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
Laboratory Diagnosis of COVID-19 Infection:
Current Issues and Challenges ASM 2020
Faeces 29% 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
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
Techniques and Test Development for COVID-19 Diagnosis .ACS 2020
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
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
Techniques and Test Development for COVID-19 Diagnosis .ACS 2020
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]