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HORIBA LIVE

WEBINAR

HORIBA –LIVE Season 2: “COVID TIMES”

WELCOME: WEBINAR hosted by HORIBA- We will start in a while

HEART TO HEART

Its All About “ TEST TEST TEST

The VTM & its Impact on TESTING

What is VIRAL TRANSPORT MEDIUM ? How should I Choose ? Any Recommendations ? How does it matter ? Collections & Transportation ?

DESIGNATION

PRESENT :ZONAL HEAD , STERLING ACCURIS

PRESENT

AFFILIATION:

HEADING OPERATIONS OF DELHI NCR & LUCKNOW

FOR STERLING ACCURIS

EDITOR & PEER REVIEWER IN MANY

INTERNATIONAL JOURNALS

EXPERIENCE

MD PATHOLOGY, SENIOR RESIDENCY (DEPT OF LAB

MEDICINE, AIIMS DELHI) WORKED AS CHIEF CONSULTANT/LAB HEAD IN NABL ACCREDITED

LABS WITH MOLECULAR IN SCOPE

AREAS OF

INTERESTHUMAN GENETICS , INFECTIVE MOLECULAR

BIOLOGY,

HEMOGLOBINOPATHIES , HEMATOLOGY

PUBLICATIONS SEVEN INTERNATIONAL PUBLICATIONS IN

REPUTED JOURNALS

DR PRASHANT NAG

Specimen

collection &

transport in

COVID

Dr PRASHANT NAG

ZONAL HEAD STERLING ACCURIS

ACCEPTABLE SPECIMEN

(CDC)

– For initial diagnostic testing for SARS-CoV-2, CDC recommends collecting and testing an upper respiratory specimen. The following are acceptable specimens:

– A nasopharyngeal (NP) specimen collected by a healthcare professional; or

– An oropharyngeal (OP) specimen collected by a healthcare professional; or

– A nasal mid-turbinate swab collected by a healthcare professional or by a supervised onsite self-collection (using a flocked tapered swab); or

– An anterior nares (nasal swab) specimen collected by a healthcare professional or by onsite or home self-collection (using a flocked or spun polyester swab); or

– Nasopharyngeal wash/aspirate or nasal wash/aspirate (NW) specimen collected by a healthcare professional.

– The NW specimen and the non-bacteriostatic saline used to collect the

specimen should be placed immediately into a sterile transport tube.

– Testing lower respiratory tract specimens is also an option. For patients who

develop a productive cough, sputum should be collected and tested for SARS-

CoV-2. The induction of sputum is not recommended. When under certain

clinical circumstances (e.g., those receiving invasive mechanical ventilation), a

lower respiratory tract aspirate or bronchoalveolar lavage sample should be

collected and tested as a lower respiratory tract specimen.

– For providers collecting specimens or within 6 feet of patients suspected to be infected with SARS-CoV-2, maintain proper infection control and use recommended personal protective equipment, which includes an N95 or higher-level respirator (or facemask if a respirator is not available), eye protection, gloves, and a gown, when collecting specimens.

– For providers who are handling specimens, but are not directly involved in collection (e.g. self-collection) and not working within 6 feet of the patient, follow Standard Precautions; gloves are recommended. Healthcare personnel are recommended to wear a form of source control (facemask or cloth face covering) at all times while in the healthcare facility.

– Self collection

https://www.cdc.gov/coronavirus/2019-ncov/infection-control/control-recommendations.htmlhttps://www.cdc.gov/coronavirus/2019-ncov/lab/biosafety-faqs.htmlhttps://www.cdc.gov/coronavirus/2019-ncov/hcp/infection-control-recommendations.html

SWAB

– Use only synthetic fiber swabs with plastic or wire shafts.

– Do not use calcium alginate swabs or swabs with wooden shafts, as they may

contain substances that inactivate some viruses and inhibit PCR testing.

– CDC is now recommending collecting only the NP swab, although OP swabs

remain an acceptable specimen type. If both NP and OP swabs are collected,

they should be combined in a single tube to maximize test sensitivity and limit

use of testing resources.

NP SWAB & OP SWAB

– NP swab: Insert minitip swab with a flexible shaft (wire or plastic) through the nostril parallel to the palate (not upwards) until resistance is encountered or the distance is equivalent to that from the ear to the nostril of the patient, indicating contact with the nasopharynx. Swab should reach depth equal to distance from nostrils to outer opening of the ear. Gently rub and roll the swab. Leave swab in place for several seconds to absorb secretions. Slowly remove swab 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 minitip is saturated with fluid from the first collection. If a deviated septum or blockage create difficulty in obtaining the specimen from one nostril, use the same swab to obtain the specimen from the other nostril.

– OP swab: Insert swab into the posterior pharynx and tonsillar areas. Rub swab over both tonsillar pillars and posterior oropharynx and avoid touching the tongue, teeth, and gums.

TRANSPORT

– Molecular detection methods do not require replication competent virus, but

preservation of nucleic acid is essential.

– Swabs should be placed immediately into a sterile transport tube containing

A. 2-3mL of either viral transport medium (VTM),

B. Amies transport medium, or

C. sterile saline

TRANSPORT

– Store specimens at 2-8°C for up to 72 hours after collection. If a delay in testing

or shipping is expected, store specimens at -70°C or below.

TRANSPORT OF SAMPLE

WITH VIRUS

Typically, liquid media are composed of buffers to control pH,

– protein to stabilize the virus,

– and often other substances to control osmolality or onto which the viruses can

adsorb.

– Proteins, such as bovine serum albumin, gelatin, skimmed milk, normal serum,

or complex broth bases, are used as protective substances

THANKSYES YOU ARE FEELING

RIGHTMOLECULAR TECHNIQUESARE GOING TO BE MAINSTAYOF DIAGNOSTIC [email protected]

mailto:[email protected]

END OF THE SESSION S ONE

The world of MOLECULAR & SEROLOGY TESTING

Technologies of Molecular Testing? What are the recommendations ? What should I know before I begin ? How to Interpret results ?

DR GEETHIKA REDDY

• M.B.B.S, Nanjing Medical University, China.

• M.D (Microbiology), Kasturba Medical College, Manipal

University

• Consultant Microbiologist and Head, Hospital Infection

Control at Medicover group of hospitals , Hyderabad

• Assistant Professor, Dept. of Microbiology, Maheshwara

Medical College, Telangana,

• In charge for serology in central laboratory

• Infection control certificate course from Infection control

academy of India (IFCAI )

DR GEETHIKA KAIPA

ASSISTANT PROFESSOR

CONSULTANT MICROBIOLOGIST AND HEAD HOSPITAL INFECTION CONTROL

SARS COV 2 is a large single

stranded positive sense RNA Virus

Compromises of four structural

proteins

Nucleocapsid protein

Spike protein

Envelope protein

Membrane protein

That create the viral envelope

Preanalytical

Post Anaytical

Analytical

Ordering right test

Right patient

Right sample collection

Right time

Nasopharyngeal swab

Throat swab

Sputum

BAL

Viral culture

Serological methods

Molecular methods

Specimens that can be tested

Specimens

Respiratory

URT

Nasopharyngeal swab

Throat swab

LRT

Sputum

BAL

ET aspirate

Other

Blood

Stool

Urine

TYPE OF SAMPLE SENSITIVITY REFERENCE

Oropharyngeal swab 32% Phelan etal 2020:Novel coronavirus originating in wuhan china; challenges for global health

governance.JAMA 2020

Nasopharyngeal swab 63% Yi-Wei Tang , Jonathan E. Schmitz , David H. Persing , and Charles W. Stratton : The

Laboratory Diagnosis of COVID-19 Infection:

Current Issues and Challenges ASM 2020

Sputum 72% Wenling Wang, PhYanli Xu, MD Ruqin Gao, MD; et al Detection of SARS-CoV-2 in Different

Types of Clinical Specimens

BAL 93% Wenling wang, PhYanli XU,MD Ruqin GaoMD;Etal Detection of SARS-COV2 in Different

types of Clinical Specimens

Blood 1% Yi-Wei Tang , Jonathan E. Schmitz , David H. Persing , and Charles W. Stratton : The



Faeces 29% Yi-Wei Tang , Jonathan E. Schmitz , David H. Persing , and Charles W. Stratton : The



RT-PCR

Truenat beta cov test

Cepheid xpert xpress SARS-COV2

Cobas 6000/8800

LAMP/RT-LAMP

TMA

CRISPR

ICMR

approved

N E

Rdrp ORF

Target genes

Reaction growth curve crosses the threshold line by 35 cycles

for E gene and both Rdrp and ORF or either Rdrp and ORF

Reaction mixture (target DNA)

Master mix

DNA Primer(oligonucleotide)

Four nucleotide triphosphates

Thermostable DNAPolymerase(Taq

polymerease)

Buffers and also magnesium chloride

Thermal cycler (a device that can

change temperatures dramatically

in a very short period of time)

STEPS IN PCR

Step 1:

Denaturation

dsDNA to ssDNA

Step 2:

Annealing

Primers onto template

Step 3:

Extension

dNTPs extend 2nd strand

Initial

denaturation

90o – 95o C 1-3 min

Denature 90o – 95o C 0.5-1 min

Primer

annealing

45o – 65o C 0.5-1min

Primer

extension

70o – 75o C 0.5-2min

Final extension 70o – 75o C 5-10min

Stop reaction 4o C or 10 mM

EDTA

Hold

25 – 40 cycles

SOURCE : Linda J. Carter Linda V. Garner Jeffrey W. Smoot Yingzhu Li Qiongqiong Zhou Catherine J.

Saveson Janet M. Sasso Anne C.Gregg Divya J. Soares Tiffany R. Beskid Susan R. JerveyCynthia Liu*Assay

Techniques and Test Development for COVID-19 Diagnosis .ACS 2020

Acrometrix covid19 RNA control to monitor

and validate the molecular methods

The new quality control product is

formulated with synthetic RNA transcripts

that contain N, S, E and ORF1ab regions of

SARS-COV-2 genome

Assay

validation

Preanalytic Analytic Post analytic

Clinical and

analytical

validation

Establish and

document

clinical validity

•Test ordering

•Informed consent

•Specimen

collection

•Accessioning

barcode scanning

•Processing

•Storage,

transportation

•Order

verification

•Nucleic acid

isolation

•Calibration

•Controls

•QC/QA

•Result

verification

•Interpretation

•Verification

•Resulting to LIS

•Reporting

•Treatment

decision

•Data retention

QUALITY MANAGEMENT IN ALL PHASES OF MOLECULAR TESTING

Analysts should be trained and familiar with the testing

procedure and interpretation

False negative results may occur due to :

inadequate viral load is present

Improper collection , transport and handling of sample

If excess DNA/RNA template is present

Pippeting errors

Source : ICMR-NIV pune standard operation for

detection of covid 19 in suspected human cases by rRT-

PCR

THE GAME CHANGERs

WHY So ???

Lets discuss

Fast

Accurate

Easy to use

No Clean rooms

No large work space

No specialised environment is needed

NOTE : ICMR recommends the BSL-2 laboratory

for handling the test

Chip based real time PCR for beta

coronaviruses

Samples : nasal and throat swab

Storage : 2 to 300c

procedure: 30-60mins

Capacity : 16 to 24 samples

First line screening test

Source : Linda J. Carter Linda V. Garner Jeffrey W. Smoot Yingzhu Li Qiongqiong Zhou Catherine J. Saveson

Janet M. Sasso Anne C.Gregg Divya J. Soares Tiffany R. Beskid Susan R. JerveyCynthia Liu*Assay


Sample : respiratory specimen

TAT: 30MINS

Acute or early infection

LIMITATIONS :

Time for the onset of illness

Quality of specimen

How it is processed

Sensitivity -34 to 80 %

False positivity : other human corona virus

WHO does not

recommend Ag

detection

ENCOURAGED

FOR RESEARCH

PURPOSE

Two weeks after the onset of symptoms

Strength of the antibody detection depends on :

Age

Nutritional status

Medications

Infections (hiv )

Diagnosis : recovery phase

Source :Linda J. Carter Linda V. Garner Jeffrey W. Smoot Yingzhu Li Qiongqiong Zhou Catherine J. Saveson

Janet M. Sasso Anne C.Gregg Divya J. Soares Tiffany R. Beskid Susan R. JerveyCynthia Liu*Assay


Cannot diagnose disease in acute stage

Cannot differentiate recent present and past

infection

opportunity for disease transmission

Missed clinical intervention

cross reaction with other human coronaviruses

Useful for Active case finding and

surveillance

WHO does not recommend antibody

detection

Two specimens negative 24hours apart : cure

Source : International federation for clinical chemistry and

laboratory medicine

Days Total AB Ig M Ig G RT-PCR

0 – 7 days 38.3% 28.7% 19.1% 66.7%

8 – 14 days 89.6% 73.3% 54.1% 54%

15- 39 days 100% 94.5% 79.8% 45.5%

Source : antibody responses to SARS cov in patients with novel coronavirus

Juanjuan zhao etal

RT-PCR IgM IgG Interpretation

+ - - Window period

+ + - Early stage

+ + + Active infection

+ - + Late/recurrent

- + - Early stage/FN

- - + Past

infection/Recovery

- + + Recovery/ FN

Giuseppe Lippi* and Mario Plebani etal The critical role of laboratory medicine during

coronavirus disease 2019 (COVID-19) and other viral outbreaks, CCLM

LDH

CK levels

Serum potassium

Source: jingyuan,rougong zou, zhaogin wang correlation between viral clearance and outcomes of 94 covid 19 infected discharged patients

Comes back to

normal levels

HOW AND WHAT TO DO?

Pool testing of the samples :

Test RT-PCR

2 to 5 samples

Low prevalence areas

NOT RECOMMENDED:

Health care workers

Close contact

Hot spots >5% positive cases

ADVANTAGES :

Time saving

Increases the testing capacity

Reduces need of man power and cost

History Clinical features Radiological

diagnosis

RT-PCRAB testing

Other parameters

Cbc, ferettin ,D-dimer

Right diagnosis

END OF THE SESSION S

What is ASSURANCE to Patient they are infection free ? Is ISOLATION enough ?

Is Clinical Remission enough to DISCHARGE ?

No INVESTIGATIONS ? Any BASELINE Testing for MILD / MODERATE COVID positive patients ?

Will CBC + CRP + Chest X Ray support Clinically Symptom free COVID 19 infected patients ?

For feedback kindly send mail at- [email protected]

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