Working with the laboratory

1
Working with the laboratory

• IDSP training module for state and district
  surveillance officers
• Module 6

2
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

3
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

4
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

5
Role of laboratories in disease surveillance

• Early diagnosis of diseases under surveillance
• Epidemiological investigation
• Rapid laboratory confirmation of diagnosis
• Implementation of effective control measures

6
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

7
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 positive laboratory identification Medical officer and Laboratory staff
More specificity
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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
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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
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Method of laboratory surveillance

• Routine passive surveillance
• Selected diseases
• Outbreak situations

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Conditions under regular surveillance
Type of disease Disease
Vector borne diseases Malaria
Water borne diseases Diarrhea (Cholera)
Water borne diseases 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
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Other conditions under surveillance
Type of surveillance Categories Conditions
Sentinel surveillance STDs HIV/HBV/HCV
Sentinel surveillance Other conditions Water quality
Sentinel surveillance Other conditions Outdoor air quality
Regular surveys Non communicable disease risk factors Anthropometry
Non communicable disease risk factors Physical activity Blood pressure
Non communicable disease risk factors Physical activity Blood pressure
Non communicable disease risk factors Tobacco, blood pressure
Nutrition
Blindness
Additional state priorities Additional state priorities Up to five diseases
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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

14
Laboratory tasks at each level for the diagnosis
of malaria
L1 L2 L3
Sample collection Smear preparation Microscopy and reporting Same as L1 Quality control for L1 Quality control for L2
15
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

16
Laboratory tasks at each level for the diagnosis
of cholera
L1 L2 L3
Stool sample collection Transport to L2 Stool sample microscopy Culture Biochemical and serotyping Transport to L3 for sensitivity Drug sensitivity and phage typing Quality control for L2 Training
17
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

18
Laboratory tasks at each level for the diagnosis
of typhoid
L1 L2 L3
Typhi-dot test Blood and stool collection for culture Transport to L2 Widal test Typhi-dot Blood and stool culture Quality assurance for L1 Quality control for L2 Special tests Training
19
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.

20
Laboratory tasks at each level for the diagnosis
of tuberculosis
L1 L2 L3
Sputum collection Smear preparation Microscopy and reporting Same as L1 Quality control for L1 Transport to L3 for culture Culture and sensitivity testing Quality control
21
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

22
Laboratory tasks at each level for the diagnosis
of measles
L1 L2 L3
Collection of blood and urine samples Transport to L3 Same as L1 Virus culture in designated labs. Serology (?)
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Polio

• Laboratory criteria for diagnosis
• Isolation of wild polio virus from stool

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Laboratory tasks at each level for the diagnosis
of polio
L1 L2 L3
Sample collection and transport to designated laboratories as per National Polio Surveillance Programme (NPSP) guidelines Sample collection and transport to designated laboratories as per NPSP guidelines Virus culture in designated laboratories
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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
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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 4oC L3
CSF Within 5 days of onset 4oC L3
Autopsy (Brain, lung, liver) In the event of death 4oC L3
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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
28
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

29
Sample collection for the laboratory diagnosis of
Japanese encephalitis
Sample Period of Collection Storage for 24 to 48 hours Transport
Serum Within 6 days of onset 4?C L3 In cold chain
CSF Within 6 days of onset 4?C L3 In cold chain
Autopsy (brain, lung, liver) In the event of death 4?C L3 In cold chain
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Laboratory tasks at each level for the diagnosis
of Japanese encephalitis
L1 L2 L3
Collection of blood for serology and culture Transport to L3 Same as L1 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)
31
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
32
Laboratory tasks at each level for the diagnosis
of plague
L1 L2 L3
Assist in sample collection Staining and microscopy Transport sample to L3 laboratory No reporting(Wait confirmation) Culture, serology and confirmation to be performed in a designated L4/L5 laboratories
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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

34
Laboratory tasks at each level for the diagnosis
of leptospirosis
L1 L2 L3
Collection of blood and urine Transport to L2 Serology by latex agglutination/ IgM ELISA DGM Transport samples to L3 labs for culture Culture MAT and serovar identification
35
Laboratory tests for water samples

• Most Probable Number (MPN) method for coliform
  bacteria
• H2S strip method for fecal contamination
  assessment

36
Laboratory tasks at each level for the assessment
of water quality
L1 L2 L3
Collection of samples Rapid test- (H2S strip) Collection of samples Rapid test- (H2S strip) MPN test Same as L2 Quality control for L2
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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

38
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

39
Quality assurance
Quality assurance
Internal quality control (Continuous,
concurrent control of laboratory work)
External quality assessment(Retrospective and
periodic assessment)


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Internal quality control

• Test request and specimen collection
• Test processing
• Temperature
• Reagent
• Maintenance of equipment
• Reporting and using test results

41
External quality assessment

• Within the state IDSP system
• L1 by L2
• L2 by L3
• Through external agency
• External quality assurance scheme for selected
  tests

42
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 Refer to the medical officer of the primary health centre for specific laboratory action
Cough lt or gt 3 weeks Refer to the medical officer of the primary health centre for specific laboratory action
Loose watery stools Refer to the medical officer of the primary health centre for specific laboratory action
Acute jaundice Refer to the medical officer of the primary health centre for specific laboratory action
Unusual syndromes Refer to the medical officer of the primary health centre for specific laboratory action
43
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
44
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
45
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
46
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
47
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
48
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
49
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 CaryBlair 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
50
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
51
Laboratory investigations by the PHC/CHC medical
officer /laboratory technician for typhoid
/fever gt 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
52
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
53
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
54
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
55
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
56
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
57
Laboratory investigations by the PHC/CHC medical
officer /laboratory technician for tuberculosis
/cough gt 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
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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
59
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
60
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
61
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)
62
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
63
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
64
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
65
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
66
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
67
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

68
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

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Sample laboratory register
ID no Name and address of patient Age Sex Prov. Diag. Lab tests ordered Lab results Date sent to L2 Result from L2 Date of result



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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

71
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

72
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