Working with the laboratory
IDSP training module for state and
district surveillance officers
Module 6
Learning objectives (1/3)
• List
 L1 and L2 laboratories in the district
 L3 laboratories in the state
 L4 and L5 laboratories in the country
• Understand the need of L1 and L2
laboratories to arrange for logistical support
• Identify what action is to be taken be the
technician for sample collection in response
to the diagnosis made by the medical officer
Learning objectives (2/3)
• List tests to be performed in L1 and L2
laboratories
• Identify quality assurance processes within
the laboratory network
• Understand bio-safety issues
• Identify transport modalities of samples to
higher levels
Learning objectives (3/3)
• Understand training needs of laboratory
personnel
• Keep track of the flow of samples
• Draw a flow diagram for reporting of the
laboratory investigations
Role of laboratories in disease
surveillance
•
•
•
•
Early diagnosis of diseases under surveillance
Epidemiological investigation
Rapid laboratory confirmation of diagnosis
Implementation of effective control
measures
Factors influencing laboratory
confirmation in surveillance
• Advance planning
• Collection of appropriate and adequate
specimens
• Correct packaging
• Rapid transport
• Ability of laboratory to accurately perform tests
• Bio-safety and decontamination procedure
Types of case definitions in use
Case definition Criteria used
Who does it
Syndromic
Clinical pattern
Paramedical personnel and
members of community
Presumptive
Typical history and
clinical examination
Medical officers of primary
and community health
centres
Confirmed
Clinical diagnosis by a Medical officer and
medical officer and
Laboratory staff
positive laboratory
identification
Laboratory network for the Integrated
Disease Surveillance Project
Laboratories
Description
L1
• Peripheral laboratories and microscopic
centres
L2
• District public health laboratory
L3
• Disease based state laboratories
L4
• Regional laboratories and quality control
laboratories
L5
• Disease based reference laboratories
Risk groups, biosafety levels, practices
and equipment
BSL
Laboratory type
Laboratory practices
Safety equipment
P1
Basic teaching,
research
Good microbiological
techniques
None
Open bench work
P2
Primary health
services;
diagnostic
services, research
Good microbiological
techniques,
protective clothing,
biohazard sign
Open bench plus
biological safety cabinet for potential
aerosols
P3
Special diagnostic
services, research
As BSL 2 plus
special clothing,
controlled access,
directional airflow
Biological safety cabinet and/or other
primary devices for all activities
P4
Dangerous
pathogen units
As BSL 3 plus
airlock entry, shower exit,
special waste
Class III biological safety cabinet, positive
pressure suits, double ended autoclave
(through the wall) and filtered air
Method of laboratory surveillance
• Routine passive surveillance
 Selected diseases
• Outbreak situations
Conditions under regular surveillance
Type of disease
Disease
Vector borne diseases
•Malaria
Water borne diseases
•Diarrhea (Cholera)
•Typhoid
Respiratory diseases
•Tuberculosis
Vaccine preventable diseases
•Measles
Disease under eradication
•Polio
Other conditions
•Road traffic injuries
International commitment
•Plague
Unusual syndromes
•Meningo-encephalitis
•Respiratory distress
•Hemorrhagic fever
Other conditions under surveillance
Type of surveillance
Categories
Sentinel surveillance •STDs
•Other
conditions
Regular surveys
Conditions
•HIV/HBV/HCV
•Water quality
•Outdoor air quality
•Non
•Anthropometry
communicable •Physical activity
disease risk
•Blood pressure
factors
•Tobacco, blood pressure
•Nutrition
•Blindness
Additional state priorities
•Up to five diseases
Diagnosis of malaria
• Laboratory criteria for diagnosis
 Detection and identification malaria parasite
microscopically
• Sample collection for microscopy
 Thick and thin blood smear
• Time of collection
 During fever or 2-3 hours after peak of
temperature
 Before patient receives anti-malarial
Laboratory tasks at each level for the
diagnosis of malaria
L1
•
•
•
Sample collection •
Smear preparation •
Microscopy and
reporting
L2
Same as L1
Quality
control for
L1
L3
•
Quality
control for
L2
Diagnosis of cholera
• Laboratory criteria for diagnosis
 Isolation of Vibrio cholera O1 or O139 from stools
in any patient with diarrhea
• Sample collection
 Transfer a portion of specimen to a cotton wool
swab
 Insert it in alkaline-buffered salt solution
 If stool specimen could not be collected take a
rectal swab and insert it in the above solution
Laboratory tasks at each level for the
diagnosis of cholera
L1
•Stool sample
collection
•Transport to L2
L2
L3
•Stool sample
microscopy
•Culture
•Biochemical
and serotyping
•Transport to L3
for sensitivity
•Drug sensitivity
and phage
typing
•Quality control
for L2
•Training
Diagnosis of typhoid
• Laboratory criteria for diagnosis
 Serology – Widal or Typhi-dot test positive
 Isolation of S.typhi from blood, stool or other
clinical specimen
• Sample collection
 Blood / stool
Laboratory tasks at each level for the
diagnosis of typhoid
L1
• Typhi-dot test
• Blood and stool
collection for
culture
• Transport to L2
L2
•
•
•
•
Widal test
•
Typhi-dot
Blood and
stool culture •
•
Quality
assurance
for L1
L3
Quality
control for
L2
Special tests
Training
Tuberculosis
• Laboratory criteria for diagnosis
 Demonstration of alcohol-acid fast bacilli in at
least two of the three sputum smears or culture
positive for Mycobacterium tuberculosis
• Sample collection for microscopy
 Three specimens
• One spot specimen
• One early morning specimen (preferably the next day)
• One spot specimen when the early morning specimen is
being submitted for examination.
Laboratory tasks at each level for the
diagnosis of tuberculosis
L1
• Sputum collection
• Smear preparation
• Microscopy and
reporting
L2
•
•
•
Same as L1
Quality
control for
L1
Transport to
L3 for
culture
L3
• Culture and
sensitivity
testing
• Quality
control
Measles
• Laboratory criteria for diagnosis
 Presence of measles virus specific IgM antibodies
 At least four fold increase in antibody titre in paired
samples
 Isolation of measles virus
• Sample collection
 Serology
• An acute phase serum specimen (3-5ml of whole blood) be
soon after onset of clinical symptoms but not later than 7 days
 Virus isolation
• Urine collected within 5 days of rash onset (1-3days best).
• Do not freeze
Laboratory tasks at each level for the
diagnosis of measles
L1
• Collection of blood
and urine samples
• Transport to L3
L2
• Same as L1
L3
• Virus culture in
designated labs.
•Serology (?)
Polio
• Laboratory criteria for diagnosis
 Isolation of wild polio virus from stool
Laboratory tasks at each level for the
diagnosis of polio
L1
• Sample collection and
transport to designated
laboratories as per
National Polio
Surveillance Programme
(NPSP) guidelines
L2
• Sample
collection and
transport to
designated
laboratories as
per NPSP
guidelines
L3
• Virus culture in
designated
laboratories
Laboratory criteria for dengue
• Isolation of Dengue virus from serum, plasma,
leucocytes or autopsy samples
• Demonstration of Dengue virus specific IgM
antibodies or four fold or more rise in reciprocal IgG
antibody titre
• Demonstration of dengue antigen in autopsy tissue
by Immunochemistry or immunoflourescence or in
serum samples by EIA
• Detection of viral genomic sequences in autopsy
tissue, serum or CSF by PCR
One or more of the above
Sample collection for the laboratory
diagnosis of Dengue
Sample
Period of
collection
Storage for
24 to 48
hours
Transport
•Serum
•5 days after
onset
•+ 4oC
•L2
•Plasma
(Citrated blood)
•Within 5 days of
onset
•CSF
•Within 5 days of
onset
•+ 4oC
•L3
•Autopsy
•In the event of
•(Brain, lung, liver) death
Laboratory tasks at each level for the
diagnosis of Dengue
L1
L2
L3
• Collection of blood
for serology and virus
isolation
• Transport to L2
• Serology by ELISA
or rapid methods
• Transport to L3 for
culture
• Culture to be
performed in a
designated
laboratories (which
needs to be defined
as a disease specific
L3 or L4 / L5
laboratories)
• Serology by IgM ELISA
and rapid tests
• Quality control for L2
Laboratory criteria for the diagnosis of
Japanese encephalitis
• Demonstration of Japanese encephalitis virus
specific IgM antibodies
• Detection/isolation of antigen/virus
• Demonstration of viral antigen in the
autopsied brain tissue by the fluorescent
antibody test
Sample collection for the laboratory
diagnosis of Japanese encephalitis
Sample
Period of
Collection
•Serum
•Within 6 days of
onset
•CSF
•Within 6 days of
onset
•Autopsy (brain,
lung, liver)
•In the event of
death
Storage for 24
to 48 hours
•+4C
Transport
•L3
•In cold
chain
Laboratory tasks at each level for the
diagnosis of Japanese encephalitis
L1
Collection of blood
for serology and
culture
• Transport to L3
•
L2
• Same as L1
L3
• Serology to be
performed in a
designated labs.
(which needs to be
defined as a disease
specific L3 or L4 / L5
labs. due to the
problem of
availability of kits)
Laboratory criteria for the diagnosis
of plague
• Gram staining on smear taken from bubo,
blood or lung aspirate
• Detection of Y. pestis F1* antigen by direct
fluorescent antibody testing or by other
standardized antigen detection method
• Isolation from a clinical specimen
• A significant (equal or more than 4-fold)
change in antibody titre to the F1 antigen in
paired serum specimens
* Fraction 1. Glycoprotein from the capsule. Elisa technique
Laboratory tasks at each level for the
diagnosis of plague
L1
• Assist in sample
collection
L2
• Staining and
microscopy
• Transport
sample to L3
laboratory
• No reporting
(Wait
confirmation)
L3
• Culture, serology
and confirmation
to be performed
in a designated
L4/L5
laboratories
Leptospirosis
• Laboratory criteria for diagnosis
 Isolation from blood or other clinical materials by culture
 Positive serology, preferably Microscopic Agglutination Test
(MAT) using a panel of Leptospira strains
• Sample collection
 Blood
• During first week of illness collect, second sample to be
collected after about a week
 Urine
• Urine should be collected after second week of illness and
transported immediately in sterile container
Laboratory tasks at each level for the
diagnosis of leptospirosis
L1
L2
•Collection of blood •Serology by
and urine
latex
agglutination/
•Transport to L2
IgM ELISA
•DGM
•Transport
samples to L3
labs for culture
L3
•Culture
• MAT and serovar
identification
Laboratory tests for water samples
• Most Probable Number (MPN) method for
coliform bacteria
• H2S strip method for fecal contamination
assessment
Laboratory tasks at each level for the
assessment of water quality
L1
• Collection of
samples
• Rapid test- (H2S
strip)
L2
•
•
•
Collection of
samples
Rapid test(H2S strip)
MPN test
L3
• Same as L2
• Quality control
for L2
Functions of L1 laboratory technicians
• Collection of samples for investigations
• Perform the laboratory tests assigned to L1 labs




Microscopy for malaria
Microscopy for tuberculosis
Typhi-dot test for typhoid fever
H2S test for water quality
• Transport relevant sample to L2 laboratories for
culture and serological investigations
• Assist Rapid Response Teams in sample collection
• Participate in External Quality Assurance conducted
by L2 laboratories
Functions of L2 laboratory technicians
• Perform all tests performed by L1 laboratories
• External Quality Assurance for L1 laboratories
• Perform the tests assigned to L2 laboratories
 Culture and sensitivity for cholera
 Serological test for typhoid, Dengue, Leptospirosis
 MPN test for water quality
• Transport relevant samples to L3 laboratories
• Transport 5% of tested samples to L3 for testing and
quality assurance
• Reporting test results to L1 laboratories for samples
received from L1 laboratories
• Reporting tests result weekly to district
Quality assurance
Quality
assurance =
Internal
quality control
(Continuous, concurrent
control of
laboratory work)
+
External
quality
assessment
(Retrospective and
periodic assessment)
Internal quality control
• Test request and specimen collection
• Test processing
 Temperature
 Reagent
 Maintenance of equipment
• Reporting and using test results
External quality assessment
• Within the state IDSP system
 L1 by L2
 L2 by L3
• Through external agency
 External quality assurance scheme for selected
tests
Action to be taken by the multi-purpose
worker in the field
Syndrome
Action
•Fever
•Blood smear for all patients
•Acute flaccid paralysis
•2 stool samples at interval of 24
hours transported to the medical
officer of the primary health
centre in reverse cold chain
•Fever with rash, altered
sensorium or bleeding
•Refer to the medical officer of
the primary health centre for
specific laboratory action
•Fever more than 14 days
•Cough < or > 3 weeks
•Loose watery stools
•Acute jaundice
•Unusual syndromes
Laboratory investigations by the PHC/CHC
medical officer /laboratory technician
for Dengue
When to collect
sample
•Single case of probable dengue
•First 10 cases in outbreak situations
What specimens
to be collected
•5ml of blood for serology
•5ml of blood in citrate for virus isolation
(If recommended by rapid response team)
Processing at the
CHC by the
technician
•Serum separation
Storage
•Serum and blood in refrigerator.
•If delay in transportation, store in –20C
Transportation
•As quickly as possible within 24 hours in reverse
cold chain to the district laboratory
Laboratory investigations by the district
and state laboratories for Dengue
Processing at
district / medical
college / sentinel
laboratories
•Serology - IgM Elisa / rapid test
•Platelet count for hospitalized patients
Storage
•–20C
Transportation
•1st and 2nd serum and blood sample sent to
state / reference laboratory
Processing at
state / national
laboratories
•Virus isolation and antigen detection
•HAI and neutralization to detect rise in
antibodies
•Quality control of the IgM Elisa of the district
Laboratory investigations by the PHC/CHC
medical officer /laboratory technician
for Japanese encephalitis /fever with altered
consciousness
When to collect
sample
•Single case of probable Japanese encephalitis
•First 10 cases in outbreak situations
What specimens
to be collected
•5ml blood for serology
•CSF in hospitalized cases: Serology and virus
isolation
Processing at the
CHC by the
technician
•Serum separation
Storage
•Serum and CSF in refrigerator.
•If delay in transportation, store in –20C
Transportation
•As quickly as possible within 24 hours in reverse
cold chain to the state reference laboratory
Laboratory investigations by the district
and state laboratories for Japanese
encephalitis
Processing at
district / medical
college / sentinel
laboratories
•NIL
Storage
•–20C
Transportation
•CSF and serum sent to state / reference
laboratory
Processing at
state / national
laboratories
•IgM Elisa for CSF and serum
•HAI / neutralization for detection of rise in
antibody titres.
•Virus isolation and antigen detection in CSF
Laboratory investigations by the PHC/CHC
medical officer /laboratory technician
for malaria or fever
When to collect
sample
•Single case of fever
What specimens
to be collected
•Blood smear
Processing at the
CHC by the
technician
•Staining and microscopy
Storage for
quality assurance
•All positive
•10% negative
Transportation
•NIL
Laboratory investigations by the district
and state laboratories for malaria
Processing at
district / medical
college / sentinel
laboratories
•As in primary health care centre for cases seen
at the district hospital
Storage
• As in primary health care
Transportation
•NIL
Processing at
state / national
laboratories
•NIL
Laboratory investigations by the PHC/CHC
medical officer /laboratory technician for
cholera /loose watery diarrhea
When to collect
sample
•Case of probable cholera
•First 10 cases in outbreak situations
What specimens
to be collected
•Fresh stools or rectal swab in Cary–Blair medium
Processing at the
CHC by the
technician
•NIL
Storage
•In refrigerator
Transportation
•As soon as possible
•No need of cold chain if within 24 hours
Laboratory investigations by the district
and state laboratories for cholera
Processing at
district
•Culture, identification and sensitivity
Storage
• Positive isolates at + 4oC
Transportation
•Sealed stab culture of positive isolates to state
reference laboratory
Processing at
state laboratory
•Confirmation of serotype / phage typing
•Antibiotic sensitivity
•Quality assurance
Laboratory investigations by the
PHC/CHC medical officer /laboratory
technician for typhoid /fever > 7 days
When to collect
sample
•One case of probable typhoid
•First 10 cases in outbreak situations
What specimens
to be collected
•5ml blood in citrate
•5ml blood for serology (2 samples at one week
interval if the first sample is negative and if
requested by the district laboratory)
Processing at the
CHC by the
technician
•Serum separation
•Typhi dot test
Storage
•In refrigerator (Serology)
Transportation
•1st and 2nd serum sample and blood sample to be
sent to the district laboratory
Laboratory investigations by the district
and state laboratories for typhoid
Processing at
district
•Serology - Widal in paired sera if first is negative
•Blood, stool and bone marrow culture,
identification and sensitivity
Storage
•At + 4oC
Transportation
•10% of positive and negative specimens to be
sent to state for quality assurance
Processing at
state laboratory
•Blood culture
•Identification
•Sensitivity
Laboratory investigations by the
PHC/CHC medical officer /laboratory
technician for hepatitis/ acute jaundice
When to collect
sample
•During outbreaks only
•First 10 cases only
What specimens
to be collected
• 5ml blood for serology
Processing at the
CHC by the
technician
•Serum separation
Storage
•At - 20C deep freezer
Transportation
•In reverse cold chain to the state/reference
laboratory
Laboratory investigations by the district
and state laboratories for hepatitis
Processing at
district
•NIL
Storage
•At - 20oC
Transportation
•Reverse cold chain to the state / reference
laboratory
Processing at
state laboratory
•IgM Elisa for HAV and HEV
Laboratory investigations by the
PHC/CHC medical officer /laboratory
technician for measles / fever with rash
When to collect
sample
•During outbreaks only
•First 10 cases only
What specimens
to be collected
•5ml blood for serology
•30 ml urine for virus isolation
(If required by the rapid response team)
Processing at the
CHC by the
technician
•Serum separation
Storage
•In refrigerator
Transportation
•Immediately to the district laboratory within 24
hours, with reverse cold chain
Laboratory investigations by the district
and state laboratories for measles
Processing at
district
•Measles IgM Elisa
Storage
•- 20oC
Transportation
•10% of positive, all negative and urine samples
to be sent to the state / reference laboratory
Processing at
state laboratory
•Urine virus isolation
•Antigen detection
•Quality assurance of the positives
•Test of negative for rubella
Laboratory investigations by the PHC/CHC
medical officer /laboratory technician for
tuberculosis /cough > 3 weeks
When to collect
sample
•All probable cases of tuberculosis
What specimens
to be collected
• 3 sputum specimens
•Spot/early morning/spot
Processing at the
CHC by the
technician
•Smear staining and microscopy
Storage for
quality assurance
•10% of positives
•All negatives
Transportation
•Sputum to the state laboratory for culture
sensitivity testing
Laboratory investigations by the district
and state laboratories for tuberculosis
Processing at
district
•Smear, microscopy
Storage
•10% of positives and all negatives to be kept for
quality assurance
Transportation
•NIL
Processing at
state laboratory
•For quality assurance: Blinded samples sent to
districts
Laboratory investigations by the PHC/CHC
medical officer /laboratory technician for
acute flaccid paralysis
When to collect
sample
•A single case of acute flaccid paralysis
What specimens
to be collected
•2 stools specimens at 24 hour interval
Processing at the
CHC by the
technician
•NIL
Storage
•In refrigerator
Transportation
•With 24 hours
•Reverse cold chain
•National polio laboratory
Laboratory investigations by the district
and state laboratories for acute flaccid
paralysis
Processing at
district
•NIL
Storage
•NIL
Transportation
•Reverse cold chain
Processing at
national polio
laboratories
(ONLY)
•Virus isolation
•Identification
•Quality assurance by the reference laboratory
and WHO
Laboratory investigations by the district
and state laboratories for HIV/HBV
Processing at
district
•Only at the voluntary counseling and testing
sites or blood transfusion centres
•Testing as per the recommendations of the
National AIDS Control Organization (NACO)
Storage
•- 20oC
Transportation
•All positive specimens to the state laboratory
Processing at the
state processing /
national
reference
laboratory
•Confirmatory tests (Western blot)
Laboratory investigations by the
PHC/CHC medical officer /laboratory
technician for plague
When to collect
sample
•From probable cases
•Samples to be collected by the rapid response
team
What specimens
to be collected
•Aspirate from the bubo
•Sputum from pneumonic plague cases
•5 ml blood sample for serology
Processing at the
CHC by the
technician
•NIL
Storage
•NiL
Transportation
•Immediately to the state/ national reference
laboratory with P3 facilty
Laboratory investigations by the district
and state laboratories for plague
Processing at
district
•At the medical college level only
•Smear, microscopy of aspirate / sputum for
bacilli
Storage
•+ 4oC
Transportation
•All samples by reverse cold chain in reverse cold
chain to the nearest reference laboratory as
specified by the rapid response team
Processing at the
state processing /
national
reference
laboratory
•Isolation of bacteria by culture
•Antigen detection
•Direct fluorescent antibody testing of smears
(for anti-F1 antibody)
•PCR test
Laboratory investigations by the
PHC/CHC medical officer /laboratory
technician for leptospirosis
When to collect
sample
•From probable cases
What specimens
to be collected
•5ml blood for serology
Processing at the
CHC by the
technician
•Serum separation
Storage
•At +4C
Transportation
•Immediately by reverse cold chain to the district
Laboratory investigations by the district
and state laboratories for leptospirosis
Processing at
district
•Rapid agglutination kit
Storage
•+ 4oC
Transportation
•To the state
Processing at the
state processing /
national
reference
laboratory
•Microscopic Agglutination Test (MAT) for
identification of serovars
Laboratory investigations by the health
workers and medical officer of the PHC
for non communicable diseases
When to collect
sample
•When surveillance is conducted
What specimens
to be collected
•Blood sample
Transportation
•To designated laboratories
Testing site
•District laboratories
•Medical college laboratories
•Identified laboratories
Test to be done
•Blood sugar, serum cholesterol, triglycerides
Laboratory data management
• Recoding
 Details of specimens received
 Tracking of samples
 Results of tests performed
• Analysis and interpretation of tests
• Timely and accurate communication of
results
Information to be recorded on each
specimen/ accompanied with each
specimen
Name, age, sex
Address in detail
Reporting unit referring the sample
Syndromic diagnosis
Date of onset of illness
Nature of sample, date of collection, date of
receipt and condition of sample
• Investigation requested
• Whether convalescent specimen or not
•
•
•
•
•
•
Sample laboratory register
ID no
Name and
address of
patient
Age
Sex
Prov.
Diag.
Lab tests
ordered
Lab
results
Date
sent
to L2
Result
Date of
from L2 result
The L form
• Weekly reports from laboratories to the
district surveillance officer
 Prepared on the basis of the laboratory register
 Filled by nodal person in the laboratory
 Sent every Saturday of each week
• Zero/NIL reporting
• Electronic link between
 District public health laboratory
 District surveillance unit
Points to remember (1/2)
• Categorization of labs - List of L1 and L2
labs in the districts & List of Disease wise L3
labs in the state
• List of tests that can be done at L1 and L2
labs
• List of diseases that can be confirmed only
by L3 labs
Points to remember (2/2)
• Sourcing the consumables required by the labs
• Samples that have to be collected for specific
disease
• Bio Safety and waste management
• Quality assurance