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