Viral Transport Media (VTM) Definition, Principle, Preparation, Application

Definition of Viral Transport Media (VTM)

A viral transport medium (VTM) is an essential nutrient that is used to transport and sustain the viability of samples (viruses) to an laboratory for analysis and identification of the specimen.

• The transport media used by viruses are particularly important because they are more susceptible than other infectious agents.
• The likelihood of success in isolating increases when between collection and the inoculation of the sample is lower and when the sample is contaminated with the most virus particles feasible.
• To maximize your chances of finding the virus present in the specimen, the is best taken at an early stage in the course of the patient’s illness.
• Reliable and successful isolation of viruses is possible with the help of a suitable medium for transport is utilized.
• The count of isolation also increases when specimens with viral infections are sent to an in-person laboratory or the reference laboratory.

Principle of Viral Transport Media (VTM)

• The viruses vary in their structure, composition the morphology, size and stability. For viruses that infect people, those loosely packed enveloped viruses are typically more prone to instability than thick viruses. 
• However certain enveloped viruses, like herpes simplex virus (HSV) are extremely stable in the nonfrozen condition when placed in an appropriate holding environment. 
• It is generally advised that clinical specimens to be used for virus isolation are placed in a cell culture in the bed or put in a refrigerator on ice that is wet and inoculated into the cell culture in the shortest amount of time feasible. 
• In reality, the majority of common viral agents are able to have the ability to survive both keeping at room temperature as well as transport over long durations (days) when they are placed in an appropriate transport medium.
• Perhaps a more thorough and better diagnosis of certain infectious diseases could be made by being more widely understood that viral samples could be taken from the patient and brought to the lab for tests. 
• The virus’s infectivity is, in reality, diminish over time, and typically the rate of decay is dependent on temperature, which means that stability is increased through cooling. 
• Thus, the viral specimens must be kept at room temperature or incubation temperatures when a refrigerator is readily available. 
• The likelihood of successful isolation increases in the event that the period between the collection and the inoculation of the culture is not extended and when the sample contains the most virus-like particles feasible. 
• To ensure that there is a sufficient quantity of virus present found in the samples, they is recommended that it be taken as early as possible in the course of the patient’s disease. 
• If a suitable medium for transport is utilized, efficient and solid isolations can be achieved when the viral specimens are taken to the local laboratory or to the reference laboratory.
• Transport mediums are not always necessary to isolate viruses at the level of the laboratory. In certain circumstances the virus may be carried in the specimen in itself. Examples include the cerebral spinal fluid, urine blood, blood samples, cleansings in the respiratory tract and stool. 
• Many viruses are isolated from the urine of patients with disease when they are eliminated from the urinary system. 
• Similar to viruses, they are discovered in the cerebral blood, spinal fluid, and stool in certain phases of certain infections. These kinds of substances are able to be transported without using an exogenous transport medium.

Viral transport media composition (VTM)

• The components of appropriate virus transport mediums are developed to create an isotonic solution with protective proteins and antibiotics that control contamination by microbial organisms, and one or more buffers that maintain the pH. 
• It isn’t a necessity; many highly effective transport media have hypertonic solutions of sucrose. 
• Liquid transport media are utilized predominantly for transporting swabs or material released from an swab collection. 
• Liquid media can be used in conjunction with other samples if activation of the virus is likely and the resulting dilution is acceptable.

The specimen must be taken within the shortest time possible following the beginning of symptoms, usually within 3-7 weeks. The sample contains the greatest quantity of the virus present at time of the collection. To ensure that the virus remains infected the specimen must be placed in a safe transport medium.

Features of Viral transport media

A perfect viral transport media contains several of the following characteristics;

• It could preserve the activeness of the virus at room temperature.
• It is non-toxic to cell cultures and wouldn’t interfere with the appearance of viral cytopathic reactions.
• It will have a long shelf time (either in the frozen or non-frozen form).
• It is suitable to both culture isolations as well as specific tests, such as an immunofluorescence test or enzyme immunoassays.

In order to attain all the characteristics mentioned above The majority of VTMs include a protein-based protective component as well as antimicrobial agents to reduce the risk of contamination and buffers to keep that pH. Certain VTMs have mineral ions or ions which aid in maintaining the viability of the samples.

Preparation of Viral Transport Media (VTM)

A variety of viral transportation media are available commercially that can be directly purchased and utilized under the conditions specified. One such medium is the COPAN Universal Transport Media and the Eagle Minimum Essential Medium (E-MEM). But, other media are able to be made locally in order to maintain the appropriate circumstances and the composition. The composition and the preparation of various viral transports differ according to whether the sample is taken from animals or human beings.

For specimens from humans

This is the process of making locally-made VTMs that can be used for throat and nasal swabs of human beings:

1. Add 10g of veal infusion broth along with 2g bovine albumin fraction to sterilized distillate water (to 400ml).
2. Add 0.8 milliliters of gentamicin sulfate (50 mg/ml) and 3.2 mg amphotericin A (250 mg/ml)
3. Sterilize with the process of filtration.

The following is a complex procedure for the making of a greater quantity of VTMs for human beings:

1. Inactivate 500mL of the fetal bovine serum (FBS) through heating it for thirty minutes within a water bath in a temperature of 56.0degC +/+/- 1.0degC. Make use of commercially activated FBS If there is.
2. Add 50mL of amphotericinB and add 50mL of gentamicin into the activated FBS and then sterilize the solution by filtering it with an 0.20 up to 0.45 um unit of filtering (150mL the filter).
3. Add 10mL of the FBS to 500mL of Hanks Balanced Salt Solution (HBSS).
4. Add 2mL of the Gentamicin/Amphotericin B mixture to the previously formed HBSS with FBS.
5. Distribute the solution to bottles and them to the bottles. Mix the mixture by inverting the bottle.
6. Label the bottle with the date of manufacture additions, the date of expiration, and the date.
7. Divide 3mL of the medium into conical, sterile screw-capped tube (such like 16x100mm tube). Make sure lids are tightly sealed when the medium has been dispensed. Label containers with the required details.
8. Store at 2-8degC.

To obtain specimens from animals:

1. To collect and transport of animal specimens, two kinds of VTMs can be used.
2. The medium of transport 199 can be extensively utilized to collect and transport of clinical specimens of all kinds.
3. Another medium, the PBS-Glycerol transport media, which is a glycerol-based medium offers longer-term stability to specimens that are not immediately possible.
4. It is suitable for egg inoculation however it is not appropriate for inoculation of tissue cultures.

Preparation of Transport medium 199

1. Choose a tissue-culture medium 199 that contains 0.5 percent bovine serum albumin (BSA).
2. For 1 litre of the above, add the following: 1 litre of benzylpenicillin (2 x 10 IIU/litre) streptomycin (200 mg/litre) and polymyxin B (2 x 10 IIU/litre) and Gentamicin (250 mg/litre) as well as Nystatin (0.5 x 10106 mg/litre) ofloxacin chloride (60 mg/litre) and sulfamethoxazole (0.2 grams/litre).
3. Sterilize through filtration, then disperse the sterilized solution in 1.0-2.0 volumetric volumes of ml in screw-capped tubes.

Preparation of PBS-Glycerol transport medium

1. Make the phosphate buffer saline by adding 8 grams of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4 and 0.24 grams of KH2PO4. Add distilled water into this mixture until you have 1 Liter.
2. Prepare the mix in an autoclave then mix it 1:1 using sterile Glycerol to create 1 Liter.
3. To 1 litre, add the following: the benzylpenicillin (2 x 10 IIU/litre) and streptomycin (200 mg/litre) and polymyxin B (2 x 100 IIU/litre) as well as the gentamicin (250 mg/litre) and Nystatin (0.5 x 10106 IIU/litre), ofloxacin chloride (60 mg/litre) and sulfamethoxazole (0.2 grams/litre).
4. Dispense 1.0-2.0 milliliters of medium for transport into sterilized screw-cap screw-cap vials.
5. The vials should be kept at -20 degrees Celsius until you need them or stored at the room temperature for brief periods of one to two days.

Instruction for use of Viral transport medium tube with swab

1. Sterilization of the sample tube and swab is carried out prior to when the medical institution or the patient is used.
2. Note the pertinent sample information on the tube and then inject a suitable amount of solution for the sample.
3. In accordance with different sampling requirements the sample swabs were taken in the appropriate locations. Holding the handle while gently placing the swab inside the sample area, slowly turning the swab around three times, before gently removing it.
4. The sample is placed into the sampling tube, the sample swab is taken from the tube. After that, the tube cover is tightened after which the sample gets then sealed for the final sampling.
5. The clinical specimens that have been collected should be delivered in the laboratory no later than 48 hours after 4. They should not be shipped back to laboratory until more than 48 hours. They must be stored at the temperature of -70 or lower. The specimens that are sent to the laboratory must be isolated and inoculated as soon as is possible. They can be kept for up to up to 48 hours. If the inoculum isn’t inoculated, it must be kept at a temperature of -70 degrees or less.
6. The standard sampling methods are:

A) nasal swab: gently massage the swab’s head through the nostrils of nasal passages, then slowly let it go after some time. Clean the other nostrils using another swab. Submerge the head of the swab in the sample and then dispose of the tail.

B) pharyngeal swabs: wipe the bilateral tonsils of the pharyngeal and the posterior wall of the pharyngeal cavity with swabs. Immerse the head of the swab into the sample fluid, and take out the tail.

C) gargle: gargle using 10mL normal saline. Rinse the head and create your “Oh” sound, let the saline spin through the pharynx and remove the lotion using the empty sample tube of 50mL.

D) nasal lotion: the patient reverted to his position and their head was slightly slanted back while injecting 50mL normal Saline into the nostril using a pipette. He instructed patients to transmit K for closing the pharyngeal apex and then allow the patient to lower their head, and release normal saline. Collect the lotion using a empty 50mL sample tube and repeat the procedure to clean the nostrils.

E) nasopharyngeal aspirates: Tracheal and bronchial secretions can be extracted using this method. Mucus is taken out of the nasopharynx using a collection device attached to the negative pressure pump. First, the head of the collector is placed in the nose cavity and then the negative pressure is switched on. The collector’s head is then sucked up and slowly pulled out. Then, it is able to collect mucus and wash the collector with 5mL of liquid for three times.

F) autopsy specimens: autopsy tissue samples were taken and samples separated if necessary. The specimens comprised autopsy tissue.

G) samples of mycoplasma, chlamydia and Ureaplasma samples: males: use the sterile cotton swabs for rotating around the urethra

Precautions/cautions

1. After the sample is taken the disposable sampler must be examined as soon as possible, then moved immediately at a low temperature of 2-8°C. The samples taken are stored for up to 2-8 hours at low temperature for up to 48 hours. The storage for long-term use must be maintained at a temperatures, lower than 20 C and kept at 70 C and -196 C.
2. This product is prohibited to be used for sampling bacteria samples. The preservative liquid itself is a source of antibiotics that inhibit the growth of bacteria.
3. Avoid direct contact with patients who are being sampled. Also, take the wet swabs away prior to collecting samples from the patients.
4. The sampling process should be conducted in strict accordance with the method of sampling, to ensure that the location of sampling is precise and the amount of sample is even. If not the quality of the sampling will be compromised.
5. The product shouldn’t be utilized until the expiration date has passed or the packaging of the product is damaged.

Viral transport media uses (VTM)

• The transport mediums for viral infections are utilized to collect and transport viruses-laden specimens.
• They also ensure the survival of other microorganisms such as mycoplasma, the chlamydiae, as well as the ureaplasma.
• VTMs can also increase the amount of virus particles present in an individual sample.
• Liquid transport media is used mostly for transporting swabs and substances released in the medium by an swab collection.
• It makes it easier to collect and transport of samples to areas where facilities for refrigeration are not readily available.

Limitations of Viral Transport Media (VTM)

• A certain amount of growth in contaminants could be seen over the long time of transportation.
• Following transportation, the sample is to be inoculated into the correct medium as soon as is possible. For the best results, the period between the time of sample collection and the time of inoculation onto the culture medium must be minimized to a minimum.
• Certain viruses may be more susceptible to labile than other viruses and the VTMs could be just as effective.
• Certain antibiotics and other chemicals that are that are used in VTMs can alter the acidity of the solutions, and this can affect the viability of the organism.

viral transport media examples

• MDS viral transport medium: This is a special-formulated medium to collect and transport viruses. It is intended to preserve the virus sample’s viability and virulence. MDS Viral Transport Medium, Hanks’ Balanced Salt Solution, is composed of a protective protein antibiotic to prevent microbial contamination as well as buffers to regulate the pH.
• Universal Transport Medium: This FDA-cleared collection and transport media is suitable for the collection, transportation, preservation and long-term freezer storage of clinical specimens containing virus for viral molecular diagnostic testing. The transport medium is packaged in a plastic tube with a screw cap and keeps organisms alive for up to 48 hours at room temperature or refrigerated.
• Remel viral transport media: Remel, a leading microbiology brand, is world-renowned. We are committed to microbiology as a trusted supplier of high-quality media and diagnostic products that can be used in clinical, industrial and research laboratories.

Media	Components
Swab-tube combination Culturette Virocult	Swab-tube combination containing modified Stuart medium, antibiotics Collection swab, plastic transtube, small sponge saturated with phosphate buffer, D-glucose, lactalbumin hydrolysate, chloramphenicol, and cycloheximide
Cell culture medium MEM	MEM with amino acids, fetal bovine serum, buffers, antibiotics
Broth-based media Nutrient broth Tryptic soy broth Tryptose phosphate broth	Beef extract, peptone Tryptone, soytone, dextrose, sodium chloride, dipotassium phosphate Tryptose phosphate broth, 0.5% BSA or 0.5% gelatin, antibiotics, phenol red (optional)
Veal infusion brot	Veal infusion, proteose peptone, sodium chloride
Bentonite containing media Bentonite transport	HB597 tissue culture powder, Tris buffer, EDTA, bentonite, antibiotics, with or without rabbit serum-coated bentonite
Richards Viral Transport	Phosphate buffer, organic buffers, sucrose, amino acids, bovine serum, phenol red, antibiotics
Leibovitz-SPG	Leibovitz #15, sucrose, phosphate buffer, BSA, antibiotics
Carr-Scarborough Viral/ chlamydial transport	Phosphate-buffered sucrose, L-15 medium, glutamic acid, bovine albumin, gentamicin

References

• https://www.cdc.gov/coronavirus/2019-ncov/downloads/Viral-Transport-Medium.pdf
• Johnson, F. B. (1990). Transport of viral specimens. Clinical Microbiology Reviews, 3(2), 120–131. doi:10.1128/cmr.3.2.120