Viral Transport Media (VTM)- Principle, Preparation, Uses, Limitations

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. Transport media for viruses are especially important as they are more labile than other infectious agents.

The probability of successful isolation increases when the time interval between collection and inoculation of the culture is less and when the specimen contains as many virus particles as possible.

In order to maximize the amount of virus in the specimen, the sample collection should be done early in the patient’s illness.

Successful and reliable isolations of viruses can be accomplished when a suitable transport medium is used.

The isolation count also increases when viral specimens are sent to either a local laboratory or a reference laboratory.

VTMs are prepared with the idea of providing appropriate conditions of temperature, pH, and nutrients.

The constituents of an appropriate viral transport media are selected to create an isotonic solution containing proteins to protect the viral structure, antibiotics to control microbial contamination, and one or more buffers to control the pH.

VTMs are prepared with the idea of sustaining the viability of the viral culture or specimens for tests like the nucleic acid amplification test (NAAT) while preventing the drying of the sample.

Viral agents vary widely in composition, stability, structure, morphology, and size. The loosely packaged enveloped viruses obtained from human samples are generally more labile than the non-enveloped dense viruses. Because they are labile to the environmental conditions, the viability of these viruses must be protected by using different solutions. Some enveloped viruses, such as herpes simplex viruses (HSV), are surprisingly stable in the non-frozen state when placed in a suitable holding environment. However, most of the viruses do require a colder temperature. The infectivity of viruses decreases over time, and generally, the decay rate is a function of temperature, so that stability is enhanced by cooling. Thus, viral transport media are prepared with the idea of providing appropriate conditions of temperature, pH, and nutrients. The constituents of an appropriate viral transport media are selected to create an isotonic solution containing proteins to protect the viral structure, antibiotics to control microbial contamination, and one or more buffers to control the pH. Viral transport media are prepared with the idea of sustaining the viability of the viral culture or specimens for tests like the nucleic acid amplification test (NAAT) while preventing the drying of the sample.

An ideal viral transport medium would possess many of the following characteristics:

• It would preserve the activity of the virus, even at room temperatures.
• It would be nontoxic to cell cultures and not obscure the appearance of viral cytopathic effects.
• It would have a long shelf life (either in a frozen or non-frozen state).
• It would be applicable for both culture isolations and direct tests such as enzyme immunoassays or immunofluorescence.

In order to achieve all the above-mentioned characteristics, viral transport media usually have a protective protein component, antimicrobial agents to prevent the chances of contamination, and buffers to maintain the pH of the solution.

Some VTMs also have additional ions and minerals that aid in maintaining the viability of such specimens.

The protective protein component is usually derived from animal sources, such as bovine serum albumin (BSA) or fetal bovine serum (FBS). These proteins help to stabilize the viral structure and prevent degradation by proteases.

The antimicrobial agents are usually a combination of antibiotics and antifungals, such as gentamicin, amphotericin B, benzylpenicillin, streptomycin, polymyxin B, nystatin, ofloxacin hydrochloride, and sulfamethoxazole. These agents help to inhibit the growth of bacteria and fungi that might contaminate the specimen or interfere with the viral detection.

The buffers are usually based on phosphate or bicarbonate salts that help to maintain the pH of the solution within a range suitable for viral survival. The pH of the solution should be close to neutral (around 7.0) or slightly alkaline (around 7.4).

The ions and minerals are usually derived from balanced salt solutions, such as Hanks Balanced Salt Solution (HBSS) or Eagle Minimum Essential Medium (E-MEM). These solutions provide essential electrolytes and nutrients for viral metabolism and function.

The composition of different viral transport media may vary depending on the type of virus, the source of specimen, and the method of detection. However, the general principles of providing protection, inhibition, and stability remain the same.

Depending on the source and type of specimen, different viral transport media (VTM) can be used to collect and transport viruses from humans and animals. Some VTMs are commercially available and ready to use, while others can be prepared locally with specific ingredients and procedures. The following are some examples of VTMs for human and animal specimens:

For human specimens:

• COPAN Universal Transport Media (UTM): This is a commercially available VTM that contains a buffered salt solution with gelatin, proteins, antibiotics, and antifungal agents. It can be used for the collection and transport of respiratory specimens such as nasal and throat swabs. It is compatible with various molecular and culture-based methods for virus detection and isolation.
• Eagle Minimum Essential Medium (E-MEM): This is another commercially available VTM that contains a balanced salt solution with amino acids, vitamins, proteins, antibiotics, and antifungal agents. It can also be used for the collection and transport of respiratory specimens such as nasal and throat swabs. It is suitable for both culture and molecular methods for virus detection and isolation.
• Locally made VTM: This is a VTM that can be prepared locally with the following ingredients and procedure:
  • Add 10 g veal infusion broth and 2 g bovine albumin fraction V to sterile distilled water (to 400 ml).
  • Add 0.8 ml gentamicin sulfate solution (50 mg/ml) and 3.2 ml amphotericin B (250 µg/ml).
  • Sterilize by filtration.
  • Aliquot 3 ml of the medium into individual sterile conical screw-capped tubes (such as 16x100 mm tubes).
  • Store at 2-8°C.
  Alternatively, a larger volume of VTM can be prepared with the following ingredients and procedure:
  • Inactivate 500 ml of fetal bovine serum (FBS) by heating for 30 minutes in a water bath at 56.0°C +/- 1.0°C. Use commercially inactivated FBS if available.
  • Add 50 ml of amphotericin B, add 50 ml of gentamicin to the inactivated FBS and then filter sterilize the solution through a 0.20 to 0.45 µm filter unit (150 ml filter unit).
  • Add 10 ml of the FBS to 500 ml of Hanks Balanced Salt Solution (HBSS).
  • Add 2 ml of the Gentamicin/Amphotericin B mixture to the previously formed HBSS with FBS.
  • Disperse the solutions to bottles and cap the bottles. Mix by inverting the bottle.
  • Label the bottle along with the date of production, additives, and expiration date.
  • Aliquot 3 ml of the medium into individual sterile conical screw-capped tubes (such as 16x100 mm tubes). Keep lids tightly closed after the medium is dispensed. Label the containers with the necessary information.
  • Store at 2-8°C.

For animal specimens:

• Transport medium 199: This is a widely used VTM for the collection and transport of clinical specimens from all animal species. It can be prepared locally with the following ingredients and procedure:
  • Take a tissue culture medium 199, containing 0.5% bovine serum albumin (BSA).
  • To 1 liter of above add: benzylpenicillin (2 x 106 IU/liter), streptomycin (200 mg/liter), polymyxin B (2 x 106 IU/liter), gentamicin (250 mg/liter), nystatin (0.5 x 106 IU/liter), ofloxacin hydrochloride (60 mg/liter), and sulfamethoxazole (0.2 g/liter).
  • Sterilize by filtration and distribute in 1.0–2.0 ml volumes in screw-capped tubes.
• PBS-Glycerol transport medium: This is a glycerol-based VTM that provides longer-term stability of specimens where cooling is not immediately possible. It is suitable for egg inoculation but not suited for tissue culture inoculation. It can be prepared locally with the following ingredients and procedure:
  • Prepare the phosphate-buffer saline by adding 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na2HPO4, and 0.24 g of KH2PO4. Add distilled water to this mixture to make 1 liter.
  • Autoclave the prepared mixture and mix 1:1 with sterile glycerol to make 1 liter.
  • To 1 liter of above add: benzylpenicillin (2 x 106 IU/liter), streptomycin (200 mg/liter), polymyxin B (2 x 106 IU/liter), gentamicin (250 mg/liter), nystatin (0.5 x 106 IU/liter), ofloxacin hydrochloride (60 mg/liter), and sulfamethoxazole (0.2 g/liter).
  • Sterilize by filtration and distribute in 1.0–2.0 ml volumes in screw-capped tubes.

Viral transport media (VTM) are used for various purposes related to the collection, transport, and analysis of specimens containing viruses or other microorganisms. Some of the common uses of VTM are:

• Viral diagnosis and isolation: VTM are used to collect and transport specimens from different body sites, such as eye, feces, genital, respiratory, skin, and tissue, that might contain viruses of clinical importance. These specimens are then inoculated into cell cultures or eggs for viral isolation and identification. VTM help to preserve the viability and infectivity of the viruses during transport and storage.
• Viral detection and quantification: VTM are also used to maintain the integrity and stability of viral nucleic acids or antigens for molecular or immunological tests, such as nucleic acid amplification tests (NAAT), enzyme immunoassays (EIA), or immunofluorescence assays (IFA). VTM help to prevent the degradation or loss of viral nucleic acids or antigens by providing a suitable buffer, protein, and preservative.
• Viral surveillance and research: VTM are also useful for collecting and transporting specimens for viral surveillance and research purposes. For example, VTM can be used to monitor the prevalence and diversity of influenza viruses in different populations or regions. VTM can also be used to collect and store specimens for future studies on viral epidemiology, evolution, or pathogenesis.

VTM are not only used for viruses but also for other microorganisms that require similar conditions for viability and stability. These include mycoplasma, chlamydiae, and ureaplasma, which are often associated with respiratory or genital infections. VTM can also be used for some bacteria that are fastidious or difficult to culture, such as Bordetella pertussis or Neisseria meningitidis. However, separate specimens should be collected if bacterial or fungal cultures are also requested, as the antibiotics in VTM might inhibit their growth.

Viral transport media (VTM) are designed to preserve the viability and infectivity of viral specimens during collection and transport. However, they also have some limitations that should be considered when using them for viral diagnosis and research. Some of the limitations are:

• Variability in viral stability: Different viruses have different levels of stability and susceptibility to environmental factors such as temperature, pH, humidity, and light. Some viruses, such as enveloped viruses, are more fragile and prone to degradation than others, such as non-enveloped viruses. Therefore, the effectiveness of VTM may vary depending on the type and strain of virus present in the specimen. Some viruses may require specific additives or conditions to maintain their integrity in VTM.
• Risk of contamination: VTM contain various components such as proteins, buffers, antibiotics, and antifungals to protect the viral specimens from degradation and microbial growth. However, these components may also introduce potential sources of contamination or interference in the subsequent viral detection or isolation methods. For example, some antibiotics may inhibit the growth of certain cell lines used for viral culture, or some proteins may interfere with the binding of antibodies or probes used for viral identification. Therefore, it is important to use appropriate quality control measures and validation procedures when using VTM for viral diagnosis and research.
• Limited shelf life: VTM have a limited shelf life and should be stored and used according to the manufacturer`s instructions or standard protocols. The shelf life of VTM may depend on factors such as the composition, packaging, storage temperature, and exposure to light. Generally, VTM should be stored at 2-8°C or frozen at -20°C or lower until use. However, repeated freezing and thawing may reduce the quality and performance of VTM. Therefore, it is advisable to use fresh or aliquoted VTM whenever possible and avoid prolonged storage or transport of VTM at room temperature or higher.
• Limited availability: VTM are commercially available from various suppliers and manufacturers. However, due to the high demand and limited supply of VTM during outbreaks or pandemics of viral diseases, there may be shortages or delays in obtaining VTM for clinical or research purposes. Therefore, it may be necessary to prepare VTM locally using available materials and protocols. However, locally prepared VTM may not have the same quality and consistency as commercially available VTM and may require additional testing and validation before use.

These are some of the limitations of VTM that should be taken into account when collecting and transporting viral specimens for diagnosis and research. Despite these limitations, VTM are still valuable tools for preserving and enhancing the recovery of viral agents from various types of specimens. By following proper guidelines and protocols for the use of VTM, the quality and reliability of viral diagnosis and research can be improved.

We are Compiling this Section. Thanks for your understanding.