6

INTEGRATED DISEASE
SURVEILLANCE PROJECT

TRAINING MANUAL FOR

STATE & DISTRICT
SURVEILLANCE OFFICERS

LABORATORY METHODS FOR

CONFIRMATION OF DIAGNOSIS, COLLECTION,
STORAGE, TRANSPORTATION OF SPECIMEN

Module – 6
93
 CONTENTS

1. Introduction 95
2. Specific Instructional Objectives 95
3. Module Structure at a Glance 95
4. Salient Points to Remember 96
5. Frequently Asked Questions 96
6. Group Activities 96
7. Handout on Role of Laboratories 100

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1. INTRODUCTION
This section covers
• Action to be taken by the HW, MO & lab assistant of the PHC, lab technician at
district lab
• Collection, Preservation and Transportation of specimen
• Tests that should be conducted at various laboratories

2. SPECIFIC INSTRUCTIONAL OBJECTIVES

At the end of the session the participants would be able to

List the L1 and L2 labs within the district; disease based L3 labs in the state and
L4 and L5 labs in the country

Understand the needs of the L1 and L2 labs (equipment, glassware, consumables,
reagents and kits) and arrange for the logistic support to the labs.

Identify what action is to be taken by the lab technician for sample collection in
response to the diagnosis made by the MO

List the tests that can be performed at L1 and L2 labs

Identify the quality control process within the lab network

Should be able to understand bio-safety issues

To identify – transport modalities of sample to higher levels.

Understand the training needs of the lab personnel.

Keep a track of flow of samples

To draw a flow diagram for reporting of the lab investigations
3. MODULE STRUCTURE AT A GLANCE
DURATION OF SESSION 2 HOURS
S.No CONTENT METHODOLOGY DURATION TEACHING
AIDS
1 Role of lab in disease surveillance. Lecture 30 mins Power point
Core conditions under surveillance
Organization of the labs
2 Disease specific action.Sample Module reading 45 mins Handouts and
collection, storage and Discussions reading material
transportation; List of lab and questions
investigation at L1 and L2 lab
3. Reporting formats Lecture 20mins OHP / PP
4. Summation Discussion, Group 25 mins Handouts
activity / Exercise

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4. SALIENT POINTS TO REMEMBER
 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
 Sourcing the consumables required by the labs
 Samples that have to be collected for specific disease
• What sample
• Quantity
• Collection criteria
• Transport and storage condition
 Bio Safety and waste management
 Quality assurance
5. FREQUENTLY ASKED QUESTIONS

Who should initiate action for sample collection?

What samples are to be collected for specific disease?

To which level of lab should the samples be sent

If the specific lab is not in a position to perform the test what action is to taken

Who is responsible for providing the consumables etc to the lab? And from
where they are to be obtained?

In outbreak situation - action to be taken to involve the lab

Training needs of the L1 and L2 lab technicians
6. GROUP ACTIVITIES

Reading the handouts

Discussions on the role and activity of lab

Exercises

96
Exercise 1: Tick the action on each functionary outlined below

INVESTIGATION
DATA ENTRY
COLLECTION

COLLECTION

FEEDBACK
RESPONSE
ANALYSIS
TESTING
SAMPLE
DATA
LEVEL FUNCTIONARIES

SUB DISTRICT LAB ASST

LEVEL MO (PHC / CHC)
DISTRICT DISTRICT LAB
LEVEL DIST HOSP
MEDICAL COLLEGES
RRT
STATE LEVEL STATE SURVEILLANCE CELL
L3 LAB
PRIVATE LABS
SECTOR

Exercise 2: Fill in sample to be collected

Disease under Sample to be collected Test at L1 lab Test at L2 lab
surveillance

Malaria

Tuberculosis

Cholera

Typhoid

Leptospirosis

Polio

Dengue

JE

Measles

Plague

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Exercise 3: Yes / No
Malaria Microscopy at L2

Tuberculosis Microscopy at L1

Measles Serology at L1

Dengue Serology at L1

Dengue Serology at L3

JE Serology at L1

Typhoid Culture at L2

Cholera Culture at L2

Exercise 4: Lab requirements: fill in

Equipments

Glass ware

Reagents for culture

Biological reagents:
Antiserum and
standard cultures

Stains for malaria and

TB microscopy

Rapid response action

Exercise 5: List the purpose for which the form is used

Lab request form

Lab form L1

Referral form

Exercise 6: Transportation of specimens (where and how)

Stool for Cholera culture

Blood for Typhoid culture

Serum for Leptospirosis

Nasopharyngeal swab
for measles virus
isolation

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 TASKS AT EACH LEVEL; *IMPLIES AS PER THE GUIDELINES OF THE
VERTICAL PROGRAMMEMES
Disease under Tasks at Level 1 Level 2 Level 3
surveillance

Malaria* 1.Blood Sample collection 1.Same as L1 QC of L2

2.Smear preparation 2.QC of L1
3.Microscopy and reporting

Tuberculosis 1. Sputum collection 1. Same as L1 1.Culture and Sensitivity testing

2.smear preparation 2.QC of L1 2.Quality control
3.Microscopy and reporting 3. Transport to L3 for culture
4.Transport to L3 for culture

Cholera 1.Stool sample collection 1.Stool sample 1.Training

2.Transport to L2 2.Microscopy 2. Drug sensitivity and Phage typing
3.Culture 3.QA of L2
4.Biochemical & serotyping
5.Transport to L3 for sensitivity

Salmonellosis 1.Blood and stool collection for culture 1.Widal test 1.Training
2.Typhidot test 2. Typhidot 2.QA
3.Transport to L2 3. Blood and stool culture 3.Special tests
4. QA of L1

Leptospirosis 1.Collection of blood and urine 1.Dark Field Microscopy 1.Culture

2.Transport to L2 2.Serology by latex agglutination/ 2.MAT test & Serovar identification
IgM ELISA
3.Transport to L3 for culture

Polio* Sample collection and transport to Sample collection and transport to Sample collection and transport
designated labs as per the NPSP guidelines designated labs as per the NPSP to designated laboratories as per
guidelines NPSP guidelines

Dengue 1.Collection of blood for serology and for 1.Serology by Elisa/ or rapid method 1. Culture to be performed in a
virus isolation 2.Transport to L3 for culture designated lab (which needs to be
2.Transport to L2 defined as a disease-specific
L3 or L4/L5 labs
2.Serology by IgM ELISA and rapid
tests
3.QC for L2 lab

Japanese 1.Collection of samples for serology and 1.Same as L1 1.Serology to be performed in a

encephalitis culture designated lab (which needs to be
2.Serum separationTransportation of defined as a disease-specific L3 or
samples to L3 L4/L5 labs due to their problem of
availability of kits

Measles 1. Collection of blood and urine samples Same as L1 1. Virus culture in designated lab.
2.transport to L3

Anthrax 1. Information to L2 1.Specimen Collection and 1.Culture

transportation to L3

Rickettsial diseases 1. Collection of blood and transportation of 1. Weil-Felix test 1Weil-Felix test
serum to L2 2.transport to L3 2. Confirmation of diagnosis to be
performed in a designated lab (which
needs to be defined as a disease-
specific L3 or L4/L5 labs

Plague 1.Assist in sample collection Staining and microscopy Culture, serology and confirmation to
Transport sample to L3 lab be performed in a designated L4/
no reporting L5 labs

Hepatitis Collect and send sera to L2 Rapid test for Hepatitis B, C if 1.QC of L2
availableTransport sample for 2. Serology for all Hepatitis markers
A,D, E and other markers to L3

Water Quality 1.Collection of samples 1.Collection of samples 1. Same as L2

2.Rapid test-H2S strip 2.Rapid test- H2S strip 2. QC for L2
3. MPN test

*Since plague is a notifiable disease, the sample should be collected and sent to referral laboratories:
1. Central Plague Laboratory, Zoonosis Division, National Institute of Communicable Diseases, 22, Sham Nath Marg, Delhi-110054, Tel: 011-23912901,
011-239123148
2. Plague Surveilance Unit, NICD, NTI Campus, 8-Bellary Road, Bangalore, Tel: 080-23446723.

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7. HANDOUT ON ROLE OF LABORATORIES IN IDSP
7.1 Introduction
Laboratory support to disease surveillance activities has been recognized as an
essential component of any surveillance programmeme both for communicable as well
as non-communicable diseases.
Laboratory based disease surveillance will be the third level of surveillance in IDSP.
The laboratories will assist in passive routine surveillance of selected diseases and
active surveillance in case of outbreak investigations. The laboratories will participate
in:

Early confirmation of diseases under surveillance

Epidemiological investigation

Rapid laboratory confirmation of the diagnosis

The implementation of effective control measures
The laboratory network for IDS will be established at four levels of functions. It will
include both private and government labs:
♦ Peripheral Laboratories and Microscopic centers - L1 Labs
♦ District Public Health Laboratory - L2 Labs
♦ Disease Based State Laboratories - L3 Labs
♦ Regional Laboratories IDSP and Quality control Laboratories - L4 Labs
♦ Disease based reference Laboratories - L5 Lab
* a glossary should specify the participating laboratories at each level. At the peripheral
level e.g. PHC lab, Microscopy centers, CHC labs and private labs (if any). Similarly at
each of the other levels the final list of category of participating laboratories can be
included only when an understanding has been reached with each of them.
The laboratory results are used to accurately diagnose the patient so that appropriate
therapy can be given, as well as verify the cause of suspected outbreaks. As a Public
Health Manager, one needs to identify what samples are to be collected and where
they have to be sent, so that there is minimum delay in confirming the diagnosis.
The initial investigation involves two important processes: collection of information on
suspect cases and collection of clinical specimens for laboratory diagnosis.
Successful laboratory confirmation of a disease depends on:

Advance planning

Collection of appropriate and adequate specimens

Correct packaging and rapid transport to an appropriate laboratory

The ability of the laboratory to accurately perform the diagnostic tests

Proper bio safety and decontamination procedures to reduce the risk of further
spread of the disease
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Consider the logistic requirements for sampling equipment and supplies, specimen
handling and transport to the laboratory (timing, route, transmit temperature
requirements, shipping procedures, and documentation), and decontamination
procedures in advance.

7.2 METHOD OF LABORATORY SURVEILLANCE

Laboratories will be participating in:

Routine passive surveillance of the selected diseases: In case of fever and cough of
more than 3 weeks the field worker would initiate the sample collection as per the
respective programme requirements (Tuberculosis). In other syndromic presentations
the health worker would refer the case to the MO of the PHC for further investigation.

In case of an outbreak situation, the MO with the help of the surveillance team would
initiate action including sample collection. Once a suspected outbreak has been
detected and reported, an epidemiological investigation would be quickly organized.
Several key issues will be discussed, and decisions agreed upon before the team goes
to the field. Ultimately these decisions will guide the materials and procedures required
for efficient specimen collection and their transport to the laboratory for testing.

Core Conditions under surveillance in IDSP

(i) Regular Surveillance:
Vector Borne Disease : 1. Malaria
Water Borne Disease : 2. Acute Diarrhoeal Disease (Cholera)
: 3. Typhoid
Respiratory Diseases : 4. Tuberculosis
Vaccine Preventable Diseases : 5. Measles
Diseases under eradication : 6. Polio
Other Conditions : 7. Road Traffic Accidents
(Linkup with police computers)
Other International commitments: : 8. Plague
Unusual clinical syndromes : 9. Meningoencephalitis/Respiratory
(Causing death / hospitalization) Distress, Hemorragic fevers, other
undiagnosed conditions
(ii) Sentinel Surveillance
Sexually transmitted diseases/Blood borne : 10. HIV/HBV, HCV
Other Conditions : 11. Water Quality Monitoring
: 12. Outdoor Air Quality
(Large Urban centers)
(iii) Regular periodic surveys:
NCD Risk Factors : 13. Anthropometry, Physical activity, Blood
Pressure, Tobacco, Nutrition, Blindness
(iv) Additional State Priorities : Each state may identify up to five additional conditions for
surveillance.

101

The number of core diseases is limited to improve quality of surveillance and
to reduce workload on the peripheral health worker.

The list will be reviewed and modified according the needs of surveillance at
least once in 2 years.
7.3 ORGANIZATION OF LABORATORIES
Stratification of laboratories
The laboratory network for IDSP will be established at five levels of functions based on
location of lab, expected functions and facilities available. A national agency will be
designated as the coordinated agency, which shall also undertake monitoring and
quality assessment of the laboratories at peripheral and intermediate levels. All
laboratories will supervise their immediate lower level labs. * peripheral laboratories
may include PHC, block PHC, CHC and select private laboratories (if any). Similar listing
in the glossary for other level labs. The list at other levels can only be finalized after
discussionswith the core group because it would require the consent of the individual
labs i.e. L3, L4and L5 labs
• Peripheral Laboratories - L1 labs
• District hospital/Public Health Laboratory - L2 labs
• Disease Based State Laboratories - L3 labs
• Regional Quality Control Laboratories - L4 labs
• Disease based National reference Laboratories - L5 labs

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 TASKS AT EACH LEVEL
Disease under Tasks at Level 1 Level 2 Level 3
surveillance

Malaria 1.Sample collection 1.Same as L1 QC of L2

2.Smear preparation 2.QC of L1
3.Microscopy and reporting

Tuberculosis 1. Sputum collection 1. Same as L1 1.Culture and Sensitivity testing

2.smear preparation 2.QC of L1 2.Quality control
3.Microscopy and reporting 3. Transport to L3 for culture
4.Transport to L3 for culture

Cholera 1.Stool sample collection 1.Stool sample Microscopy 1.Training

2.Transport to L2 2.Culture 2. Drug sensitivity and Phage typing
3.Biochemical & serotyping 3.QA of L2
4.Transport to L3 for sensitivity

Salmonellosis 1.Blood and stool collection for culture 1.Widal test 1.Training
2.Typhidot test 2. Typhidot 2.QA
3.Transport to L2 3. Blood and stool 3.Special tests
culture
4. QA of L1

Leptospirosis 1.Collection of blood and urine 1.Dark Field Microscopy 1.Culture to be performed in a
2.Transport to L2 2.Serology by latex agglutination/ designated lab (which needs to be
IgM ELISA defined as a disease-specific L3 or
3.Transport to L3 for culture L4/L5 labs
2.MAT test & Serovar identification to
be performed in a designated lab
(which needs to be defined as a
disease-specific L3 or L4/L5 labs

Polio Sample collection and transport to Sample collection and transport to designated labs as per the NPSP
designated labs as per the NPSP guidelines designated labs as per the NPSP guidelines
guidelines

Dengue 1.Collection of blood for serology and for 1.Serology by Elisa/ or rapid method 1. Culture to be performed in a
virus isolation 2.Transport to L3 for culture designated lab (which needs to be
2.Transport to L2 defined as a disease-specific
L3 or L4/L5 labs
2.Serology by IgM ELISA and rapid
tests
3.QC for L2 lab

Japanese 1.Collection of samples for serology and 1.Same as L1 1.Serology to be performed in a

encephalitis culture designated lab (which needs to be
2.Serum separationTransportation of defined as a disease-specific L3 or
samples to L3 L4/L5 labs due to their problem of
availability of kits

Measles 1. Collection of blood and urine samples Same as L1 1. Serology by IgM ELISA virus culture
2.transport to L3 in designated lab.

Anthrax 1. Information to L2 1.Specimen Collection and 1.Culture

transportation to L3

Rickettsial diseases 1. Collection of blood and transportation of 1. Weil-Felix test 1Weil-Felix test
serum to L2 2.transport to L3 2. Confirmation of diagnosis to be
performed in a designated lab (which
needs to be defined as a disease-
specific L3 or L4/L5 labs

Plague 1.Assist in sample collection Staining and microscopy Culture, serology and confirmation to
Transport sample to L3 lab be performed in a designated L4/
no reporting L5 labs

Hepatitis Collect and send sera to L2 Rapid test for Hepatitis B, C if 1.QC of L2
available 2. Serology for all Hepatitis markers
Transport sample for A,D, E and
other markers to L3

Water Quality 1.Collection of samples 1.Collection of samples 1. Same as L2

2.Rapid test-H2S strip 2.Rapid test- H2S strip 2. QC for L2
3. MPN test

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 REQUIREMENTS OF L1 LABS
Infrastructure Space for microscopy, work table for
test procedures, washing, sterilization
and decontamination
Equipments
Microscope, binocular with ain built liquid attachment 1 no
Autoclave for sterilization 1 nos
Autoclave for decontamination 1 No
Micropipettes 50-200 µl, 100-1000 µl One each
Centrifuge (benchtop) 1 no
Refrigerator 1 no
Water bath 1 no
Consumable Items Approx. quantity required per year
Autoclavable polypropylene items
Screw capped round bottom 5 ml vials externally
threaded 500 nos
Screw capped round bottom 12 ml vials 500
2ml screw capped Storage containers externally
threaded 1000
Test tube racks 4 nos
TT racks for 5 ml tubes 2 nos
Micropipette tips 50-200 microliters (yellow color) 1000 nos
Slide boxes for 25 slides 5 nos
Wash bottles 2 nos
Stool collection bottles (presterilized) 200 nos
Urine collection bottles (presterilized) 50 nos
Sputum collection containers 100 nos
Gloves autoclavable 200 nos
Vaccine carrier with ice packs 4
Glass items
Measuring cylinders – 100 ml, 50 ml 2 nos each
Conical flask –100 ml 3 nos
Conical flask –200 ml 4 nos
Pasteur pippettes (disposable, presterilized,
individually wrapped) 1000nos
2ml, 5ml and 10ml pipettes (glass) Each 10 nos
Others
Fine tips forceps 2 nos
Lancet 50 nos
Autoclave labels Quantity would depend on number of
tests and would vary. The lab in charge /
MO could give the annual requirements
to the DSO who would procure it through
the identified modalities.

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Reagents
Stain for Malaria and TB
Typhidot test kit
H2S testing kit
Stationary, records and report forms
Requirements of L2 labs
Infra Structure Space for washing, sterilization,
decontamination, Media reparation,
worktables, Safety cabinets, culture
facility, ELISA, other serology and office
Equipments Number required
Autoclave for sterilization 1
Autoclave for decontamination 1
Hot air oven 1
Incubator 1
Water bath 1
Weighing balance 1
Microscopes (Binocular) 2
Vertical Laminar flow cabinet 1
ELISA reader and washer 1
Micro plate shaker 1
Refrigerators 2
-20 deep freezers 1
Centrifuge 1
Cyclomixer 1
Hot plate 1
Distilled water still 1
pH meter 1
Variable volume Micro pipettes 50-200 µl, 100-1000 µl One each
LPG cylinders, regulators and burners 2
Needle shredder 1
Mini Incinerator 1
Consumables and Glass Ware Approximate / annum
Disposable syringes with needles 1000
Polypropylene storage vials 500 each
10 ml and 15 ml ml test tubes (glass/ PP) 500 each
Sterile sputum containers 250
Sterile Urine containers 100
Sterile stool sample containers 200

105
2, 5, 10 ml pipettes (glass) 50 each
Pasteur pipettes presterlized individualy wrapped 250
Measuring cylinders – 50,100, 250, 500, and 1000 ml Each 2
Flasks – 60, 100, 250, 500 & 1000 ml Each 5
Petri dishes 100
Reagent bottles – 60 and 100 ml Each 4
Beakers – 50, 100, 200 ml Each 6
Blood culture bottles (100 ml) 50
Centrifuge tubes – 15 ml 25
Funnel – dia 4 and 6 inches Each 3
Lancet 25
Inoculating loops 12
Test tube racks (18 hole) 10
Micropipette tips 100 microlitre 500
Variable Micropipette tips 100 – 1000 microlitre 500
Slides & Slide Boxes 4
Gloves – varying sizes Each 50
Tourniquet 3
Surgical masks 500
Discarding bags 500
Vaccine carrier with ice packs 4
Absorbent material - cotton Quantity has to be determined based
on anticipated number of cases /
annum. (Variable from dist to dist).
The lab in charge / MO could give the
annual requirements to the DSO who
could procure it through the identified
modalities.
Labels
Glass marking pens
Adhesive tape
Scissors
Scalpel and blades
Forceps
Rubber teats

Thermometer

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 MEDIA AND REAGENTS
Dehydrated Culture Media Stains - JSB, Ziehl Neelson’snelson, Grams,
Mac Conkey, and broth Waysons,

TCBS agar Kits – Typhidot Dengue water testing,

Hepatitis, Weil Felix,

Biochemical test media Chemicals for reagent and media preparation

Cary Blair media

Nutrient Agar

Mueller Hinton agar Miscellaneous

Selenite F enrichment broth Hand disinfectant – Savlon, dettol

Antibiotic discs Disinfectant solution – Sodium

Specimen collection bottles Hypochlorite 4%

XLD MediaPeptone Autoclave and Hot air oven indicator tapes

Kovac’ s reagent Autoclave spore strips

Oxidase disc Stationery:

HCL H2SO4 Lab request forms, Records etc

NaOH

Widal reagents Packing material like aluminium for

Anti sera – Cholera Special Brown Paper, Twine ball

Standard reference material such

as standard bacterial strains

The District surveillance officer is responsible for the L1 and L2 labs within the district.
Functions of L1 lab technician:(The MO to assist in blood collection)
1. Collection of samples for investigations
2. Perform the quality Laboratory tests assigned to the L1 level.
a. Microscopy for Malaria
b. Microscopy for TB
c. Typhidot test for Typhoid fever
d. H2S test for water quality.
3. Transport of relevant sample to L2 lab for culture and other serological
investigations
4. Assist rapid response team in sample collection

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5. Participate in external quality assurance conducted by L2 lab
Functions of L2 lab technician
1. Perform all the tests performed by L1 lab
2. Quality assurance of L1 lab
3. Perform lab test assigned to L2 level
a. Culture & sensitivity tests for Cholera,
b. Serological tests for Cholera, Typhoid, Dengue, Leptospirosis
c. MPN test for water quality
4. Transport relevant samples for testing at L3 lab
5. Transport 5% of tested samples to L3 for testing and QA
6. Reporting test result to L1 lab for sample received from L1 lab
7. Reporting test result weekly to DSO
7.4 Biosafety in Laboratories
Good laboratory technique is fundamental to laboratory safety. Important concepts
to have lab safety are listed below.
♦ Entry / access to laboratory area
♦ Have a biohazard sign (Fig) displayed on the doors of the rooms where
infectious agents are handled.

Fig: Biohazard sign.

♦ Entry to laboratory working area should be only for laboratory persons.
♦ Doors to the laboratory should be kept closed.
♦ No smoking, eating, or drinking is allowed in laboratory area.
Personal Protection
♦ While working in the laboratory always wear lab coat.
♦ Have all the personnel protective equipments ready & use them as per the
procedures strictly for highly infectious diseases outbreaks.

108
♦ Wear gloves for all procedures that may involve direct or accidental contact with
blood / infectious materials.
♦ After use, gloves should be removed carefully without touching infected surface,
disposed off in container containing disinfectant solution. Hands should be
washed with soap & water.
♦ Laboratory personnel must wash their hands after handling infectious
materials/ performing test procedures and before they leave the laboratory
working area.
♦ Laboratory coat should not be worn outside the laboratory area i.e canteen,
library, and toilet or staff common room.
♦ Eating, drinking, applying cosmetics and handling contact lens are strictly
prohibited in the laboratories.
♦ Laboratory coat used/unused should not be placed in the same cupboard with
street clothes or food articles etc.
♦ Lab personnel should receive suitable vaccination e.g. Hepatitis B.
General procedural precautions
♦ Mouth pipetting must be strictly avoided.
♦ Materials / articles must not be held in the mouth. Do not lick / wet labels for
sticking.
♦ All technical procedures should be such that they minimize the formation of
aerosols and droplets. In the district laboratories do not perform any procedure
that generates lots of aerosolization unless there is an access to biological
safety cabinet.
♦ Do not use hypodermic needles and syringes for pipetting devices.
♦ All spills, accident or exposure to infectious materials must be reported to
laboratory in charge and a record should be maintained.
♦ Display written procedures for the clean up of all spills.

109
Laboratory working areas
• Keep the laboratory area neat, clean and free of materials that are not required.
• Decontaminate the working surface after any spill and at the end of the working
day using 1 % Sod. hypochlorite.
• All contaminated materials, specimens, cultures, must be decontaminated in the
laboratory premises before final disposal or cleaning for reuse.
• If there are windows in laboratory area, they should have arthropod / mosquito &
fly proof mesh.
Bio safety Management
• Have one person responsible for bio safety activitiesà Biosafety officer.
• Health checks up of laboratory staff at regular intervals.
• Immunization against diseases which are feasible must be given regularly,
especially Hepatitis B.
• Bio safety officer should train lower staff regularly.
Laboratory Designs and Facilities
• Design
• Enough space should be available
• Smooth easily cleanable walls, ceiling and floors which should be impermeable to
liquids and resistant to chemicals and disinfectants.
• Ample illumination should be available for safe conduction of laboratory procedures.
• Regular, continuous and dependable, quality water supply should be available
which is important for laboratory techniques.
• Wash basins with running water if possible should be provided in each laboratory
room preferably near the exit door.
• Suitably equipped first aid box should be available in the district laboratory.
• Control programmeme for rodents and insects in the laboratory should be there.
Laboratory Equipment
 Ensure the adequate equipment be provided and that they are used properly.

Essential biosafety equipment are
• Pipetting aids to avoid mouth pipetting
• Screw capped tubes and bottles.
• Autoclaves to decontaminate infectious material wastes.

110
 • Plastic disposable pasteur pipettes, when ever possible should be used.
• Equipments should be validated before being taken for use and then
revalidation should be done at regular intervals.
7.5 Waste Management
What is Waste?
Any thing which has to be discarded is called waste. The laboratory organisms require
appropriate handling. The most common documented transmission of infection from
waste to health care worker is through contaminated metallic waste.

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Hospital/Laboratory Waste Management
• Material required
• Waste disposal color coded bags with biohazard symbol. blue, red, black and
yellow.
• Trolley baskets for holding the bags.
• Autoclave for decontamination of waste on site.
• Disinfectant solution (Sodium hypochlorite solution.).
• Incinerator if possible (Optional).
• Soap for hand washing and towel for drying hands.
• Gloves.
• Puncture proof containers plastic / metal with a biohazard symbol.
Follow management at every step from the site of generation
• Segregation.
• Collection.
• Transportation.
• Storage.
• Treatment to disinfect.
• Final disposal.
• Segregate waste into the prescribed categories at the point of generation.
• Color coded bags as per international norms. (Table-3.1)

112
Methodology

In the district lab, the lab waste handling is an essential job which needs to be under
supervision of biosafety officer. Broad guidelines to be followed are:

• Segregate the different category of waste at the point of generation.

• Discard infectious wastes (non sharp) if possible in disinfectant solution or

autoclave to render it non-infectious.

• Discard sharp waste i.e. needles, blades etc in a puncture proof containers.

• After the container is 2/3 filled, it should be autoclaved/ shredded and land filled
for disposal.

• If nothing is available for disposal deep bury (as per standard guidelines) in a
secure area.

Categories

All waste should be decontaminated (chemically/autoclaving) before final disposal/

reuse.

• Non contaminated waste which can be reused or recycled, disposed off as general
house hold waste.

• Contaminated sharps disposed off in puncture proof containers fitted with cover,
labeled as infectious.

• Contaminated reusable materials for decontamination by autoclave, thereafter

washing and reuse/ recycle.

• Contaminated disposable material for autoclaving & disposal.

• Contaminated material for direct incineration.

Quality control

• Check that proper quality bags are purchased.

• Autoclave monitoring & maintenance.

• Disinfectant quality check.

Contaminated infectious materials for autoclaving and reuse

• No pre cleaning to be done.

• Transfer material to autoclave.

• Autoclave at 121°C / 15 lbs pressure for 45 minutes.

113
• If cleaning is required, do washing as prescribed.

• Re use.
Contaminated infectious waste for disposal

• Autoclave in leak proof container. i.e. autoclavable colour coded plastic bags.

• Place material in a transfer containers / trolleys with bags.

• Transport to incinerator.

• If reusable transfer containers are used they should be disinfected and cleaned
before they are returned to laboratories.

• Discarding jars preferably unbreakable should be used and they should have
suitable disinfectant (Sodium hypochlorite 1%) freshly prepared each day.

Sodium Hypochlorite Solution Preparation

Dilution of sodium hypochlorite solutions (part of stock solution: parts of water)

Note:- Alaways prepare diluted hypochlorite solution fresh every day. If sodium
hypochlorite is not available an alternative calcium hypochlorite (1%) can be used which
needs to be prepared as follows

CALCIUM HYPOCHLORITE SOLUTION

7.6 Quality Assurance

General principles and procedures:

Quality assurance is the sum total of all activities that are undertaken to ensure
generation of reliable and accurate results / data. It is concerned with the organizational
process and the conditions under which laboratory activities are planned, performed,

114
monitored, recorded and reported. The basis for any clinical laboratory quality control
programme is a written QC policy. This policy should include a description of:
a. The control materials to be used with each test
b. Frequency of use,
c. Required documentation as well as criteria for the acceptability of patient’s results.
The quality control policy must also state that when results are unacceptable, any
patient’s results that were done with those QC specimens cannot be reported. It
should identify a set of remedial actions that should be instituted.
Following are the objectivities of Laboratory Quality Assurance

Maintain efficiency through standard procedures.

Maintain accuracy, consistency and reliability of results and data.

Prevent risks

Detect deviations and Correct errors

Accreditation of the laboratory
It is the responsibility of the head of the laboratory to establish, implement and ensure
compliance. However, Laboratory Quality Assurance (LQA) is the responsibility of all
laboratory personnel. The DSO and the MO should ensure that the lab maintains all
records pertaining to the quality checks.
Quality assurance comprises of internal quality control and external quality assessment.
Internal Quality Control
For qualitative tests, the quality control rules are simple.
1. The positive control must be positive and the negative control must be negative.
For the patient’s results to be valid a quality assurance programme is in place to
ensure the reliability of test results
2. Testing should be processed in an orderly, reproducible fashion in a setting where
environmental variation will not affect the result
3. All extraneous variables must be excluded
4. Records are available to document
To achieve and maintain IQC the following should be checked and followed:
1. Test Request and Specimen Collection
Tests should be ordered on a request form that includes the patient’s name and a
unique identifier, the time of collection, and the nature of the specimen. It is good
practice to keep a log of all specimens collected, including the time of collection
and, in the case of bacteriologic specimens, the source of the specimen. All
specimen containers and tubes must also be marked with the patient’s name and
unique identifier, preferably at the time and place of collection.
2. Test Processing
• Temperature monitoring: The temperature in the testing environment, as
well as in refrigerators, freezers, incubators, water baths involved in testing
or storing reagents or media, should be monitored daily and the results

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 recorded. If storage conditions for reagents or media are disturbed, as in a
power outage with a rise in storage temperature, the reagents must be
considered unreliable and discarded, even though the expiration date has not
yet been reached.
• Reagents used: All working solutions, prepared from more concentrated stock
solutions, should be labeled when they are prepared and the expiration date
written clearly on the label if appropriate.
• Maintenance of equipments: For reliable results, all microscopes, centrifuges
and other laboratory equipment must be properly maintained. In taking
responsibility for the validity of the test information produced in the office
laboratory an equipment log should be kept that keeps track of maintenance,
problems found, and any corrective action taken. Refrigerators, freezers,
incubators, and heating baths are a class of equipment that can directly affect
test reliability and should thus be included in the general maintenance
programme.
Small pieces of equipment, such as pipettes, should be stored or marked in such a
way that there is little chance of inadvertently using the wrong size (e.g., a 1-mL
pipette instead of a 0.5-mL pipette), and damaged pieces should be discarded.
The pipetting and diluting step is one of the most critical in many procedures and
requires extreme care on the operator’s part.
3. Reporting and Using Test Results
The other important phase of laboratory testing is accurately reporting the results
in a timely manner with appropriate interpretation and application of the test
results.
All laboratory results should include information similar to that required for ordering
the test: patient identification, time and date of specimen collection, body site of
collection, and name of test. In addition, the test result report needs to contain:
test results including the units of measurement, reference intervals (normal values),
time and date of analysis, Reports should be written on a form designed for that
purpose.
Good laboratory practice requires that laboratories routinely check for clerical errors
prior to the release of laboratory results. Usually this calls for a second person to
read the test result on the report to see if it is reasonable
Timeliness in reporting is just as much a quality issue as are the accuracy and
precision of testing.
External Quality assurance at 2 levels

Within the state IDSP system

Through an external agency not associated with the system
Within the state IDSP system

Quality control of the peripheral laboratories will be the responsibility of the district
public health laboratory. Confirming 5-10% of laboratory results of PHC laboratory
should be done at the district laboratory.

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Quality control of district labs (L2) is the responsibility of L3 labs. Confirming 5-10%
of laboratory results of L2 laboratory should be done at the state laboratory
Laboratory manuals for work on specific diseases will be made available to peripheral
laboratories, both private and government, under IDSP programme. Training, on job
evaluation and retraining will be provided to the laboratory personnel as part of quality
assurance.
External quality assurance by an agency not associated with the system
The Institution conducting EQAS will be identified by the government and intimated to
the lab. The lab will follow instructions of the National EQAS center.
Activities under EQAS Program
The laboratory diagnoses on the listed communicable diseases and non-communicable
diseases (NCDs to be lab tested only at the state lab level) will be evaluated in this
programme.
Frequency of Tests and Number of tests will be decided by the agency
Evaluation of the labs and action to be taken will be decided The final report will identify
laboratories as consistent poor performers, poor performers correcting errors,
inconsistent performers, good performers with subsequent poor performance and
consistent good performers. A critical evaluation enumerating the causes and reasons
for such categorization will be part of the final report. This helps the state take
appropriate corrective action.
Specimen collection and transport
Peripheral blood smears, sputum for smear and culture, blood for serology, blood, stool,
pharyngeal / nasopharyngeal swabs and urine for culture and water for water quality
test are the common specimens to be collected under IDSP. In outbreak conditions, the
Rapid Response Team will order for appropriate samples. (At L1 level the lab assistant
is trained in collection of blood by fingerpicks for smear preparation, collection of blood
by venepuncture would require the assistance of the medical officer).
General instructions
1. Use universal precautions for handling all specimens.
2. Whenever possible, collect all culture specimens prior to administration of any
antimicrobial agents.
3. All specimens must be appropriately labeled with the requisition number from
the corresponding requisition. The request form will include the patient name,
hospital /op number, date and time of collection, specimen type and tests
requested. A requisition needs to accompany each different specimen type.
4. Specimens should be in tightly sealed, leak proof containers. Specimens should
not be externally contaminated. Specimens grossly contaminated or compromised
may be rejected.
5. If unable to transport specimens promptly, refrigerate serum, urine, respiratory
samples and stool specimens. Leave blood culture bottles at room temperature.

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7.7 Sample Collection*
Summary of Sample Collection and Transportation
SPECIMEN COLLECTION TRANSPORTATION
Blood for smears Capillary blood from finger prick. Transport the slides within 24 hours.
Make smear, fix the same in They must not be refrigerated.
methanol or other fixative.
Blood for culture Venous blood Collect into blood culture bottles
0.5 – 2 ml for infants (With Glucose broth or Bile salt broth)
2 – 5 ml for children Transport in erect position, and with
5 – 10 ml for adults enough cushion to prevent lysis of cells.
Wrap tubes with absorbent cotton to soak
any spillage. If specimens can reach a lab
within 24 hours, then it can be sent in
ambient temperature. Else cold chain at
4*C.
Serum Venous blood is collected and Sera should be transported at 4 – 8*C and
placed in a sterile test tube. Can last at this temperature for up to 10
Let the specimen clot for 30 days.
minutes at ambient temperature.
Then place in a cool box for clot
retraction at 4 – 8*C, for a
minimum of 1 – 2 hours. This is
then centrifuged @ 1000 G for
10 mts1 . Separate the serum
from the clot.
CSF LP under aseptic conditions. Transportation does not need any special
Collect the CSF in sterile tubes. media. If one is suspecting bacteria, then
transport at ambient temperature as
relevant pathogens do not survive well at
low temperatures
If one is suspecting viruses, then they may
be transported at 4 – 8*C. for up to 48
hours.
Faeces Collect freshly passed stool (8gm) Transport in Cary Blair Medium at 4-8*C.
In children, rectal swabs may be Process within 1 – 2 days.
used.
Post mortem samples Biopsy of relevant tissues Fixed specimens can be transported at
Place in formalin for histopathology ambient temperatures
Place in Transport media for Specimens in transport media may be
microbiological testing. transported within 24 hours at ambient
Place in sterile saline for isolation temperature.
of viral pathogens Specimens in sterile saline should be
transported at 4-8*C within 48 hours.
*For details, refer to NICD/Manual of laboratory techniques.

(a) Collection of blood for serological investigations

Performing vene puncture:
 Gloves should be worn. Use sterilized disposable syringes and needles
 For avoiding soiling, a piece of linen with a layer of dressing pad may be placed
below the forearm before commencing venepuncture.
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 After collecting 5 ml of blood aseptically, it should be carefully transferred from
the syringe without squirting in to a sterile screw capped plastic leak proof speci-
men container. The containers should be labeled before commencement of
venepuncture. If the vial has anticoagulants, then a second person wearing gloves
should help in shaking the vial for mixing the blood well with the anticoagulants.
 After blood is collected, the tourniquet is removed and the needle is withdrawn.
The patient is given a dry sterile cotton swab to press over the site of venepunc-
ture. Elbow may be flexed to keep the cotton swab in place till the blood stops.
Any blood spill is carefully wiped with 70% ethanol.
 All the swabs and cotton pieces are placed in plastic bags for disposal. If the
outside of the vial is visibly contaminated with blood, it should be cleaned with
10% freshly prepared sodium hypochlorite solution.
(b) Cerebrospinal Fluid (CSF) Specimen Collection
The specimen must be taken by a physician experienced in the procedure. CSF is
used in the diagnosis of viral, bacterial, parasitic, and fungal meningitis.
Materials required
• Lumbar puncture tray which includes:
• Sterile materials: gloves, cotton, towels or drapes.
• Local anaesthetic, sterilized needle, syringe.
• Skin disinfectant: 10% povidone iodine or 70% alcohol.
• Two lumbar puncture needles, small bore with stylet (sterilized).
• Six externally threaded sterile screw-cap tubes and tube rack
Method of collection
Only experienced clinician should be involved in the collection of CSF samples. CSF is
collected directly into the separate screw-cap sterile tubes. Separate tubes should be
collected for bacterial and viral processing.
• Make the patient lie on the bed in left lateral position. Ask the patient to flex the
neck (so that the chin touches the chest) hip and the knee joint.
• Using the iliac crest as the reference point, palpate the joint space between the
4th and the 5th lumbar vertebrae and identify the surface anatomy.
• Disinfect the site meticulously with 10% povidone iodine or 70% isopropyl alcohol
by swabbing the skin concentrically from the centre of the site outwards. Let the
disinfectant evaporate. Do not repalpate the site again.
• Infiltrate the local area with the local anaestheic and wait for 4-5 minutes for the
effect to appear before performing lumbar puncture

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• Insert the sterile lumbar puncture needle between the 4th and 5th lumbar verte-
brae to a depth of 4-5 cm, withdraw the stylet. Fluid flows freely through the needle.
• Between 1 and 2 ml of CSF is collected in each of the 3 tubes, one for culture, one
for biochemical analysis and one for cytology.
Note: - Haemorrhagic CSF sample is not recommended for serological testing.
Handling and transportation
• In general, send the specimens to the laboratory and process as soon as possible.
• Transport CSF specimens for bacteriology at ambient temperature, generally with-
out transport media. Never refrigerate the CSF, as many of the bacterial patho-
gens do not survive well at low temperatures.
• CSF specimens for virology do not need transport medium. They should be trans-
ported at 4-8°C.
(c) Stool Sample Collection
Stool specimens are most useful for microbiological diagnosis if collected soon after
onset of diarrhoea (for most viruses < 48 hours and for bacteria < 4 days after the
onset of illness), and preferably before the initiation of antibiotic therapy. If required,
two or three specimens may be collected on consecutive / alternate days. Stool is the
preferred specimen for culture of bacterial, viral, and parasitic diarrhoeal pathogens.
Rectal swab samples may also be used in case of infants, debilitated patients or while
carrying out direct endoscopic visualization of a lesion or any other situation where
voided stool sample collection is not feasible. In general, rectal swabs are not recom-
mended for the isolation of viruses. As far as possible, do not collect stool samples
from a bedpan.
Materials for collection
• Clean, dry, leak-proof screw cap container (which has not been priorly washed
with a disinfectant) and tape
• Appropriate bacterial transport media for transport of rectal swabs from infants
Method of collection of stool sample
• Refer to general instructions for collection of stool sample as described above
Method of collection of rectal swab
• Moisten the rectal swab in sterile normal saline
• Introduce the swab inside the anal sphincter and go upto 2-4 cm inside rectum.
• Rotate the swab upto 90 degrees and withdraw the swab.
• Put the swab in any of the transport media like VR fluid / Cary Blair medium by
inserting the swab completely into the media.

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• Break off the excess wooden portion of swab stick and screw cap the bottle of
transport media.
• Store at room temperature till transported to the nearest lab (if cholera is sus-
pected) or else keep it in fridge (4 deg centigrade) if salmonella / shigella is sus-
pected.
• Label the bottle of transport media.
Handling and transport
Two transport media, V.R. Fluid (for cholera) and Cary Blair’s are commonly used for
isolation of common bacterial enteropathogens like Salmonella, Shigella, and Esch coli
including Vibrios. If the specimens are collected in transport media like Cary Blair’s,
then it should reach nearby laboratory in 2-3 days time and samples can be kept at
room temperature. If rotavirus or any other viral etiology is suspected then stool speci-
men can be kept in fridge (4°C-8°C) - till it is transported to the nearby laboratory.
(d) Respiratory Tract Specimen Collection
Specimens are collected from the upper or lower respiratory tract, depending on the
site of infection. Upper respiratory tract pathogens (viral and bacterial) are found in
throat and nasopharyngeal specimens. Lower respiratory tract pathogens are found
in sputum specimens.
Materials required
• Transport media - bacterial and viral.
• Throat swabs (Dacron and cotton swabs).
• Tongue depressor.
• Nasal speculum.
• 20-50 ml syringe
• Sterile screw-cap test tubes and wide-mouthed clean sterile containers (minimum
volume 25ml).
Method of collecting a Throat Swab
• Hold the tongue down with the tongue depressor. Use a strong light source to
locate areas of inflammation and exudate in the posterior pharynx and the tonsil-
lar region of the throat behind the uvula.
• Rub the area back and forth with a cotton or Dacron swab.
• Care must be taken to sample the posterior pharyngeal wall at the end to avoid
gagging by the patient.
• Withdraw the swab without touching cheeks, teeth or gums and insert into a ster-
ile screw-cap test tube containing appropriate transport medium required.

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• Break off the top part of the stick without touching the tube and tighten the screw
cap firmly.
• Label the specimen containers.
• Complete the laboratory request form.
Method of collecting per-nasal and post-nasal swabs
• Seat the patient comfortably, tilt the head back and insert the nasal speculum.
• Insert a flexible calcium alginate/Dacron swab through the speculum parallel to
the floor of nose without pointing upwards. Alternately, bend the wire and insert
it into the throat and move the swab upwards into the nasopharyngeal space.
• Rotate the swab on the nasopharyngeal membrane a few times, remove it care-
fully and insert it into a screw-cap tube containing transport medium.
• Break off the top part of the stick without touching the tube and tighten the screw
cap firmly.
• Label the specimen tube.
Method of collecting nasopharyngeal wash/aspirate
• Have the patient sit with the head tilted slightly backward.
• Flush a plastic catheter or tubing with 2-3 ml of VTM/sterile normal saline.
• Instill 1-1.5 ml of VTM (viral transport medium)/sterile normal saline into one nos-
tril.
• Insert the tubing into the nostril parallel to the palate and aspirate nasopharyn-
geal secretions.
• Repeat this procedure with the other nostril.
• Collect 1-2 ml in a sterile vial and transport in cold chain at 2-8°C
(e) Collection of Sputum Sample
Materials required
Select a good wide- mouthed sputum container, which is disposable, made of clear
thin plastic, unbreakable and leak proof material.
Method of collection
• Instruct the patient to inhale deeply 2-3 times, cough up deeply from the chest and
spit in the sputum container by bringing it closer to the mouth.
• Make sure the sputum sample is of good quality. A good sputum sample is thick,
purulent and sufficient in amount (at least 2-3 ml).

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Handling and transport
If the specimen is collected in the field and can not be immediately processed, it should
be transported to the laboratory within 3-4 days of collection. The specimen should be
collected in the containers meant for the purpose and lid tightly secured, properly la-
belled and to be kept away from the sun and heat. These can be placed in a special
box, which can withstand leakage of contents, shocks and other conditions incident to
ordinary handling practices. These boxes should be kept in the cool conditions and
then transported to the laboratory.
(f) Collection of Sputum Sample For Culture
Materials required:
• An ideal container is a wide mouthed, screw cap sterile, Universal bottle, or polypro-
pylene bottle.
Method of collection (as described above):
• It is best to obtain a sputum specimen early in the morning before the patient has
taken food, since food particles in smears make them difficult to examine.
• Patients should produce specimens either outside in the open air or away from
other people and not in confined spaces such as toilets. A good sputum specimen
consists of recently discharged material from the lower respiratory tract, with mini-
mum amounts of oral or nasal material. Ideally, a sputum specimen should have a
volume of 3-5ml, although smaller quantities are acceptable if the quality is satis-
factory. An early morning specimen, after thorough cleaning of mouth, is preferred.
Handling and Transport:
• Specimens should be transported to the laboratory as soon as possible after col-
lection. If delay is unavoidable, the specimens should be refrigerated
(g) Post Mortem Specimen Collection
Need to be collected during outbreak situation when causative agent in not known.
Strict precautions, including respiratory protection from aerosolized particles, must
be taken when carrying out post-mortem specimen collection in communicable dis-
ease outbreaks. Collect the specimens as soon as possible, preferably within 24 hour
since viral titres decline while bacteria multiply rapidly after death. Experienced medi-
cal personnel may only accomplish thorough post-mortem examinations.
Materials required
• Barrier precautions: double gloves, sterile gown, eye goggles, mask
• For collecting blood and other fluids, refer to corresponding sections for materials
• Aseptic surgical and biopsy instruments for collecting tissue specimens
• Fixatives: saline formalin for histology
• Sterile saline, appropriate viral and bacterial transport media
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• Sterile containers, sterile screw cap tubes or vials, glass slides and slide box
• Disinfectant such as household bleach diluted 1:10 in water.
Method of collection
• Use a separate sterile instrument for each tissue specimen from affected sites
(several fragments with 1-2 grams of each is sufficient). Smaller, but adequate,
specimens may be taken with a biopsy needle.
• Place different tissues in separate sterile containers containing the relevant
medium (sterile saline for preparation of tissues for immunofluorescence micros-
copy; and microbiological transport media for the isolation of bacterial and viral
pathogens, fixatives for histopathology).
• Label all containers and tighten the screw caps firmly.
• Blood may be taken from the heart cavities.
• If cerebral malaria is suspected, make several smears from the cerebral cortex
on glass slides to detect Plasmodium falciparum. Label the slides and transport
in a slide box.
Handling and transportation
• Fixed specimens can be stored and transported at ambient temperature.
• Tissue specimens for isolation of bacterial pathogens can be transported at
ambient temperature in transport media for up to 24 hours.
• Transport tissue specimens for isolation of viral pathogens in viral transport
medium or sterile saline at 4-8°C for 24-48 hours. For longer periods, freeze and
store at -70°C.
• If rabies is suspected and brain samples are collected, freeze unfixed specimens
immediately after collection. Formalin-fixed samples are also useful and may be
transported at ambient temperature.
(h) Collection of Specimens for Culture
Collection time: In the acute phase of the disease
Specimen to be collected: depends on the Syndrome / presumptive diagnosis: Ty-
phoid/ Cholera: Stool, Blood,
Respiratory specimens (Sputum, Throat swabs, & nasopharyngeal swabs)
Method of collection: refer disease wise.
Disease wise - Sample collection and transportation

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Malaria
Laboratory Criteria for Diagnosis
Diagnosis of malaria is done by detecting and identifying malaria parasites microscopi-
cally in blood films.
Sample collection for microscopy
Sample to be collected: peripheral finger-prick blood smear i.e. thick and Thin Blood
smears
Time of collection
During fever or 2-3 hours after the peak of temperature.
Before the patient receives antimalarial drugs.
Tuberculosis
Laboratory criteria for diagnosis
Demonstration of acid-fast bacilli in at least two of three sputum smears or culture
positive for Mycobacterium tuberculosis.
Specimen collection (refer to method for collection as described above)
• A screw capped, wide mouthed, clean, disposable plastic container is used to
collect sputum specimens from suspected cases.
• Three specimens should be collected for diagnosis as follows.
One spot specimen when the patient first attends the health service.
One early morning specimen (preferably the next day)
One spot specimen when the early morning specimen is being submitted for ex-
amination.
• A good sputum sample should be thick, purulent and of sufficient quantity (at least
5ml).
For Quality assurance, all positives and 1% of negatives are to be sent to L2 lab for
cross checking.
Typhoid
Laboratory criteria for diagnosis:
Serology – Widal/Typhi-dot test positive
Isolation of S.typhi from blood, stool or other clinical specimen.

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Sample collection:
Blood and serum
• Collect aseptically 5ml of blood for serology (It may be necessary to collect 2
samples at 1 week interval if the first sample is negative and if requested by the
L2 Lab). Refer to method for collection of blood as described earlier.
• Collect aseptically 5ml of blood in BHI broth for culture (refer to method for
collection of blood as described earlier) if it is not possible to collect 2 blood
samples, culture can be done from the clot after separation of serum for serology.
Faeces for culture
On some occasions it may be necessary to collect stool samples. Request the speci-
men from the patient. The patient must be provided with a wide necked container to
pass the faeces. The container must not contain any disinfectant. The patient must be
instructed not to contaminate the faeces with the urine.
Transfer a portion of specimen using cotton wool swab, into Cary Blair medium and
transport it to the L2 lab within 48 hours. Cold chain is not required.
Cholera
Laboratory criteria for diagnosis:
Isolation of Vibrio cholera O1 or O139 from stools in any patient with diarrhoea
Collection of sample: Faeces for V. Cholera isolation
 Follow general instructions for stool sample collection (as described above)
 Transfer a portion of specimen to a cotton wool swab; insert it in Cary Blair’s trans-
port medium. (The medium can be obtained from L2 lab every three months)
 If stool specimen could not be collected take a rectal swab and insert it in the
above solution.
 Complete the lab request form.
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
Collection of samples
Blood: During first week of illness collect
• 2 ml of venous blood with anticoagulant (Heparin or EDTA) using sterile precau-
tions, for dark-field microscopy (method of collection of blood as described earlier)

126
• 3 ml of blood for serology using a sterile, dry syringe to avoid haemolysis (method
of collection of blood as described earlier).
Urine: Urine should be collected after second week of illness and transported immedi-
ately in sterile container (acidic urine is inhibitory to leptospires).
(Second serum sample to be collected after 5 days if required, for demonstrating rising
titre or sero conversion).
Handling and Transport
• Store serum in refrigerator. If there is delay in transportation, store the serum in
deep freezer at –20 o C.
• Transport serum as quickly as possible, within 24 hours to L2 lab.
Measles
Laboratory criteria for diagnosis:

Presence of measles virus specific IgM antibodies

At least fourfold increase in antibody titre in paired samples

Isolation of measles virus
7.8 Specimen Collection, Processing and Transportation
Blood sample for serology:
An acute phase serum specimen (3-5ml of whole blood) should be taken soon after
onset of clinical symptoms but not later than 7 days.
Samples for virus isolation:
(i) Urine Specimens:
Urine should be collected within 5 days of rash onset (1-3days best) First morning
mid stream voided specimens are preferable. Urine should be collected in sterile
wide mouthed containers.
NOTE: Do not freeze the urine specimen. The entire specimen should be stored
at 4°C or (Icepack) till transported to the Virus Isolation lab - L3 lab.
(ii) Respiratory Specimens (nasopharyngeal swab, pharyngeal and throat swab):
The specimens should be collected within 5days after the onset of rash. Follow
general instruction for swab collection (as described earlier). The tip of the swab
is put into a vial containing 1.5-2ml of transport medium and the applicator stick is
broken off.
The sterile cotton swabs can be prepared in the L1 / L2 labs.
The VTM will be supplied by the L3 lab on request

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Storage of Samples:

The samples should be stored in the refrigerator. If there is a delay in transport, the
serum and nasopharyngeal swab in VTM should be stored in the -20 deep freezer. The
urine sample should not be frozen.

Transportation:

All samples should be transported maintaining cold chain.

The carrier should be marked as ‘Potentially Hazardous’.

If any delay in transportation, samples should be stored at 4 o C

Transport the specimen always ‘in person’ to L3 lab

Dengue

Laboratory criteria for diagnosis:

Any one or more of the following:

1. Isolation of dengue virus from serum, plasma, leucocytes, or autopsy samples

2. Demonstration of Dengue virus specific IgM antibodies or fourfold or more rise in

reciprocal IgG antibody titre

3. Demonstration of dengue antigen in autopsy tissue by Immunochemistry or

immunoflourescence or in serum samples by EIA

4. Detection of viral genomic sequences in autopsy tissue, serum or CSF by PCR

Sample Collection
Sl. Sample Period of Method of Qty Storage Trans Trans
No. Collection Collection for 24 to Temp
48 hrs
1 Serum 5 days after Collect Intravenous 3 – 5 ml +4°C L2 Cold
onset blood chain
2 Plasma Within 5 Collect Intravenous blood 3 – 5 ml L3
(Citrated days of in a citrated tube
blood) onset
3 CSF Within 5 Collect CSF by lumbar 2 ml
days of puncture in a sterile vial
onset
4 Autopsy In the event Collect in a sterile Small
(brain, lung, of death container quantity
liver)

* 5 to 10 representative samples should be sent to L3 Lab for virus isolation and serotyping in case of
outbreaks.

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Japanese Encephalitis: (Fever with Altered Consciousness)
Laboratory criteria for diagnosis
Laboratory diagnosis of JE is done by the following methods:
♦ Demonstration of JE virus specific IgM antibodies
♦ Detection/isolation of antigen/virus
♦ Demonstration of viral antigen in the autopsied brain tissue by the fluorescent
antibody test
Presumptive
Detection of an acute phase anti-viral antibody response through one of the following:
Elevated and stable serum antibody titres to JE virus through haemagglutination- inhi-
bition or IgM antibody to the virus in serum/CSF by ELISA
Confirmed
Detection of JE virus, antigen, or genome in tissue, blood or other body fluids by immu-
nohistochemistry or immunoflourescence or PCR.
Sample Collection
Sl. Sample Period of Method of Qty Storage Trans Trans
No. Collection Collection for 24 to Temp
48 hrs
1 Serum/ Within 6 days Collect Intravenous blood 3 – 5 ml +4°C L3 Cold
plasma of onset as per the guidelines chain
2 CSF Within 6 days Collect CSF by lumbar 2 ml
of onset puncture in a sterile vial
3 Autopsy In the event Collect in a sterile Small qty
brain of death container
Tissue

Plague
Plague is a zoonotic disease caused by a bacterium Yersinia pestis, transmitted be-
tween rodents and human beings through infected rodent fleabites.
Suspected Plague:
1. Clinical symptoms that are compatible with plague, e.g. fever sepsis syndrome,
lymphadenopathy and/or acute pneumonitis in a person who resides in or recently
traveled to a plague –endemic area
2. If gram negative and/or bipolar –staining coccobacilli are seen on a smear taken
from affected tissues, e.g.,
 Bubo (bubonic plague)

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 Blood (septicemia plague)
 Tracheal/lung aspirate (pneumonic plague)
Presumptive:
 Y.pestis F1 antigen detected in clinical materials by direct fluorescent antibody
testing, or by other standardized antigen detection method or
 Isolate from a clinical specimen or
 A single serum specimen is found positive for diagnostic levels of antibodies to
Y.pestis F1 antigen, not explainable on the basis of prior infection or immunization
Confirmed:
 Isolate identified as Y.pestis by phage lysis of cultures; or
 A significant (equal or more than 4-fold) change in antibody titre to the F1 antigen
in paired serum specimens. or
 PCR positive
Clinical samples to be collected
 Blood, Serum, bubo aspirate and sputum.
 Organ for culture & smears - Lungs, Liver and spleen of Rodents
 The clinical samples are to be collected during the acute phase of illness. 8 to 10 ml
of blood to be collected for blood culture and serology.
 Storage: All clinical specimens should be stored at 4ºC.
Transport of Clinical Samples
The methanol fixed smears are to be sent to the L2 in a slide mailer. The clinical samples
and rodent tissue samples for culture, serology and PCR are to be transported to L3
laboratory maintaining cold chain.
Collection Procedure - The kit for collection of sample to be brought by Surveillance
team (Kit containing the following: 10-20 ml syringe, 18 –20 gauze needle, petridish,
sterile swab stick, clean dry vials, sterile normal saline, Cary Blair Transport medium)
Collection of Bubo aspirate
 Sterile the surface of the bubo with tincture iodine
 Draw a few ml of sterile physiological saline in a 10 –20 ml syringe fitted with 18 or
19 gauge needle
 Puncture the bubo and apply suction. If bubo fluid could not be aspirated, inject
saline into the bubo and aspirate again.
 Make smears with the bubo aspirate

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 Transfer the bubo aspirate in the Cary Blair Transport Medium for transportation
to the laboratory.
Sputum, Throat swab, and blood follow universal procedure and precautions as
described earlier
 Ask the patient to expectorate into a sterile, wide mouthed container such as
petridish
Precautions in handling specimens
As these specimens are known or thought likely to contain infectious substances, the
following precautions should be applied
 Strict aseptic technique (gowns, gloves, masks)
 Wash hands before and after the collection of material
 Place the specimen aseptically in an appropriate sterile container
 Tightly close the container
 Label and date the container
Packaging/Transportation of Specimen:
In accordance with currently accepted bio-safety norms, Y.pestis is listed under Bio-
safety 2 level.
The following practices, should be observed
 A watertight primary receptacle
 A watertight secondary recetable
 An absorbent material, which must be placed between the primary receptacle
and the secondary packaging. The absorbing material must be adequate to ab-
sorb the entire contents of all primary receptacles.
 Itemized list of contents must be enclosed
 Outside package must be marked with identification of the infectious substance,
volume of contents, name, and telephone number of sender
Hepatitis
Hepatitis surveillance is sentinel based. However, while investigating a case of syn-
drome of jaundice, hepatitis needs to be investigated on a routine basis also.
Laboratory criteria for diagnosis:
Hepatitis A: IgM anti HAV positive
Hepatitis B: Positive for HbsAg or IgM anti-HBc
Hepatitis C: Positive for anti-HCV
Hepatitis D: Positive for HbsAg or IgM anti-HBc Plus anti-HDV
Hepatitis E: Positive for anti-HEV

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L1 lab: Collect serum samples (as described earlier) (Using bio-safety guidelines) and
transport to L2 labs in cold chain.
L2 laboratory
 Perform Rapid tests for
o HbS Ag, - Latex agglutination and Dip stick ELISA
o Hepatitis C - Immunochromatic test
 Transport to L3 under cold chain for Hepatiits A, D and E serology
Testing of Water Samples
In the laboratory there are two simple procedures of testing water samples for faecal
contamination:
1. Most Probable Number (MPN) method for coliform bacteria – using the multiple
tube fermentation technique in this method the MPN of total coliform bacteria,
faecal coliform bacteria (or the thermo tolerant coliforms) present in the water
sample is determined, along with the presence/absence of Escherichia coli.
2. H2S-Strip method: This is a simple, reliable and easy-to-perform (by even un-
trained personnel), Presence/Absence test for bacteriological quality Which works
on the principle that there is a close correlation between faecal contamination and
the presence of hydrogen sulphide (H2S) producing bacteria and, that faecal pollu-
tion of water can be deduced by demonstration of H2S production. It has been
claimed, by various workers, that the H2S-strip method shows >90% agreement
with the conventional MPN test described above.
General instructions for collection, storage and transport of water samples for bac-
teriological examination:
1 Care must be taken to ensure that samples are representative of the water being
examined.
2 Care must be taken to see that no accidental contamination occurs during sam-
pling: The sample collector must wash his/her hands well using carbolic soap, just
prior to collection of samples.
3 Test the water samples as soon as possible after collection. If delay of more than
3 hours is expected, transport samples to laboratory under cold-chain conditions.
4 A volume of water sample adequate to carry out all the tests should be collected:
generally, 200-250 ml of water sample should be collected.
5 Samples for bacteriological examination shall be collected in clean, sterilized, nar-
row mouthed neutral borosilicate glass bottles or Autoclavable plastic (poly pro-
pylene) bottles with screw cap lids.
6 Collection of samples from taps: Available in the manual available with the labora-
tory.

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Materials for collection

• Glass bottles with securely fitting stoppers or caps having an overhanging rim.
Both the bottle and the cap/stopper should be adequately sterilized. The bottle
should be able to hold at least 200ml of water. Alternatively, autoclavable plastic
bottles with a tight screw capped lid may be used.

• Sodium thiosulphate (0.5ml of 10% solution or a small crystal), in case collecting

sample from chlorinated source of water.

Method of collection

Sampling from a tap or a pump or a pump outlet

• Remove from the tap any attachments that may cause splashing and using a clean
cloth, wipe the outlet removing any dirt.

• Turn on the tap at maximum flow rate and let the water flow for 1-2 minutes. Close
the tap

• Sterilize the tap for a minute with flame: a gas burner or a cigarette lighter may be
used.

• Carefully turn on the tap and allow the water to flow for 1-2 minutes at a medium
flow rate.

• Open a sterilized bottle with its protective cover on. While holding the cap and the
protective cover face downwards (so as to prevent entry of dust that might carry
microorganisms), immediately hold the bottle under the water jet, and fill.

A small airspace should be left to facilitate shaking at the time of inoculation prior
to analysis.

• Place the stopper in the bottle or screw on the cap and fix the brown paper pro-
tective cap in place with the string.

Sampling from a watercourse or reservoir

• Holding the bottle by the lower part, submerge it to a depth of about 20cm; with
the mouth facing slightly upwards; if there is a current, the bottle mouth should
face towards the current.

Sampling from dug wells or similar sources

• With a piece of string attach a stone of suitable size to the sampling bottle.

• Take a 20m length of clean string rolled around a stick and tie onto the bottle string.
Open the bottle as described earlier.

• Lower the bottle, weighted down by the stone, into the well, unwinding the string
slowly. Do not allow the bottle to touch the sides of the well.

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• Immerse the bottle completely in the water and lower it down to the bottom of
the well.
• Once the bottle is judged to be filled, rewind the string round the stick to bring up
the bottle. If the bottle is completely full, discard some water to provide airspace.
• Stopper or cap the bottle as described earlier.
Handling and transport
• Test the water sample within 3 hours of collection during which time it can be kept
at ambient temperature. If more delay is expected, pack the water sample in ice
for transport to the laboratory.
• The refrigerated sample should be tested within 24 hours.
SUMMARY OF SAMPLE COLLECTION / TRANSPORTATION/
LAB INVOLVED IN TESTING
Disease Specimen Quantity Condition Storage Trans Test
collected/by for temp temp done
whom collection at

Malaria Peripheral blood Thick and thin Clean Room Room L1

smear for smear in single slides temp temp
microscopy by slide
PHN / lab asst

TB Sputum for 5 – 10ml Clean RT RT L1

microscopy containers

Sputum for 5 – 10ml Sterile RT RT L3

culture by
VHN / lab

Typhoid Blood for 5 ml Sterile +4oC RT L1

serology (Typhidot)

Blood for Serology, 5 ml in Cary Blair Sterile +4oC RT L2 (Widal)

culture & Sensitivity medium
by MO / Tech

Cholera Stools for culture Clean

by VHN containers +4oC RT L2

Leptospirosis Blood for serology 5 ml Sterile +4oC RT L2

by MO / tech

Polio Stool for culture 2samples Clean +4oC Cold L3

by VHN 24 hrs apart chain

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 Disease Specimen Quantity Condition Storage Trans Test done
collected / by for temp temp at
whom collection
Measles Blood for serology by 5 m from Sterile +4oC Cold L3
MO / Tech 5 to 10 cases > 24hrs chain
at –20oC
Urine for culture by 15 ml Sterile +4oC Cold L3
VHN chain
Naso pharyngeal In VTM Sterile +4oC Cold L3
swab by Tech > 24hrs chain
at –20oC
Dengue Blood for serology 5 ml Sterile +4oC Cold L2- rapid
by MO > 24hrs chain test L3 -
at –20oC ELISA
(IgM)
Blood for culture by 5 ml Sterile +4oC Cold L3
MO > 24hrs chain
at –20oC
JE Blood for serology by 5 ml Sterile +4oC Cold L3 - ELISA
MO > 24hrs chain
at –20oC
Hepatitis Blood for serology by 5 ml Sterile +4oC Cold L3 - ELISA
MO > 24hrs chain
at –20oC
Water Water samples by 50 – 100 ml from Sterile RT RT L1 – H2S
quality tech different sources L2 MPN
Plague Blood for smear by Drop Clean slide RT RT L2
MO

7.9 Recording and Reporting

Inputs from laboratory in the form of accurate and timely data are the most important
activities in disease surveillance, without which, all the efforts in building the system
would go waste. The laboratory data management includes:
• Recording details of specimens received
• Record of tracking of samples
• Recording results of tests performed
• Analysis and interpretation of tests
• Timely and accurate communication of results
7.10 Recording of Data:
Information to be recorded on each specimen/ accompanied with each specimen:
• Name, age, sex,
• Address in detail,
• From whom referred
• Syndromic diagnosis
• Date of onset of illness

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• Nature of sample, date of collection, date of receipt, condition of sample,
• Investigation requested
• Whether convalescent specimen or not
From the lab request form, relevant information is written in the lab register after
assigning a laboratory number.
SAMPLE LAB REGISTER
Date:
ID no Name and Age Sex Provisional Lab Lab Date Result Date
address of Diagnosis tests results sent to from of
patient ordered L2 L2 result

On every Monday, the laboratory is expected to send a weekly report on the tests
performed along with results in the prescribed form to the District Surveillance Officer.
Even if no test was performed, a nil report is a must to ensure regularity in reporting.
Diseases of public health importance like Cholera, Dengue Fever, Diphtheria, Japanese
Encephalitis, Leptospirosis, Plague, Whooping Cough, etc must be reported to the
District Health Authorities immediately.
Specimen referral form
Referring lab:
Lab No:
Name and address of patient
Age
Sex
Provisional Diagnosis
Lab tests ordered
Date of Collection
Date of dispatch

Remarks
Lab I/c

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