5. Clinical Specimen Sources
Be prepared to collect specimens before you leave for the
field
• Suspected cases
• Symptomatic cases
• Contacts
• Including people living or working with suspected cases
9. How to Manage Kits
• Store specimen collection kits in a dry, cool place
• Store specimen collection kit where it will be
accessible after office hours and on weekends
10. Specimen types
• Basically whether seasonal, avian, swine or pandemic,
the samples for laboratory diagnosis are the same-
respiratory specimens in Viral Transport Media (VTM)
Upper Respiratory tract specimens:
• Nasal swab
• Nasopharyngeal swab (specimen of choice for MERS)
• Nasopharyngeal aspirate
• Nasal wash
• Throat swab
12. What to Collect
Preferred specimens
From an Ambulatory patient
• Nasopharyngeal swab and
• Throat swab
• Can be collected into the same VTM
From an Intubated patient
• Lower respiratory aspirate
14. What is Viral Transport
Medium?
• Used in the collection of samples for viral isolation and
testing
• Prevents specimen from drying out
• Prevents bacteria and fungi growth
15. Storing VTM
• Sterile collection tubes containing 2-3
ml of VTM
• Tubes can be stored at 4-8 °C until use
• Tubes can be stored for short periods
of time at Room Temperature
• Keep records of when the VTM tube
was received and its expiry date
16. When to Collect
Respiratory Specimens
• As soon as possible after symptoms begin
• Before antiviral medications are administered
• Even if symptoms began more than one week ago
• Collect additional specimens when required or
indicated
16
18. • Before collection - need to reassure and explain the
procedure
• Children may need to be restrain
• Child’s parents or guardian must be made aware that
the child may become distressed
• Parents should not be in room- may interfere, more
risk, no PPE
19.
20.
21.
22.
23.
24.
25.
26. Nasopharyngeal Swab
1. Insert dry swab into nostril and
back to nasopharynx
2. Leave in place for a few seconds
3. Slowly remove swab while slightly
rotating it
4. Put tip of swab into VTM tube,
breaking applicator’s stick
5. Use a different swab for the other
nostril, same procedure as above
27. Throat Swab
• Tilt the patient’s head back and gently depress the
tongue with a tongue depressor
• The tonsillar areas and the posterior pharyngeal wall
should be rubbed with the polyester swab to dislodge the
epithelial cells
• Care should be taken not to touch the tongue and the
lateral walls of the buccal cavity to avoid contamination
with commensal bacteria
• After collection, break the shaft of the swab and place
immediately into a VTM tube
30. Specimen Tracking
System
Maintain a record to track:
• Identification number
• Subject information
• Specimen collection date
• Specimen collection location
• Diagnostic test results
33. How to Store Specimens
For specimens in VTM:
• Transport to laboratory as soon as possible
• Store specimens at 4 °C before and during transportation within 48
hours
• Do not store in standard freezer – keep on ice or in refrigerator
• Avoid freeze-thaw cycles
• Better to keep on ice for a week than to have repeat freeze and
thaw
34. Specimen forwarding
Three main purpose
To maintain specimen viability
To prevent leakage outside the package
To prevent cross contamination
*All samples for virus diagnosis must be sent in cold
chain
36. Packing Specimens for
Transportation
• Use three packaging layers
• First layer should be water tight
• Use absorbent material in all layers
• No more than 500 mL should be in the specimen
container
37. • Keep specimens at 4 ºC
• Fill a cooler with ice packs or coolant packs
• Include an itemized list of specimens with identification
numbers and laboratory instructions
Packing Specimens for
Transportation
38. Transporting Specimens
• Refer to WHO guidelines for the safe transport of
infectious substances and diagnostic specimens
• Inform and Coordinate with the laboratory
• NHL telephone: 01- 371957, 01- 371925
44. Transmission of infection
• Through the inhalation of infected aerosols
and droplets
• Infected formites are involved less
frequently
• Highly communicable (99% chance of
acquiring disease in non immune person)
45. Clinical Features
• Incubation period approx: 1 week to 10 days
• Clinical features are
fever ,
cough, coryza 3-4days
conjunctivitis
Koplik’s spots (50-90%)
Rash 4—5 days
46. Koplik’s spots
• Appear on the buccal mucosa
• Shortly before rash onset
• Small irregular red spots with a bluish white
speck in the centre
50. Measles Rash
• First appear on the forehead or neck or
behind the ears
• Lesions are red macules and become
maculopapular
• By the end of second day upper extremities
and trunk
• Third day lower extremities are affected
52. Measles Rash contd
• Rash resolves in the same order first
disappearing from the face and neck
• last about 6 days
• turn brown and persists for 7-10 days
• Followed by fine desquamation
53. Complications
• Bronchitis, bronchiolitis, pneumonia and otitis
media
• Encephalitis
• Diarrhoea
• Blindness
• Death- 1/1000 cases
• Risk of death is greater for infant and adult than
children and adolescents
56. 1.Urine
• 10-20ml of urine collected in a sterile container
• First urine passed in the morning
• Collect within 3 days after the onset of rash
• Label the tube with the patient’s name,
outbreak ID number, specimen number, date of
collection and specimen type
57. 1.Urine
• Before transport, in the hospital laboratory,
they should be kept at 4-8°C.
• Urine should be sent to NHL within 24 hours
after collection (in cold box) with laboratory
request form
58. 2.Throat Swab
• Collect within 3 days after the onset of rash
• Tilt the patient’s head back and gently depress the
tongue with a tongue depresser.
• The tonsillar areas and the posterior pharyngeal
wall should be rubbed with the polyester swab to
dislodge the epithelial cells.
• Care should be taken not to touch the tongue and
the lateral walls of the buccal cavity to avoid
contamination with commensal bacteria
59. 2.Throat Swab
• After collection, break the shaft of the swab and
place immediately into a sterile leakproof container
containing viral transport medium (VTM)
• Label the tube with the patient’s name, outbreak ID
number, specimen number, date of collection and
specimen type.
• Before transport, in the hospital laboratory, they
should be kept at 4-8°C.
• Throat swab should be sent to NHL within 48 hours
after collection (in cold box) with laboratory request
form.
60. For serology
Collect within 4 - 28 days after the onset of rash
Collect 5ml of blood in a sterile plain tube
one tube is enough
Test for Measles IgM Ab for recent infection
• Label the tube with the patient’s name, age, sex, outbreak ID number,
specimen number, date of collection and specimen type
• We cannot do the samples without label on the tubes
61. Transport the whole blood specimen to NHL if it can
reach within 24 hours.
If it cannot reach NHL within 24 hours, do separation
of serum
Separate serum after clotting, and transfer into a
new sterile bottle or microvial and send to NHL.
To prevent insufficiency, collect 5 ml of blood or 2
ml of serum in a sterile bottle
For outbreak, 5 cases enough
Before transport, in the hospital laboratory, they
should be kept at 4-8°C
The specimens should be sent to NHL in cold box
with laboratory request form
62. The serum/ blood samples should not be
haemolysed samples (Prevent hemolysis of
samples – narrow needle, rapid suction, rapid
pushing blood out of syringe, wet container should
not be used)
Measles Laboratory Requisition Form must include
- Date of collection
- Date of onset of rash
- History of measles vaccination
- Patient’s address
Some of the lab forms are not filled completely.
Please fill completely. Some samples are without
lab request forms
64. Clinical and Virological Features
• Infects susceptible individuals via respiratory
route
• Nasopharyngeal secretions- principle source
• Primary replication---epi cells of nasopharynx
• IP-14-21 days
• Viremia-widespread dissemination of the virus
(blood, nasopharynx, urine, stool, synovial fl,
skin, cervix, & L/N)
• Joint symptoms-commonest complication,
appear soon after the rash faded, lasts for 3-4
days
65. Clinical and Virological Features
contd
• Patients- infectious for 3 wks----nasopharyngeal
excretion may occur up to a week before the onset
of rash & for 7-10 days thereafter
• Viremia is present about a week before the onset
of rash, and end as rubella Ab develop
• 25%- inapparent infection
• Typical rubelliform rashes may result from infection
with enteroviruses, human parvovirus B19 & some
arboviruses (Chikungunya)
67. Why is rubella infection so
concerning ?
Outcome of Rubella in pregnancy
Live birthTermination Miscarriage Stillbirth
1. Congenital Rubella Syndrome
2. Congenital Rubella Infection
3. Normal
68. What is Congenital Rubella
Syndrome (CRS)?
• A sequel of rubella infection in pregnancy
• Associated with Infection early in pregnancy
• Weeks 1- 10 – 90% CRS*
• Weeks 11-12– 33%
• Weeks 13-14– 11%
• Weeks 15-16– 24%
• Weeks > 17– 0%
* Miller E, Cradock-Watson JE, Pollock TM. Consequences of
confirmed maternal rubella at successive stages of pregnancy.
Lancet. 1982 Oct 9;2(8302):781-4. PubMed PMID:6126663.
The purpose of rubella vaccination program is
thus prevention of congenital rubella infections
which can lead to fetal deaths/loss, pre-mature
delivery or CRS
69. Congenitally Acquired Infection
• Rubella in pregnancy--fetal death and
spontaneous abortion or delivery of a severely
malformed infant, an infant with minimal
damage or a healthy infant
• Outcomes depend on gestational age at which
maternal infection occurs
• First 8 weeks of pregnancy- spontaneous
abortion in 20% of cases
• 13th
-16th
weeks of gestation- 17% of infant may
develop deafness & retinopathy
70. Congenitally Acquired Infection
contd
• Rubella virus can be recovered from most infants
with severe congenitally acquired rubella at birth
• 3 months of age----50-60% of nasopharyngeal
secretions
• 9-12 months of age---10%
• Delayed manifestation- diabetes mellitus & other
endocrinopathies, sensory neural deafness,
glaucoma and progressive panencephalitis
71. How does CRS present clinically?
PERMANENT
•Hearing Impairment
•Ophthamologic
• Cataract, Microphthalmia,
Retinopathy, Glaucoma
•Heart defects
• Patent Ductus Arteriosus
•Microcephaly
•Developmental Delay
TRANSIENT
•Thrombocytopenia
•Jaundice
•"Blueberry muffin" appearance
•Hepatosplenomegaly
•Bone lucencies
DELAYED
•Endocrinopathies
•Progressive auditory or ocular
dysfunction
Organ specificity generally related to stage of
gestational infection.
Editor's Notes
You should plan on the need to collect specimens before you leave for the field, so that you are prepared when you arrive. Prepare the needed materials beforehand.
Specimen samples need to be collected from both suspected cases and their contacts. Suspected cases are people with symptoms consistent with an influenza illness. Contacts are people with direct contact with suspected cases, such as people who live or work with them.
The first topic that we will discuss in this module is the specimen collection kit.
VTM is just one component of the specimen collection kit. Here is a checklist of all items that should be in the specimen collection kit. The items include:
- Collection vials with VTM
- Polyester fiber-tipped applicators
- Sterile saline which is 0,85% NaCl
- A sputum or mucus trap
- Tongue depressors
- Specimen collection cups or Petri dishes
- Transfer pipettes
- A secondary container
- Ice packs
- Items for collection of blood
- Personal protective equipment
- Field collection forms
- A pen or marker for labeling samples
Let’s look more closely at a few of these items. . .
The items in the specimen collection kit should be kept together, except for collection vials containing VTM. As much as possible, the specimen collection kit should be kept in a dry, cool place. In addition, it must be accessible after hours and on weekends, in case you need to leave for the field at these times.
Considerations for what specimens to collect may also depend on the status of the patient. For an ambulatory patient (a patient that can walk around), nasal swabs and throat swabs are the easiest and are appropriate to collect. Both of these can be collected from the same patient and placed into the same viral transport medium.
If the patient is intubated, aspirates are logical to collect, and can even be collected directly from the intubation tube.
Facilitator Note: There are contradictions between the current avian influenza and if it goes pandemic - this module reflects the protocol for current situation and not a pandemic of human to human transmission that might emerge. It is important to check before teaching this to see if this needs to be updated.
Viral transport medium, abbreviated as VTM, is used in the collection of samples for viral isolation and testing. VTM prevents the specimen from drying out, and it also prevents bacteria and fungi from growing.
It is important to correctly store VTM. If VTM is made in the laboratory, place 2 to 3 milliliters of VTM into sterile collection vials. The vials can be stored in the freezer at -20 ºC until use. The vials can be stored for short periods of time at 4 - 6 ºC.
Influenza virus is most likely to be detected soon after symptoms begin. Therefore, in order to increase the chances that the laboratory can detect the H5 virus, respiratory samples should be collected as soon as possible after symptoms begin and before antiviral medications are administered. However, it is not always logistically possible to collect respiratory samples early in the clinical course of disease. But you can still collect specimens even if the symptoms began more than a week ago, as it is still possible to detect virus in these specimens.
It is best to collect multiple types of specimens on multiple days.
Next we will describe how to collect a nasopharyngeal swab.
First, insert a dry drayon, rayon, or polyester swab into the nostril and back to the nasopharynx.
Leave the swab in place for a few seconds.
Then slowly remove the swab while slightly rotating it.
Throat swab collection
Stick out tongue
Press down tongue with wooden spatula
Using polyester swab stick, rub both tonsillar arches, uvula, Posterior pharyngeal wall without touching sides of the mouth
You will need to use a specimen tracking system to keep track of the specimens at all times. The particular specimen tracking system will vary by country. However, here are some guidelines. It is advisable to maintain a database that contains information about each sample, including the identification or tracking number as we just illustrated on the sample field data collection form, subject information, and when and where a sample was collected. Results from diagnostic testing should also be entered into the database.
Facilitator Note: Please adapt this content to your country.
Here is an example of a field data collection form. The particular collection form used in your country might be different. The tracking number on the upper right hand corner should be the same number on the clinical samples taken from the patient. You may want to include priority ratings of the specimens to help the laboratory determine which specimens to test first.
The top portion of the form collects demographic information: the patient’s name, address, date of birth, sex, and occupation. Information about the health status and clinical diagnoses is also collected. Finally, it is important to provide information about how the specimen was collected.
You should include a copy of the field data collection form with the specimens that you transport to the laboratory.
We are next going to discuss considerations for storing, handling, and transporting the specimens that you collect. This may be the most important task related to specimens that you as a rapid responder will perform.
Although you should send specimens in Viral Transport Medium (VTM) to the laboratory as soon as possible, it is important to properly store them before you send them to a laboratory. If you will be transporting specimens within 48 hours, you can store them at 4 °C both before and during transportation. If you will not be able to transport the specimens to the laboratory within 2 days, you need to store them at -70 °C. If this can’t be done, keep the specimens on ice or in the refrigerator as long as necessary. DO NOT put specimens in a standard freezer, as this will damage them.
It is also very important to avoid freeze-thaw cycles. Do not freeze samples if they will thaw and be frozen again, as this will destroy the virus. It is better to keep a sample on ice even for a week, than to allow the sample to freeze and thaw multiple times.
When you are ready to pack specimens for transportation from the field to the laboratory, you must use three packaging layers. This is done to protect specimens from damaged during transportation. The first packaging layer should be water tight, and all layers should be absorbent in case there are any leaks. There should be no more than 500 mL of liquid in the specimen collection container.
Just as it is important to keep specimens cold during storage, it is important to keep specimens cold during transportation. Try to keep specimens at 4 ºC. A cooler filled with ice packs can be used for this purpose, but do not use dry ice unless the specimens are double-bagged and airtight; carbon dioxide from the dry ice can inactivate the virus.
In all specimen shipments, include an itemized list of specimens, with specimen identification numbers and instructions for the laboratory.
When you send any specimens from potential cases of avian influenza from the field to a laboratory, we recommend that you follow WHO guidelines which have been outlined here for the safe transport of infectious substances and diagnostic specimens.
In addition, you may need to follow local regulations on the transportation of infectious material.
Be sure to coordinate the shipment with the laboratory. Arrangements should be made so that the laboratory is prepared to receive the specimens when they arrive.
Facilitator Note: Please adapt this content to your country.
Here is an image of a properly packed specimen ready for shipment to a laboratory. Included are the three layers of packaging, the absorbent packing material, the specimen ID, the biohazard label, the itemized list of contents, and the labeling of the outer package as UN 3373 diagnostic specimens.
If this material is not available to you, transport specimens in a cool box with ice.
