Laboratory support

for
Measles-Rubella and VPD
Surveillance

(Collection, Storage and Shipment of specimens)

Immune response to measles virus infection
IgG: Serum/DBS/OF
100%

75%
Patients positive

Virus detection: OF
50% IgM: Serum/DBS/OF

25%

Virus culture

-3 -1 0* 3 5 7 14 21 28 35 60 90
Fever DBS=Dried Blood Spot
Rash
OF = Oral fluid sample
Virus detection = RT-PCR
*Day 0 = Day of rash
Incubation
7-18 days Detection of IgM: Single serum, 4-28 days post rash onset
Virus culture: Urine, T/S, -2 to 5 days post rash
 Outline of the presentation

Introduction
Laboratory network for measles-rubella
Sample collection and transportation
Laboratory tests and interpretation of
results
 Outline of the presentation

Introduction

Laboratory network for measles

Sample collection and transportation

Laboratory tests and interpretation of

results
Measles Echoviruses
Dengue

Measles case
definition
Case with fever and maculopapular rash, with either
cough, coryza or conjunctivitis or any case which a
physician thinks is measles

HHV-6 Rubella

Parvovirus B19 Others

 Measles virus
Paramyxo virus Genus Mobillivirus.
Thermo Labile.
Antigenically stable.
Single Serotype and multiple genotypes.
Evolutionary drift - small differences in gene
sequences over a period of time.
Genetic analysis and mapping of a measles family
tree is important to molecular epidemiology and
global eradication efforts.
Not essential in initial stages of surveillance.
 Outline of the presentation

Introduction

Laboratory network for measles

Sample collection and transportation

Laboratory tests and interpretation of

results
WHO Measles Laboratory Network
-Specialised reference
-Sequencing
-Global QA
-assay assessment

- Regional QA
-Virus isolation for NMLs

-Confirmation of clinical cases by serology

-Virus isolation by some laboratories
Measles-Rubella (MR) laboratory network and their
catchment area

WHO Accredited MR lab-net

Main Functions of Laboratories
Testing and referring:
Serological confirmation of clinically suspected measles outbreaks
using validated IgM ELISA
Collection and dispatch of samples for virus isolation to Regional
Reference laboratory if cell and virus culture facility is not
established

Timely Reporting: Sender, Country program and WHO

Quality assurance:
Performs annual proficiency test
Maintain IQC standards
Refers 10% of tested samples to Regional Reference Laboratory
for validation
Participate in annual accreditation reviews
 Measles / Rubella IgM testing strategy

Suspected Measles Case Measles IgM serum

Positive Negative

Confirmed measles

Rubella IgM

Negative Positive

Discard Confirmed Rubella

 Outline of the presentation

Introduction

Laboratory network for measles

Sample collection and transportation

Laboratory tests and interpretation of

results
Samples for measles and rubella serological
diagnosis

WER: 25, 2008, 83, 225232 and MMWR: 2008; 57:657-660

 Steps in blood collection
The sample collection kit will be available at the DIO /
SMO office.

Arrange for a competent person from the nearest health

institution / clinical laboratory for collection of blood
sample by vein puncture

Complete the MR-LRF: 3 dates are important for

interpretation of the test
Date of collection of samples
Date of onset of rash
Date of last measles vaccination

Make arrangements to ship the specimen from point of

collection to the DIO / SMO office.
Contents - Specimen Collection Kit
Contents
5ml syringe and needle
5ml externally threaded
screw cap vials
Tourniquet, sterile swabs
& band aid
Gloves
Specimen labels
Zip lock plastic bags
Autoclavable disposal
bags
Measles lab request form
 Blood sample collection
Label the blood collection tubes: Epid No, Name, age, sex
and DOC.
Apply tourniquet 2 3 inch above elbow, Swab the
cubital fossa.
Draw 2 - 3 ml of blood by vein puncture.
Withdraw the needle, apply pressure at point of prick and
seal with band aid
Push the blood gently into sterile labeled vial. Do not
push blood through the needle
Discard the barrel of syringe into autoclavable disposal
bag
 Separation of serum
Leave the tube with the blood sample at room temperature
for clot formation (~ 30 mins)
Clotted whole blood can be kept in the refrigerator and sent
to the laboratory within 24 hrs (do not freeze whole blood)
If blood sample cannot be transported within 24 hrs then
serum should be separated in a laboratory:
Centrifuge available centrifuge the clotted blood to
separate the serum. Transfer serum using sterile pipette
to a sterile, labelled screw - capped vial.
Centrifuge not available - blood is held in a refrigerator
for 24 hours for clot retraction. The serum is then
carefully removed without disturbing the clot using a
sterile pipette.
Steps in Shipment of specimens
Specimens should be shipped to the laboratory as soon as
possible. Do not wait to collect additional specimens before
shipping
Place specimens in zip lock or plastic bags
Use vaccine carriers with frozen ice packs, Styrofoam boxes
or a thermos flask with wet ice
Place Laboratory request form in separate plastic bag to be
hand carried or tape to inner top of box
Arrange shipping date. Avoid weekends and holidays
When arrangements are finalized, inform the receiver of
time and manner of transport
 Specimen collection & processing

>7

labeled sterile vials

day

Collect serum in
s

Storage

ays
7 d
s
h r
24
Centrifuge Clotted Blood

Leave at
room temp.
or at 4oC for
clot retraction
Centrifuge @ 3000 RPM 10 Min
 Adequate serum samples from
Measles outbreak investigation
A Serum Sample from each case investigated
Minimum 5 or more samples from suspected
outbreak having 10 or more suspected cases
Within 4 - 28 days of rash onset
From any age group (preferably younger)
No history of measles vaccination in last 1 month
Serum volume should be >500 micro liter (0.5ml)
With no hemolysis /gross contamination
With proper documentation and labeling
Leak proof container with adequate cold chain
 Outline of the presentation

Introduction

Laboratory network for measles

Sample collection and transportation

Laboratory tests and interpretation of

results
 Testing strategy
One Serum Samples from every case investigated
Five blood samples collected from every outbreak
Transport serum samples to NML for confirmation
of outbreak
Laboratory perform IgM tests using a validated
ELISA kit (Siemens assay)

2-3 urine samples/ throat swab collected from

selected outbreaks for virus isolation
Transport samples for virus isolation to five
designated MR laboratory with molecular test
facility
Focus is on confirmation of outbreaks by serology
Classification of M/R outbreaks
based on IgM serology results
IgM results of samples Classification
collected from an of Outbreak
outbreak
> 2 measles positive and <2 Measles
rubella positive
> 2 rubella positive and <2 Rubella
measles positive
> 2 measles positive and > 2 Mixed
rubella positive
<2 measles positive or <2 Negative for
rubella positive both measles
and rubella
Sample and Communication flow

20% Pos/Neg Virus isolates

(sequencing)
NATIONAL REGIONAL SPECIALISED
LAB REF. LAB
M LAB
Within 7

on
days

Samples
th
ly

24 hrs

State /National WHO Reports

EPI / field REGION
Sample movement
Lab-Information System (MLIS)
 Measles genotyping
Molecular tests and sequencing helps genomic analysis, establish
epidemiologic linkages and create virus data bank of circulating strains

It is not recommended for confirmation of outbreaks

Detection and identification of measles virus requires a sophisticated

virology laboratory with molecular/PCR and cell culture facilities

Genotyping is possible from urine, throat swab, oral fluid &

pharyngeal washings. Recommended - throat swab/urine

Sample for genotype identification is to be done (one/two) from every

outbreaks and not mandatory from outbreak
confirmation/classification

Refer SOPs developed for quality sample collection and transportation

 Throat swab
(posterior pharyngeal swab)
Hold tongue away with
tongue depressor
Locate areas of inflammation
and exudate in posterior
pharynx, tonsillar region of
throat behind uvula
Avoid swabbing soft palate;
do not touch tongue
Rub area back and forth with
cotton or Dacron swab

WHO/CDS/EPR/ARO/2006.1
 Virologic Investigations

Storage condition
Transport Purpose/ Lab
Specimen
media Transport Pending test investigation

Throat swab VTM 2-8 0C -20 0C Isolation

NPA/ swab VTM 2-8 0C -20 0C Isolation

Urine No 2-8 0C -20 0C Isolation

Serum/ No 2-8 0C -20 0C Serology
Clotted blood 2-8 0C
Whole blood No 2-8 0C 2-8 0C Serology
Measles / Rubella genotypes (SEAR) Data: RRL Bangkok, NIV
& CDC

H1

D4,D8
BJM Ahmadabad
D8
D5
NIV Pune D4,D7,D8,B3

2B D5,G2

D5
D9
,G
2,G

( 18 Labs)
3

( 1 Lab)
Ref Lab Sequencing (India)
Updated Jan 2007
 Sample collection, storage and
sportation from reported Diphtheria and
Diphtheria and Pertussis surveillance and laboratory network, India, 2017

CMC Vellore
(Reference Lab )
MVIDH New Delhi
PGIMER Chandigarh
SPHL
Thiruvananthapuram
VPD surveillance initiated 5 states KGMC Lucknow
National laboratories 6 Choithram Hospital,
Reference laboratory 1 Indore
Importance of quality sample
Patient/Client Prep
Sample Collection

Personnel Competency
Reporting Test Evaluations

Quality
Assuranc
Sample Receipt and
e Cycle Accessioning

Record
Keeping Sample Transport

Quality Control
Testing
Analysis
 Importance of quality sample
Influence laboratory results
Poor quality sample lead to false negative
results
Wastage of resources
Improper case management and interventions
Blinds you on the reality
Diphtheria and Pertussis are fastidious
organisms
Die quickly if samples are not stored or
transported in appropriate conditions
Sample collection and
transportation

Diphtheria
Prerequisites/Conditions Diphtheria

Window period from onset 2day-4 weeks

Throat swab or pieces of

Type of specimen membrane or
nasopharyngeal swab

Number 1

Amies transport
Transport media
media/Silica gel

Storage and transportation 2-8 OC

Throat swab sample collection

Use throat swab made up of cotton, polyester or

Dacron
Check the expiry date of transport media to ensure
acceptability of the material
 Throat swab sample collection
Label the specimen collection tube/sachet with
unique identification code
patient name
date of collection
Swab the inflamed area of tonsils and posterior
pharynx. If membrane is visible then rub the swab
beneath the membrane
Dislodging of membrane can lead to severe bleeding
Piece of membrane can also be collected on the swab
Avoid touching other areas in the mouth
Demonstration: throat swab
collection
 Material required for throat swab
collection

Wooden sticks Throat swab

(disposable tongue Silica gel/Amies
depressors) transport media
Gloves Zip lock bag
Face masks Labels
Disposable bag Laboratory request form
Tissues
Cello tape
Scissors
Sample collection and
transportation

Pertussis
 Prerequisites/
Pertussis
Conditions
Window period
2-4 weeks > 4weeks-8 weeks
from onset
Nasopharyngeal swab*
Type of specimen Serum
and serum

Number 1 each 1

Regan-Lowe / Amies
Transport media transport media with Not applicable
charcoal for swab

Storage and
2-8 OC 2-8 OC
transportation
*attempt under medical specialist in a Hospital /CHC
 Nasopharyngeal swab sample collection

Obtain a thin flexible nasopharyngeal swab

made up of Dacron or nylon
Cotton and calcium alginate swabs are not to be
used
Check the expiry date
Label the specimen collection tube
 Nasopharyngeal swab sample collection
Have patient sit with head against a wall or a
support
Patients have a tendency to pull away during this
procedure
Explain the procedure to the parents or patient
Measure the distance between anterior nares
to the lower lobe of the ear of one side
Mark the swab with half of the above
measured distance
Ask the patient to blow the nose forcefully to
remove any mucous plug
Nasopharyngeal swab sample collection
Position the head slightly upwards and insert the swab
along the floor of the nose up to the distance marked
Avoid insertion of swab in upward direction
Do not force swab if obstruction is encountered before
reaching the nasopharynx
Remove swab and try the other side
Try to leave the swab in place for 5-10 seconds to
increase sensitivity
Immediately place the swab in transport media and
tighten the cap
Best is to wrap the tape around cap to prevent any leakage
Ship at 4OC
Demonstration: Nasopharyngeal
swab collection
Material required for nasopharyngeal
swab collection

Nasopharyngeal swab
Gloves Regan-Lowe transport
Face masks media/ Amies transport
Disposable bag media with charcoal
Tissues Zip lock bag
Cello tape Labels
Scissors Laboratory request form
Paper scale
 Serum sample collection
One serum sample should be obtained for
pertussis cases
Methodology would be as described for
serum sample collection for measles-rubella
Use of transport media
 Use of transport media
Plating of swab on culture media:
Within 4 hrs of sample collection:
transport media not required
>4hrs: transport media required
Transport media: just preserves the
organisms
Silica gel sachet - Diphtheria
Amies transport media
Plain - Diphtheria
With Charcoal Pertussis and Diphtheria
Regan lowe transport media - Pertussis
Transportation in Amies transport media

Insert the swab till the bottom of the media

If capped swab then throw the cap of the tube
If un capped swab then cut the shaft of the
tube to fit into the tube and cap it securely
 Transportation

With proper documentation

With no leakage
Within 48hrs
Under cold chain - 4-8 degree C
 Suspected case

Clinical examination

Meets case definition

Yes No

Sample

Yes No

Lab result

Epidemiological
Pos Neg
linkages

Yes No

Lab- confirmed Epi- confirmed Clinical confirmed Rejected

 Successful lab-investigations

Advance planning
Collection of adequate and appropriate specimens
Sufficient documentation
Biosafety and decontamination
Correct packaging
Rapid transport
Choice of a laboratory that can accurately perform the tests
Timely communication of results
 Summary

Laboratories has a critical role to play in the MR

and VPD surveillance in India
Standardisation of laboratory activities key to a
successful MR and VPD laboratory network
Serology , virology and bacterial samples are
collected and transported appropriately to the
accredited designated MR/VPD Lab-net for your
state
Thank you