collected in compliance with all biosafety precautions using

standard triple-packaging and that they are accompanied by a detailed patient history on a

proforma that can be obtained from the testing laboratory. PPE (aprons, gloves, face shields,

24

N95 respirator masks, etc.) must be worn and all biosafety precautions should be followed

while carrying out sample collection and packaging. Samples should be transported under

cold chain to the reference laboratory, with prior notice given. The triple packaging should

comprise three layers, as follows19:

• Primary receptacle: a labelled, watertight, leak-proof primary receptacle containing the

specimen. The receptacle must be wrapped in sufficient absorbent material to absorb all

fluid in case of breakage.

• Secondary receptacle: a durable, water-tight, leak-proof secondary receptacle to

enclose and protect the primary receptacle(s). Several wrapped primary receptacles may

be placed in one secondary receptacle. Sufficient additional absorbent material must be

used to cushion multiple primary receptacles.

• Outer shipping package: the secondary receptacle should be placed in an outer

shipping package that protects it and its contents from outside influences, such as

physical damage and water, while in transit. Specimen data forms, letters and other types

of documents that indicate the specimen is intended for “SARS-CoV-2 – Severe acute

respiratory disease” testing and that identify the shipper and receiver, should be taped

to the outside of the secondary receptacle.

The successful diagnosis of SARS-CoV-2 depends on the quality of the specimen and the

conditions under which the specimen is transported and stored prior to being processed in

the laboratory13.

FAQs about sample collection and transportation

What is viral transport medium (VTM)?

Viral transport medium (VTM) enables the safe transport of viruses and organisms such

as Chlamydia and Mycoplasma for further research or analysis, including conventional

cell culture methods, diagnostic tests and molecular biology techniques. Commercially

prepared VTM are available in screw-cap plastic tubes containing buffered proteins

(serum albumin or gelatin) and antibiotics. Antibiotics are usually incorporated into VTM

to suppress the growth of contaminating bacteria and fungi. Therefore, separate

samples from the same site should be collected if bacterial or fungal cultures are also

requested.

25

What are the guidelines for lower respiratory tract specimen collection?

Collection of specimens from the lower respiratory tract (LRT) is advised if the patient is

in the later stages of disease or if an upper respiratory tract specimen tests negative

despite having a strong clinical suspicion of COVID-19. LRT specimens may comprise

spontaneously produced sputum, endotracheal aspirate or bronchoalveolar lavage.

Why are both nasopharyngeal (NP) and oropharyngeal (OP) samples sometimes

collected?

If both NP and OP specimens are collected, they should be combined in a single tube

to maximize the test sensitivity, as viral presence varies in OP and NP locations during

COVID-19 infection.

What other samples can be tested for COVID-19?

Bronchoalveolar lavage, tracheal aspirate, pleural fluid, and lung biopsy samples can

be used as specimens to test for COVID-19 if they are collected under expert guidance;

these samples may be limited to patients that have more severe disease, including

people admitted to hospital and individuals who have died.

COVID-19 sampling:

COVID-19 sampling should be conducted as per prevailing guidelines. A detailed sampling

procedure is described below20

Nasopharyngeal specimen (NP) collection /Oropharyngeal (OP) (throat) specimen

collection

It is recommended to use synthetic fiber swabs specially designed for sampling

nasopharyngeal mucosa. Guidelines recommend collecting only NP specimen, although an

OP specimen is an acceptable specimen type. If both NP and OP specimens are collected,

it is recommended to combine them in a single tube for maximized test sensitivity and

rationalized use of testing resources.

20 Interim Guidelines for Clinical Specimens for COVID-19 | CDC

26

Collection of Nasopharyngeal (NP) specimen:

• Patient’s head to be tilted to 70 degrees (as per figure below )

• Swab to be inserted slowly through the nostril parallel to the palate until resistance is

encountered or to cover a distance that is equivalent to that from the ear to the nostril of

the patient, indicating contact with the nasopharynx.

• Swab to be rolled 5 times

• Swab to be removed while rotating it. Specimens can be collected from both sides using

the same swab, but it is not necessary to collect specimens from both sides if the mini tip

is saturated with fluid post first collection

• Swab to be placed, tip first, into the viral transport medium tube provided in the kit.

Nasopharyngeal (NP) sample collection20

Collection of Oropharyngeal (OP) specimen:

• Swab to be inserted into the posterior pharynx and tonsillar areas.

• Swab to be rotated over both tonsillar pillars and posterior oropharynx while avoiding

grazing of tongue, teeth, and gums.

• Swab to be placed, tip first, into the viral transport medium tube provided in the kit.

Collection of Nasal mid-turbinate (NMT):

• Use of a tapered swab is recommended

• Patient’s head to be tilted to 70 degrees.

• Swab to be inserted to less than 1 inch (about 2 cm) into the nostril (while gently rotating

the swab) so that the swab is parallel to the palate (not upwards) until resistance is met

• Swab to be rotated several times against the nasal wall and procedure to be repeated in

other nostril using the same swab.

• Swab to be placed, tip first, into the viral transport medium tube provided in the kit

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Collection of anterior nasal specimen:

• The entire collection tip of the swab to be inserted inside the nostril.

• Rotation of the swab in a circular path against the nasal wall at least 4-5 times (to firmly

sample the nasal wall)

• Collection of the specimen to take approximately 15 seconds with careful collection of

any nasal drainage that may be present on the swab

• Procedure to be repeated in the other nostril using the same swab if needed

• Swab to be placed, tip first, into the viral transport medium tube provided in the kit

Collection of lower respiratory tract (LRT) samples:

Collection of Bronchoalveolar lavage (BAL), tracheal aspirate, pleural fluid, lung biopsy (By a physician)

• Collection of at least 2-3 mL of samples into a sterile, leak-proof, screw-cap sputum

collection vial or sterile dry container

• This sampling is for patients with severe disease, including patients admitted to the

hospital and/or fatal cases

• Collection of this type of sample requires technical skills and equipment

Sputum collection:

• This type of sample to be collected from patients with a productive cough. Sputum of the

patient to be collected and tested when available for SARS-CoV-2

• Sputum induction is not recommended

• The procedure to commence with explanation to the patient regarding difference between

sputum (deep cough) and oral secretions (saliva/spit).

• Patient must be requested to rinse his/her mouth with drinking water and then

expectorate deep cough/ sputum directly into a sterile, leak-proof, screw-cap collection

vial or sterile dry container

Storage:

The respiratory specimens should be stored at 2-8°C after collection, if the samples can be

transported within 2-5 days as detailed in the table below. If a delay in testing or shipping is

expected, specimens to be stored at -70°C or below. The specimen type and storage details

are tabulated in Table 4 below.

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Table 4: Specimen type and storage details

Specimen type Collection materials Storage temperature until testing in-country laboratory

Recommended temperature for shipment according to expected shipment time

Nasopharyngeal and oropharyngeal swab

Dacron or polyester flocked swabs

2-8oC 2-8oC if ≤5 days -700C (dry ice) if >5 days

Bronchoalveolar lavage

Sterile container 2-8oC 2-8oC if ≤2 days -700C (dry ice) if >5 days

(Endo) tracheal aspirate, nasopharyngeal or nasal wash/aspirate

Sterile container 2-8oC 2-8oC if ≤2 days -700C (dry ice) if >5 days

Sputum Sterile container 2-8oC 2-8oC if ≤2 days -700C (dry ice) if >5 days

Diagnostic accuracy of different samples:

The guidelines by CDC and WHO recommend Nasopharyngeal (NP) and Oropharyngeal

(OP) samples for their high diagnostic accuracy for detection of SARS-CoV-2 by Ag-RDTs

and RT-PCR.

A recent study 21 has shown that the combined swab (NP+OP) had near 100% accuracy as

compared to NP (91.5%), OP (72.3%) and sputum (63%). Another study22 suggests that,

compared with the gold standard of nasopharyngeal swabs, pooled nasal and throat swabs

showed the best performance of detection. Alternative sampling approaches for diagnosis of

SARS-CoV-2 infection are currently under investigation. Saliva and nasal swabs may also

provide comparable diagnostic performance and are clinically acceptable alternative

specimen collection methods. Throat swabs have much lower sensitivity, positive predictive

value and are not recommended. Self-collection for pooled nasal and throat swabs and nasal

swabs were not associated with any significant impairment of diagnostic accuracy. However,

the diagnostic accuracy of samples also depends on factors including the duration of the

illness and the quality of collection.

21 Sharma K, Aggarwala P, Gandhi D,Mathias A, Singh P, Sharma S, et al. (2021)Comparative analysis of various clinical

specimens in detection of SARS-CoV-2 using rRT-PCR in new and follow up cases of COVID-19 infection: Quest for the best choice. PLoS ONE 16(4): e0249408.https://doi.org/10.1371/journal.pone.0249408 22 Nicole Ngai Yung Tsang, et al, 2021 (Lancet Infect Dis. 2021 Apr 12 doi: 10.1016/S1473-3099(21)00146-8 )

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2.5 RT-PCR testing and guidelines for COVID-19

What is a COVID-19 RT-PCR test?

The COVID-19 RT-PCR test (real-time reverse transcription polymerase chain reaction, rRT-

PCR) is used for the qualitative detection of nucleic acid from SARS-CoV-2 in upper and

lower respiratory tract specimens (such as nasopharyngeal or oropharyngeal swabs, sputum,

lower respiratory tract aspirates, bronchoalveolar lavage, and nasopharyngeal

wash/aspirate) collected from individuals suspected of having COVID-19.

SARS-CoV-2 RNA is generally detectable in respiratory specimens during the acute phase

of infection, clinical correlation with patient history and other diagnostic information is

necessary to help determine a patient’s infection.

What is involved in RT-PCR testing for SARS-CoV-2?

The oligonucleotide primers and probes used for the detection of SARS-CoV-2 were selected

from regions of the virus nucleocapsid (N) gene. The panel (two primer/probe sets) is

designed for the specific detection of SARS-CoV-2. An additional primer/probe set to detect

the human RNase P gene (RP) in control samples and clinical specimens is also included in

the panel.

RNA isolated and purified from upper and lower respiratory specimens is reverse transcribed

to cDNA and subsequently amplified in a real-time PCR instrument. During this process, the

probe anneals to a specific target sequence located between the forward and reverse

primers.

During the extension phase of the PCR cycle, the 5′ nuclease activity of Taq polymerase

degrades the probe, causing the reporter dye to separate from the quencher dye, generating

a fluorescent signal. With each cycle, additional reporter dye molecules are cleaved from

their respective probes, increasing the fluorescence intensity. Fluorescence intensity is

monitored at each PCR cycle by the real-time PCR system.

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COVID-19 testing lab in Chennai, India © FIND/YRG Care 2020

What are the different types of molecular testing platforms used for the detection of SARS CoV-

2?

There are two general types of molecular testing platforms for the detection of SARS-CoV-2:

1. Closed systems: These are proprietary testing systems, where all accessories needed

must be sourced from the test manufacturer. Closed testing systems have standardized

procedures and cannot be programmed to use test accessories sourced from a different

manufacturer. Examples of closed systems include the Abbott m2000 RealTime

System, Cepheid GeneXpert® System, Roche cobas® System and Molbio Truenat™

System. (More details are provided below.)

2. Open systems: Unlike closed systems, open systems can accommodate different kinds

of tests from multiple manufacturers. The standards such as positive control, negative

control and internal control depend on the various components of the kit manufacturers

used. Examples of open systems include: BioRad CFX Systems, Qiagen Rotor-

Gene and Applied Biosystems 7500 Fast Real Time PCR System.

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How do you choose which platform is suitable to conduct COVID-19 testing in the laboratory?

Factors to consider include:

1. Availability of the platform in the country, including reagents and servicing, e.g. is there a

local distributor for the platform/test I am interested in?

2. Appropriate technology based on testing performance, e.g. what is the performance of this

test?

3. Ease of implementation, e.g. is this platform/system already available in the laboratory?

4. Ability to accommodate testing for infections other than COVID-19, e.g. can the platform

also be used to test for tuberculosis, HIV, etc.?

Although there is no single perfect option, a laboratory can ensure efficiency of

implementation by balancing the use of an approved technology, standardization and work

optimization.

What does test performance refer to?

Test performance is an important factor when selecting specific tests and determining the most

appropriate approaches for implementation. The following should be considered when assessing

the performance of a COVID-19 test for implementation:

• Analytical sensitivity: also known as the limit of detection or the lowest

concentration of viral RNA that the test can detect.

• Analytical specificity or cross-reactivity: describes whether the test will detect

other viruses or infections that are not SARS-CoV-2, which can lead to false-positive

results.

• Clinical sensitivity: the ability of a test to correctly identify COVID-19-positive

individuals.

• Clinical specificity: the ability of a test to correctly identify COVID-19-negative

individuals.

While no test is perfect, you should ideally prioritize tests that have both high sensitivity and high

specificity, although there is a trade-off between the two. Additionally, test performance can be

affected by many factors that are independent of the test design, including the prevalence of the

disease in the population being tested. Based on our current understanding of COVID-19 and

existing clinical/public health guidance, it is best to prioritize tests with a low limit of detection

and high clinical sensitivity when selecting a test to diagnose an active infection. This will also

reduce the chance of any COVID-19 cases going undetected.

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What are closed RT-PCR systems?

Closed RT-PCR systems are described below:

Truenat assays: Truenat is a chip-based, point-of-care, rapid molecular test for the diagnosis of

infectious diseases. The technology is based on TaqMan real time RT-PCR. The Indian Council

of Medical Research (ICMR) recommends three types of Truenat assays in its revised guidelines

(dated 24 September 2020) for the screening and confirmation of COVID-19 cases:

a. Assay 1: Truenat Beta CoV E gene screening assay

b. Assay 2: Truenat SARS-CoV-2 RdRp gene confirmatory assay

*Assay 1 must be followed by Assay 2 for confirmation of the results

c. Assay 3: Truenat COVID-19 multiplex assay

Detailed guidelines on the use of multiplex assays are as follows:

a) Two-step single-plex assay

Step 1: E gene screening assay:

• All samples from suspected COVID-19 cases should be tested using this assay first.

• All negative samples should be considered to be true negatives.

• All positive samples should be subjected to confirmation by Step 2.

• Step 2: RdRp gene confirmatory assay: All samples that test positive by this assay

should be considered to be true positives.

b) Single-step multiplex assay**

This assay comprises both the screening (E gene) and confirmatory (Orf1a) targets in a

single test. All samples from suspected cases of COVID-19 can also be tested using this

assay.

• All negative samples should be considered to be true negatives.

• All samples that test positive by this assay should be considered to be true positives.

No further rRT-PCR-based confirmation is required for samples that are confirmed as true

positives by the Truenat assays.

**As single-step multiplex PCR assay kits are available, all Truenat laboratories are now

using these single-step multiplex kits.

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Internal controls in Truenat tests

• Detection of the human RNase P gene serves as a full process internal positive

control (IPC) for proper swab collection, nucleic acid extraction and PCR.

• The IPC should be positive in negative samples and may be negative or positive in

positive samples.

• If the IPC fails to amplify in a negative sample, the test is considered to be invalid.

CBNAAT closed RT-PCR test:

The CBNAAT test is carried out using the GeneXpert® System, which comprises a GeneXpert

instrument, a computer and preloaded software. The specimen is mixed with the sample buffer

and injected into a single-use disposable cartridge that contains the primers, probes (E and N2

genes) and internal controls used for RT-PCR. This cartridge is inserted into the instrument,

where the automated sample processing, RNA isolation, reverse transcription and real-time PCR

is carried out. The software uses a predefined algorithm to obtain the results. The CBNAAT test

for SARS-CoV-2 detects the N2 and E genes from the viral RNA. The test includes two internal

controls:

a) Sample processing control (SPC) The SPC is used to confirm adequate processing

of the sample and to monitor for the presence of potential inhibitor(s) of the RT-PCR

reaction.

1. The SPC also ensures that the RT-PCR reaction conditions (temperature and

time) are appropriate for the amplification reaction and that the RT-PCR

reagents are functional.

2. The SPC should be positive in negative samples and may be negative or

positive in positive samples.

3. The result is invalid if the SPC is negative in a negative sample.

b) Probe check control (PCC) The PCC confirms that all reaction components are

present in the cartridge. It also verifies reagent rehydration and that the PCR tube is full,

including monitoring for probe integrity and dye stability.

1. During the PCC, the system tests the fluorescence readings of the probes at

different temperatures, prior to the start of PCR amplification.

2. A test is aborted if the PCC is not passed.

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3. The test results are based on the detection of two target genes, according to

the preset algorithms. A recent ICMR advisory has outlined the use of this

test for the detection of COVID-19. A Biosafety Level 2 laboratory and the

availability of a functioning and calibrated BSC type 2A or 2B is

recommended. Proper biosafety measures should be taken while performing

this test.

2.6 COVID-19 immunoassays

What types of COVID-19 immunoassays are there?

Rapid diagnostic tests (RDTs) can detect either antigens (Ag) or antibodies (Ab). Both types

of tests have an important role to play in tackling COVID-19, given the current epidemic

context of community transmission.

What are antigen-based rapid diagnostic tests (Ag-RDTs)?

Antigen-based RDTs can detect the presence of SARS-CoV-2 viral proteins (antigens) in

respiratory tract specimens. Many Ag-RDTs have been developed and are commercially

available. Most are lateral flow immunoassays (LFIs), which can typically be completed within

30 minutes. In contrast to NAATs, no amplification of the target occurs, making antigen tests

less sensitive. Additionally, false-positive results (indicating that a person is infected when

they are not) may occur if the antibodies on the test strip also recognize antigens of viruses

other than SARS-CoV-2, such as other human coronaviruses.

The sensitivity of different RDTs compared with RT-PCR in specimens from URT samples

(nasopharyngeal swabs) appears to be highly variable, but specificity is reported to be

consistently high. Currently, data on performance of Ag-RDTs in clinical settings remain

limited but is growing.

Certain independent studies demonstrate heterogeneous performance compared to NAAT

in samples from the upper respiratory tract (sensitivity ranging from 0% to 94% for Ag-RDTs).

However, almost all of these Ag-RDTs have shown high specificity (>97%), and the

performance of some products exceeds minimum performance requirements in patient

35

samples with higher viral loads 23 . Further Ag-RDT validations in clinical studies are

encouraged to further qualify Ag-RDTs that are either under development or have already

been commercialized and demonstrate acceptable performance in representative field

studies.

When their performance is acceptable, Ag-RDTs could form part of a diagnostic algorithm to

reduce the number of molecular tests that need to be performed and to support rapid

identification and management of COVID-19 cases. How antigen detection would be

incorporated into the testing algorithm will depend on the sensitivity and specificity of the Ag-

RDT and the prevalence of SARS-CoV-2 infection in the intended testing population.

Higher viral loads are associated with improved Ag-RDT performance; therefore, test

performance is expected to be best around the time of symptom onset and during the initial

phase of a SARS-CoV-2 infection24. For specific guidance on Ag-RDTs, see the WHO interim

guidance on antigen detection in the diagnosis of SARS-CoV-2 infection using rapid

immunoassays 20.

23 An implementation guide “SARS-CoV-2 antigen-detecting rapid diagnostic tests” , WHO 2020 24Michael J. Mina et. Al. “Rethinking Covid-19 Test Sensitivity — A Strategy for Containment”, N Engl J Med 2020; Nov 2020

High-Frequency Testing with Low Analytic Sensitivity versus

Low-Frequency Testing with High Analytic Sensitivity21

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What is the current ICMR guidance on the use of Ag-RDTs?

ICMR issued an advisory on 4th May 2021, during the second wave of COVID-19 in India, on

the use of Ag-RDTs and guidance on testing25; it can be accessed via the link provided as a

footnote.

What is antibody testing?

Serological assays detect antibodies produced by the human body in response to infection,

for example with SARS-CoV-2. These assays can be useful in various ways. For example,

serosurveillance studies can be used to support the investigation into a past outbreak of

disease as well as supporting the retrospective assessment of the attack rate or the size of

an outbreak. As SARS-CoV-2 is a novel pathogen, our understanding of the antibody

responses it engenders is still emerging and therefore antibody detection tests should be

used with caution. Based on current evidence, WHO does not recommend the use of

antibody-detecting rapid diagnostic tests for patient care but encourages the continuation of

work to establish their usefulness in disease surveillance and epidemiologic research.

Can antibody assays be used to diagnose COVID-19 disease?

Non-quantitative assays (e.g. lateral flow assays) cannot detect an increase in antibody titres,

in contrast to semi-quantitative or quantitative assays. Lateral flow antibody-detection assays

(or other non-quantitative assays) are currently not recommended for the diagnosis of acute

disease and clinical management, while their role in epidemiological surveys is being studied.

For more information on the utility of rapid immunodiagnostic tests, please refer to the WHO

scientific brief for advice on the specific point-of-care immune diagnostic tests for SARS-CoV-

2.

Serology should not be used as a standalone diagnostic tool to identify acute cases in clinical

care or for contact tracing purposes. The interpretation of serological results should be made

by an expert and is dependent on several factors, including the stage of the disease, clinical

25https://www.icmr.gov.in/pdf/covid/strategy/Advisory_COVID_Testing_in_Second_Wave_04052021.pdf

37

morbidity, the epidemiology and prevalence within the setting, the type of test used, the

validation method, and the reliability of the results. Seroconversion (the development of a

measurable antibody response following infection) has been observed to be more robust and

rapid in patients with severe disease compared with those with milder disease or

asymptomatic infections.

Antibodies have been detected as early as the end of the first week of illness in some patients

but can also take weeks to develop in patients with subclinical/mild infection. A reliable

diagnosis of COVID-19 infection based on a patient’s antibody response will often only be

possible during the recovery phase, when opportunities for clinical intervention or interruption

of disease transmission have passed. Therefore, serology is not a suitable replacement for

virological assays to inform contact tracing or clinical management. The duration of the

persistence of antibodies generated in response to SARS-CoV-2 is still under study. It should

also be noted that the presence of antibodies that bind to SARS-CoV-2 does not guarantee

that they are neutralizing antibodies or that they offer protective immunity26.

2.7 Quality management mechanisms in the laboratory

A well-functioning quality management mechanism is an integral aspect of any diagnostic

laboratory. The implementation of a quality management system in diagnostic laboratories

ensures accurate, reliable and efficient test results, as well as standards that are on a par

with international standards. The WHO definition of quality assurance (QA) notes that QA is

a complete process whereby the quality of laboratory reports can be guaranteed, including

the right result at the right time and for the right specimen, with the right interpretation of

results.

The purpose of quality assurance, therefore, is to provide relevant, quality test results that

can be interpreted correctly. It involves activities both inside and outside the laboratory, good

laboratory practice, and proper management skills.

Quality control (QC), on the other hand, covers the part of QA primarily concerned with

controlling errors in the performance of tests and verifying test results. QC must be practical,

achievable and affordable. The broad aim of QC is that results from one laboratory should

26https://www.icmr.gov.in/pdf/covid/kits/Antibody_based_tests_12022021.pdf

38

be comparable with those from any laboratory in the world, provided the same method is

followed. QC can be implemented in two ways:

• Internal or intra-laboratory QC, which is performed by individual laboratories at their

own level. This forms the day-to-day basis of QC.

• External or inter-laboratory QC, which is performed by many laboratories at the

same time and monitored by a single reference laboratory. This is costly and entails

long-term enrolment in QC programmes.

How can QC in the laboratory be ensured?

Regardless of the type of testing being conducted, some practical steps for implementing

and maintaining a QC programme include:

1. Establishment of written policies and standard operating procedures (SOPs). These

should include corrective actions with regard to providers of materials, frequency of

testing, expected results, recording, reporting and the procedure to follow in the case

of QC failure.

2. Training of laboratory staff in all COVID-19-related testing activities and procedures,

including recording of the results and actions to be taken in case of QC failure. It is

important that test results should not be reported in cases where there is a QC failure.

3. Investigation of all QC failures and reporting of the investigation’s findings on a

corrective action form. The corrective action form should document the cause of the

QC failure, the action taken and the effect of the action. Repeated QC failures may

indicate systemic problems that require re-training of staff.

4. Complete documentation, including reporting to the national surveillance database

and maintaining records of QC results and any corrective actions taken.

5. Reviewing of QC data or proficiency testing, as per the manufacturer’s

recommendations.

Key aspects of a well-functioning testing laboratory

A well-defined organogram, SOPs, good laboratory practices, quality control, human

resources, equipment management, reagents, inventory of records, and proper

communication must be incorporated into lab practices, to assure quality. Biosafety

considerations are as per guidelines laid out by WHO, the Institutional Biosafety

Committee, and the Department of Biotechnology, Government of India, for carrying out

diagnostic work in the laboratory27.

27ibid

39

Options available for external quality assurance (EQA) for COVID-19 testing

EQA allows a laboratory’s testing performance to be compared with the performance of a

peer group of laboratories, national reference laboratories or WHO reference laboratories.

There are three EQA methods:

1. Proficiency testing (PT): an external provider sends a blinded, well-characterized

panel at regular intervals (usually quarterly) to a set of laboratories for analysis.

Manufacturer’s recommendations for where to source the EQA panels for COVID-19

testing kits should be followed. Most approved COVID-19 testing kits have initial EQA

panels for use during installation and training. These can be used as an interim EQA

procedure.

2. Rechecking or retesting: samples tested by one laboratory are retested by another

laboratory (inter-laboratory comparison). WHO recommends that specimens of the

first five positive cases and the first ten negative cases that meet the COVID-19 case

definition for testing should be shipped for confirmation to the national or international

reference laboratory for COVID-19.

3. On-site evaluation: usually performed in addition to PT or rechecking and may be

carried out when it is difficult to conduct traditional PT or rechecking/retesting. An

evaluator (e.g. a staff member from the national reference laboratory) will visit the

laboratory to check if the laboratory is meeting quality requirements, retest and verify

a set of test results, and provide advice to correct any faulty procedures. On-site visits

are also important to motivate staff and provide refresher training if needed.

Importance of QA/QC and PT in detecting errors within the laboratory

The laboratory is a complex system, with procedures that involve numerous steps and many

people that must work together seamlessly. Therefore, the quality management system

(QMS) that monitors the entire laboratory system is very important for achieving good

laboratory performance. Implementing QA/QC and PT will help with the early identification of

any errors in the system and reduce the risk of further laboratory errors. Different stages

within the testing cycle have errors that are likely to be encountered. For example,

1. Pre-analytical stage: at this stage, errors relate to the specimen itself. A faulty specimen

is a specimen that is inadequate, collected at the incorrect time, in a dirty or contaminated

container, or has been stored incorrectly (e.g. at temperatures outside the recommended

40

range). Errors may also occur in relation to incorrect or missing test requisition forms

and/or faulty labelling.

2. Analytical stage: at this stage, errors in results can occur if the test principles and

procedures are not strictly followed; if reagents, standards, or QC materials are not

prepared, mixed, or properly processed; and if performance standards are not strictly

followed.

3. Post-analytical stage: at this stage, errors may occur if reporting, checking and

verification are not correctly performed, if the interpretation of test results are not

considered seriously, and if abnormal or unexpected results are not reviewed and/or the

tests repeated.

Pre-analytical stage: faulty, inadequate, or incorrectly collected specimens

Analytical stage: flaws in test procedures or performance standards

Post-analytical stage: incorrect test interpretation or reporting of results

2.8 Guidance on COVID-19 test results Laboratory testing is most likely to benefit the public health response only through well-managed

laboratory practices that produce accurate, error-free, valid and reliable results. This section

therefore provides basic information related to the interpretation, reporting, review and notification

of COVID-19 test results.

Interpreting COVID-19 test results

Routine laboratory confirmation of COVID-19 cases is based on the amplification and detection

of SARS-CoV-2 viral RNA by rRT-PCR, with confirmation by nucleic acid sequencing when

necessary. An example of COVID-19 test results obtained using the Xpert/ Xpress SARS-CoV-2

test is shown in Table 5. The results generated by the GeneXpert System software are

automatically reviewed and are shown in the “View Results” section in each run. There are six

possible test result outcomes for COVID-19, four of which are shown in Table 5.

Table 5: COVID-19 test results interpretation:

ANALYTE - NAME -

Test sample E gene N2 gene SPC COVID-19 test result

Sample #1 +/− + +/− Positive

Sample #2 + − +/− Presumptive positive

Sample #3 − − + Negative

Sample #4 − − − Invalid/Error/No result

41

Invalid, error, no result, or presumptive-positive test results

There are several reasons why a test may give an indeterminate result (invalid, error or no result).

These include internal control failure due to the presence of PCR inhibitors and instrument failure

due to electrical or electronic problems. Presumptive-positive results occur when the test fails to

detect all the possible target genes. In such a situation, the sample should be retested; additional

confirmatory testing may also be required.

The results of closed PCR platforms are also based on PCR cycle threshold (Ct) values. Ct values

between 10 and 45 are considered to be positive for COVID-19, regardless of the Ct value of the

internal control. The test is considered negative for COVID-19 if the Ct value for COVID-19 is zero

and the Ct value for the internal control is between 14 and 30. Invalid test results (where there is

no Ct value for the gene targets and the Ct value of the internal control is either less than 14 or

more than 30) can also occur due to PCR inhibitors and instrument failure. An uninterpretable

result is most likely to occur due to errors made during the extraction step.

What should you do in the event of an uninterpretable result?

✓ Repeat the test using fresh reagents and a new transfer pipette, using the leftover sample

from the original specimen transport medium tube or a new external control tube.

✓ If the test gives an error, invalid, no result or presumptive-positive result a second time,

contact the relevant clinician and request a new sample.

Reporting of results

The results of each test must be reported accurately, clearly, unambiguously and in accordance

with the specific instructions in the examination procedures. COVID-19 test results should be

reported within 24 hours of receiving the sample to allow rapid identification and subsequent

notification of the clinician, should the sample test positive. The following details should be

included on the laboratory report form26:

• Laboratory serial number

• SRF ID

• Specimen type (e.g. nasopharyngeal swab)

• Visual appearance of the specimen (if applicable)

• Test result

• Name of the person who performed the test

• Date when the test was performed28.

26Revised_SRF_Form_22032021_1.pdf (icmr.gov.in)

42

Review of results

All test results must be reviewed and authorized against the original printout. This review must

validate the patient details and test result to ensure that the details match the report and patient

register. In emergency situations, such as the COVID-19 pandemic, results should be reviewed

and authorized as frequently as possible. In addition, cumulative test results must be analyzed

and reviewed after each batch of tests to detect any changes that may indicate procedural

problems, e.g. increased errors when certain staff members are processing the samples or

differences in performance due to different cartridge batch numbers.

Notification of results

In general, all test results, whether positive or negative, should immediately be reported to the

national authorities. Notifications should be made immediately by healthcare providers on

identification of a laboratory diagnosis of SARS-CoV-2 infection. Laboratories are urged to seek

confirmation of any unusual results by an international reference laboratory within or outside the

region. As per WHO guidance for laboratory-confirmed cases, two negative specimens at least

24 hours apart indicate recovery from COVID-19 infection. Based on initial epidemiological data,

this is most commonly 14 or more days following the resolution of symptoms. Laboratories should

follow national reporting requirements.

Key steps for result declaration:

✓ Ideally, results from real-time PCR devices should be interfaced with a Laboratory

Information Management System (LIMS) to mitigate potential transcription errors and

minimize turnaround times. Results can then be automatically sent out once reviewed,

using electronic means of transmission.

✓ In the absence of an interfaced LIMS, results should be transcribed or entered into the

approved reporting form for your laboratory.

✓ Submit the report to your supervisor for review and authorization of your results. Provide

the original printout from the instrument with the report.

✓ After review and authorization by your supervisor, disseminate the report to the relevant

clinician:

43

• Reports may be communicated via telephone during the COVID-19 pandemic given

the emergency status and to allow for the initiation of contact tracing should a sample

test positive. Confirmation of results by SMS, e-mail or fax is permissible, but this

should be guided by the national policy.

• Reports can also be sent as paper copies by courier, according to your arrangement

with the referring sites (and the national policy).

✓ Ensure that there are appropriate sample transportation systems in place for rapid

reporting of results to the referring sites.

Preventing unauthorized access to test results

Only trained laboratory staff members are authorized to perform COVID-19 tests. Where

available, results should be interfaced with an electronic LIMS and reported directly to the

requesting clinician, thereby ensuring patient confidentiality. Where an electronic LIMS is not

available or if the LIMS is not functional, the results should be shared by phone with the requesting

healthcare worker; paper-based results should be recorded in a logbook, which should be kept

safe by the laboratory staff member(s) authorized to review and release the results.

Tips to ensure restricted access to test results to ensure patient

confidentiality

✓ Restrict access to the laboratory where COVID-19 testing is taking place.

✓ Ensure that laboratory staff members are provided with unique usernames and

passwords to enter or retrieve results once they have been reviewed by the authorized

staff member(s). This will ensure traceability to the staff and limit access to the results.

✓ Limit the amount of laboratory staff authorized to review and release results.

✓ Ensure that healthcare workers are provided with unique usernames and passwords

for the retrieval of results if electronic means of transmission are used.

44

2.9 Laboratory safety measures

COVID-19 testing lab in Chennai, India © FIND/YRG Care 2020

Some biosafety prerequisites for COVID-19 testing in the laboratory

There are some essential biosafety considerations when establishing laboratory testing

procedures for COVID-19, as outlined below.

Preparing yourself for testing:

➢ Ensure that adequate personal protective equipment (PPE) is available to all laboratory

staff, including support staff.

➢ Review your facility’s standard operating procedure (SOP) on the appropriate use of PPE.

➢ Provide training for staff by appropriate professionals in the correct sequence for donning

and removing PPE.

➢ Display the WHO “How to put on and take off personal protective equipment (PPE)” poster

in areas where PPE is donned and removed.

45

While working in the laboratory:

➢ Change gowns and gloves whenever they become soiled or contaminated.

➢ Remove gowns and gloves before leaving the laboratory.

➢ Discard disposable gowns after a single use. Cloth gowns can be re-used following correct

cleaning and decontamination procedures.

➢ Always maintain good hand hygiene after working with specimens.

What is personal protective equipment?

Personal protective equipment, commonly referred to as “PPE”, is equipment recommended to

maintain biosafety when handling potentially hazardous material, including samples to be tested

for SARS-CoV-2. In general, PPE is worn to minimize exposure to hazards that cause serious

workplace injuries and illnesses. These injuries and illnesses may result from contact with

biological, chemical, radiological, physical, electrical, mechanical or other workplace hazards.

Personal protective equipment includes items such as gloves, safety glasses and shoes,

earplugs or earmuffs, hard hats, respirators, coveralls, vests and full body suits29.

These are used as follows with regard to COVID-19:

• For specimen collection: non-sterile nitrile gloves (single use only), gown, eye

protection, N95 respirator mask.

• For specimen receipt and accession: non-sterile nitrile gloves (single use only),

gown, N95 respirator mask.

• For specimen testing (non-propagative): non-sterile nitrile gloves (single use only),

gown, N95 respirator mask, Biosafety level 2 (BSL2).

29https://www.osha.gov/personal-protective-equipment

46

2.10 Biosafety management

Biosafety cabinets

WHO recommends that procedures with a high likelihood of generating aerosols or droplets are

carried out in either a certified Class II type A1 or A2 biosafety cabinet (BSC). When used

correctly, BSCs are highly effective at reducing laboratory-acquired infections. BSCs are designed

to protect the operator, laboratory and samples from exposure to infectious aerosols and splashes

that may be generated when manipulating materials containing infectious agents.

Operating and maintaining a BSC

Each BSC should have a monthly maintenance log to document routine activities, as well as a

certificate from the service technician who performs the annual service. On a daily basis, and

after every use, the vertical air pressure in the BSC must be checked and recorded in the BSC

log. Under no circumstances should you work in a BSC with incorrect vertical air pressure. The

following steps are necessary to maintain a BSC:

➢ Interior surfaces of BSCs should be decontaminated, before and after each use, with a

disinfectant that can kill all microorganisms. A solution of 10% bleach followed by 70%

ethanol should be used.

➢ At the end of the working day, a final surface decontamination should be performed

including wiping down the work surface, the sides and back of the BSC, and the interior

of the BSC glass.

© FIND / John Rae 2016

47

➢ The functional operation and integrity of each BSC should be certified to national or

international performance standards at the time of installation and regularly thereafter by

qualified technicians, according to the manufacturer’s instructions.

Disinfectants for SARS-CoV-2 testing laboratories

Based on the similarity of SARS-CoV-2 to other enveloped viruses, it is recommended that

disinfectants with proven activity against such viruses are selected for disinfecting surfaces

potentially contaminated with the virus. The following disinfectants are recommended for COVID-

19 testing laboratories:

Sodium hypochlorite (bleach): a 1% solution of sodium hypochlorite should be used to soak

items for 10–15 minutes or disinfect spills. Note that this is corrosive to metals and plastics.

Sodium hypochlorite (bleach): a 0.1% solution of sodium hypochlorite should be used to clean

instruments.

Ethanol: a 70% solution of ethanol should be used to decontaminate surfaces (including surface

and metals).

2.11 Laboratory waste management

It is crucial that laboratory waste from COVID-19 testing laboratories is handled with the utmost

care as it will include specimens from suspected or confirmed COVID-19 cases. This waste must

be managed and disposed of in the same way as all other biohazardous waste.

Some tips on laboratory waste management

1. Seal any contaminated material (such as used specimen-containers, transfer

pipettes and cartridges) in a biohazard bag at the end of each day.

2. Autoclave and then incinerate the biohazard bag. Remember, incinerating plastics

can release toxins that are harmful to breathe.

3. Consult your facility’s environmental waste policy regarding the proper disposal of

unused reagents.

48

National guidelines on COVID-19 testing This section covers India’s national guidelines around COVID-19 testing, choice of tests as per

settings of use, frequency, algorithm and overview of current available tests in the country.

3.1 Advisory on strategy for COVID-19 testing in India Recommended by the National Task Force on COVID-19, the ICMR advisory is generic in nature

and may be modified at the discretion of state health authorities. The general guidelines for

COVID-19 testing strategies are as follows30:

Routine surveillance in containment zones and screening at points of entry:

i. Choice of test (in order of priority)

a. Ag-RDT

b. RT-PCR, Truenat or CBNAAT

ii. Screening population

a. All symptomatic cases with influenza-like illness (ILI) symptoms, including

healthcare workers and frontline workers.

b. All asymptomatic direct and high-risk contacts (family and workplace, elderly ≥65

years of age, immunocompromised individuals, individuals with comorbidities, etc.)

of a laboratory-confirmed case of COVID-19 to be tested once between day 5 and

day 10 of coming into contact with the case.

c. All asymptomatic high-risk individuals (elderly ≥65 years of age, individuals with

comorbidities, etc.) in containment zones.

*Ag-RDT for containment zones: ideally, it is suggested that 100% of people living in containment

zones should be tested by Ag-RDT, particularly in cities where there has been widespread

transmission of COVID-19.

Routine surveillance in non contaminant areas:

i. Choice of test (in order of priority

a. RT-PCR, Truenat or CBNAAT

b. Rapid antigen test (Ag-RDT)*

30Testing Strategy (icmr.gov.in)

CHAPTER 3

49

ii. Screening population

a. All symptomatic (ILI symptoms) individuals with a history of international travel in

the past 14 days.

b. All symptomatic (ILI symptoms) contacts of a laboratory-confirmed case of COVID-

19.

c. All symptomatic (ILI symptoms) healthcare workers/frontline workers involved in

containment and mitigation activities.

d. All symptomatic ILI cases among returnees and migrants within 7 days of illness.

*All asymptomatic high-risk contacts (family and workplace, elderly ≥65 years of age, individuals

with comorbidities etc.) Ag-RDT is recommended as the first choice of test in order of priority.

In hospital settings:

i. Choice of test (in order of priority)

a. RT-PCR, Truenat or CBNAAT

b. Rapid antigen test (Ag-RDT)

ii. Screening population

a. All patients with severe acute respiratory infection (SARI).

b. All symptomatic (ILI symptoms) patients presenting in a healthcare setting.

c. All asymptomatic high-risk patients who are hospitalized or seeking immediate

hospitalization, such as immunocompromised individuals, patients diagnosed with

malignant disease, transplant patients, patients with chronic comorbidities, and the

elderly aged ≥65 years.

Asymptomatic patients undergoing surgical/non-surgical invasive procedures (not to be tested

more than once a week during their hospital stay).

d. All pregnant women in/near labour who are hospitalized for delivery. Points to be

noted:

i. No emergency procedure (including delivery) should be delayed for lack of

a COVID-19 test. However, a sample can be sent for testing if indicated as

above simultaneously.

ii. Pregnant women should not be referred to a COVID-19 testing facility. All

arrangements should be made by the hospital to collect and transfer

samples to testing facilities.

50

iii. Mothers who test positive for COVID-19 should be advised to wear a mask

and undertake frequent handwashing for 14 days while handling their baby.

They should also be advised on breast cleaning before feeding their

neonate. These measures are likely to reduce the likelihood of transmitting

COVID-19 to their babies.

e. All symptomatic neonates presenting with acute respiratory/sepsis-like illness.

Features suggestive of acute respiratory illness in a neonate are respiratory

distress or apnoea with or without cough, with or without fever. Neonates may also

present with non-respiratory symptoms only, such as fever, lethargy, poor feeding,

seizures and diarrhoea.

f. Patients presenting with atypical manifestations based on the discretion of the

treating physician; atypical manifestations may include stroke, encephalitis,

haemoptysis, pulmonary embolism, acute coronary symptoms, Guillain–Barré

syndrome, multiple organ dysfunction syndrome, progressive gastrointestinal

symptoms, and Kawasaki disease (in pediatric patients).

Testing on demand (state governments to decide simplified modalities):

a) All individuals undertaking travel to countries/Indian states mandating a negative COVID-

19 test at the point of entry.

b) All individuals who wish to get themselves tested for COVID-19. Tracking and contact

tracing mechanisms should be ensured by the testing laboratories, who should notify the

public health authorities.

3.2 Frequency of testing • A single, positive RT-PCR/Truenat/CBNAAT/Ag-RDT test is to be considered

confirmatory, without any repeat testing required.

• No retesting is recommended prior to discharge from a COVID-19 facility following

clinical recovery (please refer to MoHFW guidelines), including for transfer from a

COVID-19 area/facility to a non-COVID-19 area/facility.

• If symptoms develop following a negative Ag-RDT, a repeat Ag-RDT or RT-PCR should

be performed.

Points to be noted:

• WHO case definition for ILI: an individual presenting with acute respiratory infection with

fever ≥38C and cough with onset within the past 10 days.

51

• WHO case definition for SARI: an individual presenting with acute respiratory infection

with a history of fever ≥38C and cough with onset within the past 10 days and requiring

hospitalization.

• All healthcare workers and frontline workers coming into contact with suspect/confirmed

COVID-19 patients should ensure appropriate use of PPE.

• Home quarantine for 14 days is recommended for all individuals before undergoing

elective surgery to minimize their chances of COVID-19 infection prior to their procedure.

Algorithm for COVID-19 test interpretation using an Ag-RDT- redrawn with Ag-RDT

information from ICMR31

31 “Testing_Strategy”_v6_04092020.pdf (icmr.gov.in)

52

What is the advice on the feasibility of using pooled samples for molecular testing for

COVID-19?

With the exponential increase in the number of COVID-19 cases in India during April and

May 2021, particularly in some states, it became critical to increase the number of tests

conducted by laboratories where positivity rates in cases were low. Hence, it was useful to

use pooled samples for screening. A pooled testing algorithm involves the PCR screening of

a specimen pool comprising multiple individual patient specimens, followed by individual

testing (pool deconvolution) only if a pool returns a positive result. As all individual samples

in a negative pool are regarded as negative, this approach results in substantial cost savings

when a large proportion of pools test negative.

The objective of sample pooling is to increase the capacity of laboratories and enable them

to screen increased numbers of samples using molecular testing for COVID-19 for the

purpose of surveillance.

A feasibility study was conducted at DHR/ICMR Virus Research & Diagnostic Laboratory at

King George’s Medical University (KGMU), Lucknow. It was demonstrated that performing

real-time PCR for COVID-19 by pooling five samples of throat or nasal swab (200 µl/sample)

is feasible when the prevalence of infection is low. Pooling of more than five samples is not

recommended to avoid a dilution effect, which could lead to false-negative results.

The recommendations to perform sample pooling for real-time RT-PCR screening for COVID-19

are as follows (based on the KGMU study):

Use only in areas with a low prevalence of COVID-19 (initially using a proxy of low positivity

rates of 5% for COVID-19). The preferred number of samples to be pooled is five, although

two or more samples can be pooled. However, considering the greater chance of missing

positive samples that have a low viral load, the pooling of more than five samples is strongly

discouraged, unless it is for research purposes32.

32Advisory on feasibility of sample pooling (icmr.gov.in).

53

3.3 Information about COVID-19 test kit manufacturers

Table 6: List of companies that manufactures Rapid Antigen Tests (Ag-RDTs) for COVID-19 that

are validated and approved for use in India

Serial No.

Kit name Company

name

India/other

countries

Supplier name Validation

status*

1 STANDARD Q COVID-19 Ag

SD Biosensor South Korea/India

SD Biosensor Validated, approved

2 COVID-19 Antigen Lateral Test Device

LabCare Diagnostics Ltd.

India My Lab Discovery Solutions

Validated, approved

3 COVID-19 Ag Respi-Strip

Coris BioConcept

Belgium Vishat Diagnostics Pvt. Ltd, Mumbai

Validated, approved

Up to 9 July 2021, 123 Ag-RDT Kits have been validated by ICMR (including 26 revalidated kits);

47 kits were found to be satisfactory, with 3 important kits in use were listed here together with

their manufacturer. *For the complete list, please refer to Microsoft Word - Rapid Ag kits_09.07..2021

(icmr.gov.in)

Table 7: List of companies for RT-PCR testing kits for COVID-19 that have been validated and

approved for use in India

Serial No. Company name Type of kit Country

1. Roche Diagnostic, Mumbai

PCR USA

2. SD Biosensor, Delhi PCR SD Biosensor Korea, CE Approved

3. Matrix, Chennai PCR M/s GhangzhouWandfo Biotech Co.Ltd. China, CE Approved

4. Mylab, Pune PCR Indigenous approved by NIV Pune

5. Med Source Ozone Biomedical Pvt. Ltd.

PCR M/s. Hangzhon Biotechnology Co. Ltd., China, CE Approved

6. Invitrogen BioServices (ThermoFisher)

PCR M/s. Life Technologies, USA, US Approved

7. Solution One Delhi PCR M/S SeegeneInc South Korea, CE approved

8. M/S Cepheid, Delhi PCR M/S Cepheid, USA, approved by USFDA for emergency use

9. M/s AltonaDiag.Pvt Ltd, Delhi

PCR M/S Altona Diag., GmbH Germany

10. M/S Imperial Life Sciences Pvt Ltd

PCR M/S BGI Biotechnology (Wuhan) Co. Ltd China

11. M/s Qiagen India Pvt. Ltd.

SARS CoV-2 Panel

M/s Qiagen, GmbH Germany

54

Up to 6 April 2021, a total of 346 RT-PCR kits have been evaluated by ICMR validation centres,

with 162 RT-PCR kits found to be satisfactory. Eleven PCR kits are listed here, together with

their manufacturers; for the complete list, please refer to:

(https://www.icmr.gov.in/pdf/covid/kits/RT_PCR_Tests_Kits_Evaluation_Summ_06042021.pdf).

3.4 Concluding note

The second wave of COVID-19 in India has highlighted the need for more rapid and accurate

diagnosis of COVID-19 infection to help control the pandemic situation. It is anticipated that the

information presented in this document will help all categories of laboratory personnel, as well as

decision makers, to better understand COVID-19 disease, preventive measures, diagnostic tests

available for screening, specimen types and laboratory testing techniques, under various

scenarios. It is also hoped that the details provided with regards to diagnostic tests, such as

NAATs, Ag-RDTs, antibody detection tests and point-of-care tests, will facilitate decision making

about the tests needed to diagnose COVID-19.

In addition to the above, this document is a composite guide to the standard procedures and

scenarios required to establish a COVID-19 laboratory equipped with all necessary biosafety

maintenance. The COVID-19 Ready Reckoner will thus enable all laboratory personnel and

related staff members to operate in a safe environment within a COVID-19 testing laboratory, with

all important guidelines at hand.

……………………………………………………………………………………………………………………………………………………………..

55

About FIND

FIND, the global alliance for diagnostics, seeks to ensure

equitable access to reliable diagnosis around the world. We

connect countries and communities, funders, decisionmakers,

healthcare providers and developers to spur diagnostic

innovation and make testing an integral part of sustainable,

resilient health systems. We are working to save 1 million lives

through accessible, quality diagnosis, and save US$1 billion in

healthcare costs to patients and health systems. We are co-

convener of the Access to COVID-19 Tools (ACT) Accelerator

diagnostics pillar, and a WHO Collaborating Centre for

Laboratory Strengthening and Diagnostic Technology

Evaluation. For more information, please visit www.finddx.org

About PATH

PATH is a global nonprofit dedicated to achieving health equity.

With more than 40 years of experience forging multisector

partnerships, and with expertise in science, economics,

technology, advocacy, and dozens of other specialties, PATH

develops and scales up innovative solutions to the world’s most

pressing health challenges.