VIRAL

TRANSPOR
T MEDIUM
DR APARNA GOPINATH

DEOT OF MICROBIOLOGY
CONTENTS
• VIRAL TRANSPORT MEDIUM
• SPECIMENS / COLLECTION
• TRIPLE PACKAGING SYSTEM
• RABIES
• H1N1
• FEACAL PCR
REFERENCES
• Centers for Disease Control and Prevention ; SOP#: DSR-052-05
• Journal - American Society for Microbiology
• National Centre for Disease Control Directorate General of Health
Services -World Health Organization Collaborating Centre for Rabies
Epidemiology 2022
• A viral transport media (VTM) is a nutrient substance used to
carry and maintain the viability of specimens (viruses) to a
laboratory for the identification and further processing of the
sample.

• VTM typically consist of a buffered salt solution such as Hanks’ balanced salt solution buffered
with HEPES to maintain a neutral pH, protein-stabilizing agents such as bovine serum albumin or
gelatin, and antimicrobials to prevent bacterial and/or fungal overgrowth.
1. Viral transport tube

• Viral transport media is included in a sterile tube typically made of premium medical
grade plastic.

• Some tubes may be formulated to contain buffered proteins and antibiotics that
suppress the growth of potentially contaminating bacteria and fungi.
2. Viral transport cap
A securely closed transport tube is critical for protecting the sample specimen from
contamination during transport. Many tubes feature screw caps designed for
secure closure and ease of use, while other transport tubes may feature a plug top
for a secure seal.

3. Collection swab
Some viral transport media and transport systems are formulated to work
with a specific type of swab. Not all swab materials are suitable for viral
testing.
For example, nasopharyngeal swabs cannot be made with cotton as organic
materials interfere with the polymerase chain reaction (PCR) testing process..
 SPECIMEN
COLLECTION
• Swab/Scapping,Vesicle Aspirate, Small tissue pieces : Place into tube with 1-2ml VTM

• Fluids – CSF/Urine/BAL : Place into sterile leak proof container

• Solids – stools / tissues : Place into sterile leak proof container

,add 5-10ml VTM

Blood – collect with anticoagulants.

Body Sites Potential Viruses Specimen Collection

Collect conjunctival swab on Dacron or rayon

Adenovirus, enterovirus, coxsackievirus A,
swab moistened with saline and place in VTM;
Eye cytomegalovirus (CMV), herpes simplex virus
aqueous and vitreous fluid placed in a sterile
(HSV), varicella-zoster virus (VZV)
container without VTM.

Fecal specimens (preferred) in a leak-proof

Feces Adenovirus, astrovirus, CMV, norovirus, rotavirus
container; fecal swabs in VTM

Adenovirus, CMV, HSV, papillomavirus, varicella-

Genital Collect on swabs and transport in VTM
zoster virus (VZV)

Adenovirus, coronavirus, hantavirus, CMV, HSV, Nasopharyngeal aspirates or

influenza virus, parainfluenza virus, nasopharyngeal swabs ; bronchoalveolar lavage
Respiratory
metapneumovirus, measles virus, rhinovirus, for the diagnosis of lower respiratory tract
RSV, VZV infections. Swabs are transported in VTM.

Adenovirus, enterovirus, HHV6, HHV8, HSV,

Swab or aspirate fluid in vesicle and scrape cells
Skin measles virus, parvovirus B19, poxvirus, rubella
at the base of the lesion; place fluid in VTM
virus, VZV

Tissue Adenovirus, CMV, HSV Place in VTM

SPECIMEN COLLECTION VIRUSES

BLOOD 8-10ml ARBOVIRUS,ARENAVIRUS,CMV,HSV,HIV

CSF 2-5ml ; STERILE CONTAINER CMV,ENTEROVIRUSES OTHER THAN

POLIO,HSV

URINE 10.20ML MSU; STERILE CONTAINER ADENOVIRUS,CMV,HSV,MUMPS,RUBELL

A
TRIPLE PACKAGING
SYSTEM
 Primary container

• “sample” container ; leak-proof

• When shipping liquids, a secondary seal of the lid is advised
(i.e. parafilm).
• If shipping several primary containers, a means of
cushioning or separation (i.e. bubble wrap, etc.) should be
used to reduce the possibility of the containers damaging
each other.
• Assure that liquids are oriented in an upright position to
further minimize the potential for leakage.
 2. Secondary container:

• Your primary container(s) will be placed inside this container.

• The purpose of this container is to capture any leakage from the

primary containers. Therefore, this container should be properly
sealed for transport.

• Adequate absorbent materials should be placed inside this container

to absorb any leakage of liquids from primary containers.
3. Outer package:
The purpose of this package is to provide protection against physical damage
to the inner contents that may occur during the transport process. Therefore,
it is strongly advised that the outer package be a sturdy fiberboard box.

• Cushioning or spacer material (such as bubble wrap) should be placed in

the box to fill up the remaining space and keep the contents from shifting in
the box.
• A list of contents between the secondary container and outer package is
also advised.
• Orientation arrows should also be placed on the outer package if it contains
liquids.
RABIES
Collection of specimen
• All samples from suspected Rabies cases should be considered
potentially infectious.

• All specimens should be collected in a primary leak-proof container

Antemortem specimens
• all aseptic universal and standard precautions and appropriate PPE.
Saliva
• 1. Saliva (around 500 µl) should be collected using a sterile
eyedropper pipette and transferred to a small sterile container that
can be sealed securely.

• Collect three specimens in total: one specimen daily on three

consecutive days (not three specimens on the same day).
• Wet a sterile swab with tissue culture medium or physiological saline
and remove excess medium by squeezing on the sides of the vial.

• Swab under the tongue, rinse in the tissue culture medium or

physiological saline containing 2% normal horse serum (NHS) in a
small sterile container.

• Tracheal aspirates and sputum are not suitable for rabies tests.
Corneal Smear
1. Retract the eyelids with the thumb and one finger and press a clean
marked slide against the cornea.

2. Prepare two impression on each slide, applying sufficient pressure to

get the smear carefully.

3. Avoid exerting too much pressure as it may damage the eye.

4. Air-dry the impression for 10-15 mins at room temperature.
5. Treat with chilled acetone and process further for Direct Fluorescent
Antibody Test (DFAT).
Nuchal Skin
• Section of skin, 5-6 mm in diameter and ≈5-7 mm in depth, must be
taken from the nape of the neck .

• It is vital that the specimen should contain hair follicles and should be
of sufficient depth to include the cutaneous nerves at the base of
hair follicles.
• An excision or punch biopsy may be collected.
• Place the skin biopsy onto, and cover with, a piece of sterile saline-
moistened gauze. This keeps the specimen from drying out. Place the
gauze with the biopsy into a screw-top container. No fixative is
required.

• The sample can be used for DFAT/ DRIT for detecting viral antigens (by
using a cryostat to cut thin frozen sections of biopsy specimens for
making impression smears), RT-PCR for detecting viral nucleic acid,
and RTCIT for virus isolation
Serum and Cerebrospinal Fluid (CSF)
• 2-3ml (at least 0.5 ml) each of serum and CSF should be collected in a sterile
vial with all aseptic precautions.
• If no vaccine or rabies immune serum has been given, the presence of
antibody to rabies virus in the serum can be diagnosed.
• Antibody to rabies virus in the CSF suggests a rabies virus infection regardless
of the immunization history.

• Laboratory tests for antibodies includes ELISA, indirect immunofluorescence,

and virus neutralization.
• CSF samples can also be processed for RTCIT and molecular technique(NAAT-
PCR).
• All the specimens, e.g., slides and vials, must be labelled with the
number of specimens, name of the patient, animal species, type of
preservative used, etc., and should be collected only after
consultation with the laboratory.

• Permanent markers should be used. The parcels should also be

appropriately labelled, and relevant information to be enclosed for
the following )
• Human (Hydrophobia/ Paralytic): Name, Age, Gender, Treatment
taken, Exposure to animal etc. may be enclosed.
Transport
• All samples should be considered potentially infectious.
a. Nuchal skin samples are best shipped frozen (at -20 °C or below) but
can be shipped simply with ice packs (2-8 °C) as well.

b. Saliva samples should be shipped frozen (at -20 °C or below)

c. Brain samples should be frozen, or half preserved in 50% glycerol–

saline solution (if freezing is not readily available)
Brain samples should never be preserved in formalin.
H1N1
• Respiratory specimens including: bronchoalveolar lavage, tracheal
aspirates, nasopharyngeal or oropharyngeal aspirates as washes, and
nasopharyngeal or oropharyngeal swabs.

• Swab specimens should be collected only on swabs with a synthetic

tip (such as polyester or Dacron) plastic shaft.

• Swabs with cotton and wooden shafts are not recommended.

Throat Swab
Highest yield in detecting H1N1 influenza in suspected cases
Have the patient open his/her mouth wide open.
The patient should try to resist gagging and closing the mouth while the
swab touches the back of the throat near the tonsils.
The posterior pharyngeal wall and tonsils should be swabbed

Nasal Swab is collected from the anterior turbinate.

Nasopharyngeal Swab:
Insert dry swab into nostril and back to nasopharynx.
Leave in place for a few seconds. Slowly remove swab while slightly
rotating it.
Use a different swab for the other nostril. Put tip of swab into vial
containing VTM, breaking applicator’s stick (the part projecting
outside).

Both Nasal and Throat swabs can be collected into the same VTM to
increase the viral yield.
STOOL PCR
• THE BIOFIRE GI PANEL MENU
• Overall 98.5% Sensitivity and 99.2% Specificity 2
Sample Type: Stool in Cary Blair medium

• Individuals presenting with:

• Dysentery
• Diarrhea with fever, severe abdominal cramps, or signs of sepsis
• Moderate to severe disease
• Symptoms lasting more than seven days
• Immunocompromised patients with diarrhea
BACTERIA:
• Campylobacter (C. jejuni / C. coli / C. upsaliensis)
• Clostridioides (Clostridium) difficile (toxin A/B)
• Plesiomonas shigelloides
• Salmonella
• Yersinia enterocolitica
• Vibrio (V. parahaemolyticus / V. vulnificus / V. cholerae)
• Vibrio cholerae
Diarrheagenic Ecoli , Shigella
PARASITES:

• Cryptosporidium
• Cyclospora cayetanensis
• Entamoeba histolytica
• Giardia lamblia
VIRUSES

• Adenovirus
• Astrovirus
• Norovirus
• Rotavirus A
• Sapovirus (I, II, IV, and V)
Thank You !