From cold chain to ambient temperature: transport of viral specimens- a review

Abstract

The diagnosis of an aetiology is dependent on the collection, transport, and storage of the infectious sample. The transport of the sample plays a crucial role in the chain of diagnosis. It is important to maintain the biological integrity of the pathogen during the transport of the sample to achieve an accurate diagnosis. This is important, particularly for labile organisms like viruses that are inactivated easily compared to other microorganisms. Many transport media have been utilised to ensure the integrity of the virus during transport. While most of the transport media are focused on preserving the infectious properties of the virus, progress has been made to develop virus transport media to inactivate the virus and obtain the stability of the viral nucleic acid, enabling better molecular diagnosis of the virus aetiologies. This review summarises the various media used for the transport of virus samples and focuses on the need to develop virus transport media that inactivates the virus and preserves the viral nucleic acid.

Keywords:

• Ambient temperature
• inactivation of the virus
• molecular diagnosis
• polymerase chain reaction
• virus transport medium

1. Introduction

Viruses are obligate intracellular parasites, the simpler viruses made up of nucleic acids and their specified polypeptides, but complex viruses made up of lipids and carbohydrates [1]. The nucleic acid in the virus is either DNA or RNA but never both, unlike organisms. The decay rate of viruses decreases at low temperatures, and stability decreases with an increase in temperature. The virus is stable at a pH range of 7.3–7.5 [2]. The viral nucleic acid, DNA and RNA are sensitive to extreme pH. Nucleic acid structure alters at pH below three or above 10 [3]. The other factor affecting nucleic acid stability is the nuclease enzymes- deoxyribonuclease and ribonuclease. However, the stability of the virus can be maintained when transported in an appropriate medium, commonly known as the viral transport medium.

The collection and transport of the sample are major factors for the accurate diagnosis of any specimen. Viruses are stable at low temperatures (2-8 °C) and labile as temperature increases [2], and hence it is desirable to transport the samples for virus diagnosis in a viral transport medium with a cold chain [4]. However, body fluids like blood, cerebrospinal fluid (CSF), bronchoalveolar lavage (BAL), stool, urine and vitreous fluid are transported in the cold chain without a viral transport medium. Several methods have been attempted to develop a medium to transport viral samples at room temperature for both isolation and molecular diagnosis of the virus in a cost-effective manner [5].

Laboratory diagnostics play an essential role during public health emergencies. Inactivation of the infectious sample upon collection reduces the biohazard associated with the transport and handling of the sample. The inactivation of infectious samples is inappropriate for virus isolation. However, it may be used for other diagnostic procedures like molecular and serological diagnosis. Inactivation of the infectious sample also helps during public health emergencies of unknown aetiologies. The purpose of this review is to illustrate different viral transport mediums which can stabilise the virus at room temperature and also different sample collection devices for viral testing.

2. Viruses and transport medium

Viral transport medium is crucial in enhancing the stability of virus present in the infectious samples during transport [6]. The medium is generally composed of a buffer to maintain the pH of media, a protein stabilising agent, antimicrobials to prevent contamination and some containing reagents that can inactivate the virus [7]. Preferably, viral samples are transported in the cold chain, but there are transport media which can inactivate the infectious virus and preserve the nucleic acid improving molecular detection even after extended transport time. This review covers various transport media used for transporting virus samples at ambient temperature while emphasizing the need to improve research on transport media that can inactivate the virus and prevent nucleic acid degradation.

2.1. Transport medium for isolation of virus

Charcoal viral transport medium (CVTM) was compared with the dextran viral transport medium containing 0.5 g/litre of Diethylaminoethyl (DEAE)-dextran in place of charcoal, agar control medium which is CVTM minus the charcoal, throat wash control medium containing the salts in CVTM with 0.45% of bovine albumin, Amies, and dextran-Amies. Adenovirus types 4 and 7, coxsackie virus types B-5 and A-21, herpesvirus, vaccinia, parainfluenza type 2, and influenza A type PR-8 were subjected to survival studies at ambient temperatures (22–25 °C). The recovery of virus was done by inoculating the samples in HEK cell line containing L15 medium supplemented with 2% fetal calf serum (FCS) and antibiotics. The inoculated fertilized eggs and cell culture were observed for 21 days. The CVTM was superior in the recovery of virus compared other media for preservation of all the viruses under study and the recovery of viruses was possible for 21 days [8]. The charcoal media was modified to Leibovitz-Emory medium CVTM (LEM) by substituting agar with agarose and compared with CVTM. The recovery rate of HSV 2 from clinical sample was better in LEM (87%) than compared to CVTM (60%) and Amies media (45%). HSV-1 isolation rate was higher in LEM than other media [9].

A study comparing the Virocult transtubes, Tryptose phosphate broth media, Richards viral transport medium, HH medium, sucrose phosphate- glutamate (SPG) and Bartel’s transport tubes was performed. The survival of the Long strain Respiratory Syncytial Virus (RSV), Adenovirus type 2, clinical HSV isolates AO301 (type 1) and AO386 (type 2), Herpes simplex virus (HSV) type-1 strain McIntyre, HSV type-2 strain 333, was studied. The samples after inoculation in the respective media were transported at 4 °C and 22 °C. The results showed that the half-life of the various viruses at 22 °C was lower than 4 °C. The half-life of all HSV strains in Richards transport medium 3.1–15 days; 1.1–4.2 days in tryptose phosphate broth, 1.9–3.6 days in SPG, 1.7–3.7 in Virocult trans tubes and 2.9–4.2 days for HH medium. [10] At 22 °C, HSV 2 333 strain had a longer half-life in Richards transport medium than other media (p < 0.05). Among the media analysed, Richards transport medium was superior to others [10].

A comparison study was done among sample collection using flocked swabs in universal transport medium (UTM), UTM mixed with saline nasal aspirate, and unpreserved saline wash. The flocked swab in UTM was transported at room temperature. For the 72 samples in flocked swabs in the UTM, the sensitivity was 91.7% (95% confidence interval [CI] = 82.7 to 96.9%) and the specificity was and 85.3% (95% CI = 77.3 to 91.4%); the sensitivity was 98.6% (95% CI = 92.5 to 100%) and specificity was 94.5% (95% CI = 88.4 to 98%) of the saline aspirate preserved in UTM which was higher than unpreserved saline aspirates (sensitivity- 70.8% (95% CI= 58.9 to 81%) and specificity of 88.1% (95% CI= 80.5 to 93.5%)). No statistically significant difference was found between saline aspirates preserved in UTM and flocked swabs in UTM and [11]. The UTM was also compared with M4- RT transport medium for analysis of Influenza A and B, RSV, HSV 1 and HSV 2, Adenovirus, Cytomegalovirus (CMV), Parainfluenza virus (PIV) type 1 and PIV 3; Coxsackie B4, Echovirus 11, and Chlamydia trachomatis by real-time PCR and cell culture [5]. It was possible to recover RSV after 96h from UTM but not in M4 RT. There was a reduction in the cycle threshold value in M4 RT with 72h and 96h of storage at 20 °C. The recovery of CMV and parainfluenza virus 1 in M4 RT decreased with time [5]. Another study evaluated three new Viral Transport media namely CVM, MPT and Transystem manufactured by Copan Diagnostics, compared to a BBL Viral Culturette swab system and Multi-Microbe M4, a liquid transport medium by Micro Test, Inc [12]. The study was conducted to evaluate the stability of coxsackievirus B5 (CVB5), adenovirus serotype 16 (AD16), HSV 1, HSV-2, echovirus type 9 (ECV9), and PIV3 using shell vial culture [12]. The results for the different viruses at 22 °C are summarised as follows. The half-life of CVB5 was highest (38.2 days) in CVM; in M4 transport medium ECV9; PIV3, HSV1 had higher half-lives 16.7 days, 1.9 days and 2.9 days respectively [12].

An evaluation for optimal condition detection of viruses was performed by comparing swabs purchased from Copan (Brescia, Italy). The details of the swabs used in the study are plain (rayon-tipped) swabs without medium (catalogue no. 8154CIS), virus transport medium swabs in a medium-soaked sponge (VTM) (catalogue no. 147CV), flocked swabs with universal transport medium (UTM) (catalogue no. 359 C), flocked nylon fibre swabs with liquid Amies medium (E swabs) (catalogue 480CE), and swabs with Amies gel containing charcoal (catalogue E114). The viruses analysed in the study are HSV-2, echovirus type 30, influenza A/New Caledonia/20/99 (H1N1), and adenovirus type 7. The samples in the respective medium for respective viruses were incubated at 4 °C, 20-22 °C and 37 °C. There was no significant decrease in the concentration of all viral RNA concentration at 20–22 °C for 7 days [13].

Universal viral transport medium (Becton, Dickinson and Company, Sparks, MD), containing polyester swabs was analysed to study the stability of the viruses at different temperatures for the long term. To check the stability, each transport medium tube was spiked with 100 µL of 10^6.4 TCID₅₀ of Influenza A virus. At room temperature, the stability declined rapidly as indicated by the TCID₅₀ value. The half-life of the virus was 1.9 days at room temperature and the virus was undetected after 29 days. The decomposition of the viral gene was determined by the standard curve of diluted day 0 cDNA, the nucleic acid was not detected after 29 days [14].

The UniTranz-RTTM (Puritan Medical Products Company Universal Transport System) was compared with BD Universal Viral Transport System (UVT) for influenza A virus, adenovirus (AdV), HSV types 1 and 2, CMV, EV 30, RSV, PIV 3, and varicella zoster virus (VZV). The samples were spiked in both the transport media and incubated at room temperature and 4 °C for 72 h. Analysing both the media by shell vial assay and immunofluorescence staining found that UniTranz-RTTM had significantly higher recoveries than UVT for AdV, EV, and VZV. For HSV-2 and PIV there was no significant difference. UVT showed better recovery for HSV-1, influenza A, and RSV than UniTranz-RTTM.

Bentonite is a cation exchange resin. It is an adsorbing agent for electropositive and electronegative proteins, and a good stabilizer of viral infectivity [15]. The two mediums, the first consisting tissue culture HB597 medium powder (10.96 g), Trizma-base (0.8 g), Trizma-HCI (6.85 g), and EDTA (1.8 g), streptomycin (300 pg/ml), penicillin (500 U/ml), neomycin (100 pg/ml) at the final pH 7.1; the second media consisted of reagents double the concentration first media and bentonite is coated with sterile normal rabbit serum. The two media were compared with Amies transport medium modified by Leibovitz, charcoal viral transport medium, and tissue culture medium HB597 [16]. Coxsackie virus (CV) A9 was stable by 1 log₁₀ in 0.07% bentonite added to NaCl (0.85%) buffered at pH 7.1 with 0.05 M Tris buffer compared to the absence of bentonite in the diluting solution. The stability was enhanced by adding 0.005 M EDTA to the NaCl-Tris-bentonite mixture. Both the media stabilized CV-A9, CV-B5, and echovirus 11 at room temperature for 21 days with no loss in infectivity compared to charcoal and HB597 media while adenovirus type 5 was stable for 14 days in all three media. Influenza virus A was stable for 5 days without a change in titre in both bentonite media, but the other two media did not stabilise the virus. Parainfluenza type 3 virus was stable for 7 days, but the infectivity decreased after 14 days. In medium B, herpes simplex virus was comparatively labile and was stable only for 3 days in titre dropped after 7 days; rubella virus was stable for 7 days but after 14 days a sudden drop in the titre in both the bentonite media but in charcoal and HB597 (Table 1).

Table 1. Composition of transport medium for isolation of virus.

Transport media	Composition	References
Leibovitz Charcoal viral transport medium (CVTM)	sodium chloride (4.00 g), potassium chloride (0.20 g), dipotassium phosphate (1.70 g), bacteriological charcoal (10 g), and agar (4.010 g)	[8]
Leibovitz-Emory medium	Sodium chloride (4.00 g); potassium chloride, (0.20 g); dipotassium phosphate (1.70 g); bacteriological charcoal (10.00 g); and agarose (4 g).	[9]
Amies transport medium	Sodium chloride (3 g), potassium chloride (0.20 g), calcium chloride (0.10 g), magnesium chloride (0.10 g), normal potassium phosphate (0.20 g), disodium phosphate (1.15 g), sodium thioglycolate (1.00 g), charcoal (10.00 g), and agar (4.00 g).	[9]
Richards viral transport medium (ChlamydiaPort)	Phosphate buffer, bovine serum, sucrose, organic buffers, amino acids, phenol red and antibiotics	[17]
Sucrose phosphate- glutamate (SPG)	Potassium dihydrogen phosphate (3.8 mM) Dipotassium hydrogen phosphate (7.2 mM), sucrose (0.218 M), glutamate (4.9 mM) and 50 µg./ml of gentamycin	[18]
HH medium	Hanks balanced salt solution with 0.88% sodium bicarbonate, 0.2% bovine serum albumin (BSA) (Sigma), 30 mM HEPES buffer, and 50 µg/ml gentamicin with pH 7.2 adjusted with 1 N NaOH	[19]
Tryptose phosphate broth	Rehydrated broth according to the standard formula (Difco, Detroit, MI, USA), 0.5% BSA (Sigma), 50 µg/ml gentamicin, and 25 µg/ ml amphotericin B (Sigma)	[6]
Bartel’s transport tubes (Bartel’s Immunodiagnostic Supplies)	Transport medium tube containing 2 ml of Eagle MEM supplemented with 2% fetal bovine serum, 20 mM HEPES buffer, antibiotics and a monolayer of human diploid fibroblast cells.	[6]
CVM	HBSS, glutamate, sucrose, bovine serum, gelatin, colistin, vancomycin amphotericin B, and phenol red indicator	[12]
MPT	Minimal essential media (MEM) in deionized water, bovine serum, amphotericin B, vancomycin, colistin and phenol red indicator	[12]
Transystem	Distilled water, sodium chloride, disodium phosphate, magnesium chloride, calcium chloride, thioglycolate and monopotassium phosphate	[12]
M4	HBSS, sucrose, bovine serum, glutamate, gelatin, cryoprotectants, colistin, amphotericin B and vancomycin, buffered to pH 7.3 ± 0.2	[12]
BBL	Water, glucose, sodium citrate, potassium chloride, monobasic sodium phosphate, calcium chloride, potassium phosphate, magnesium sulphate, and phenol red	[12]
Stuart’s medium	Sodium glycerophosphate 10 g/L, sodium thioglycolate 1 g/L, calcium chloride 0.1 g/L, methylene blue 0.002 g/L, agar 3 g/L, pH 7.4 ± 0.2 (at 25 °C)	[20]
Universal transport medium (Becton, Dickinson and Company, Sparks, MD	HBSS, BSA, HEPES buffer (pH 7.3), L-glutamic acid, L-cysteine, amphotericin B, gelatin, sucrose, colistin, vancomycin and phenol red	[7]
Puritan UniTranz-RTTM	HBSS, sucrose, gelatin glutamic acid, HEPES and bovine serum albumin as stabilizers,	[21]
Bentonite transport medium	Medium A: Tissue culture HB597 medium powder (10.96 g), Trizma-base (0.8 g), Trizma-HCI (6.85 g), and EDTA (1.8 g), streptomycin (300 pg/ml), penicillin (500 U/ml), neomycin (100 pg/ml) at the final pH 7.1	[16]
	Medium B: The reagents double the concentration of media A and bentonite is coated with sterile normal rabbit serum

2.2. Transport medium for molecular purposes

PrimeStore® MTM is a molecular transport medium which inactivates infectious human samples. Stability of the Influenza A (A/California/04/2009) in PrimeStore MTM was found to be 30 days at 25 °C. The medium also inactivated Adenovirus type 5, Influenza A (H3N2) and Influenza A H5N1 (A/Vietnam/1203/04) viruses. The medium was cytotoxic and destroyed the membrane of cells until it was diluted 1:1000. The medium also protects the viral nucleic acid from degradation by nucleases like DNase, RNase A, and RNase T1. The medium has an internal positive control (IPC) ssRNA (0.02 pg/µL) included in PrimeStore® MTM to track the degradation of nucleic acid. Influenza A H1N1 virus was also stable at 38 °C as visualised by gel electrophoresis to day 7 when compared to no bands for the same virus in the standard viral transport medium. Similar results were obtained for rRT-PCR for Influenza A matrix gene. The MTM was compatible with commercially available bead-based and silica-based extraction kits [22]. Comparison of MTM with Universal Transport medium (UTM, Copan diagnostics) where MTM was transported at room temperature (17 °C- 22 °C) and UTM was transported at −80 °C. The sample was collected in flexible nasopharyngeal nylon flocked swabs (FLOQSwabs™, Copan Diagnostics, Murrieta, California, USA). The commonly detected viruses were Influenza, PIV, RSV, and human metapneumovirus . The PrimeStore® MTM was more specific for the detection of four viruses but less sensitive in the detection of RSV [23].

SupraSens microbial transport medium (SSTM) was tested for inactivation and transport of diagnostic samples at temperatures 15-40 °C. Severe Acute Respiratory Syndrome Virus − 2 (SARS-CoV-2) was inactivated at 15 min of contact time. The media was able to inactivate the virus even at 50% dilution as analysed by the absence of plaque formation. The samples transported in SSTM showed lower Ct value (mean Ct difference >3.50) and 70% higher detection of positive samples compared to the VTM used as reference [24].

The Flinders Technology Associates filter paper (FTA)® filter paper was used in the collection and transport of samples for the infectious bronchitis virus [25], Newcastle disease virus [26], Infectious bursal disease virus [27, 28] has been documented [29]. The FTA lyses cells like tumour cells, white blood cells, viruses and bacteria. FTA ® inactivates the virus when stored for 1-5 days. The sensitivity was found to be 1 µl of 10⁴ EID₅₀/ml of virus and no cross-amplification was found with mycoplasma and other viruses. The stability of RNA was found to be for 15 days at room temperature and a slight decrease at 41 °C after 15 days. Molecular characterisation was possible for samples stored for less than 16 days at room temperature. The field evaluation showed 100% sensitivity when compared to virus isolation of the same samples. The detection sensitivity of the Newcastle virus disease decreased by one log by 14th day and the virus was inactivated on contact with FTA® cards [26]. Infectious bursa disease virus was detected in the minced bursas or stamped bursas for 15 days at room temperature whereas at 25 °C molecular characterisation by RFLP and nucleotide sequencing was doable up to 1–3 months [27]. At room temperature, low pathogenic avian influenza virus (LPAIV) A/turkey/Germany/R617/2007 (H6N2) and highly pathogenic avian influenza A/chicken/Egypt/0879/2008 (H5N1) were stable for 5 months [25]. Samples were inactivated by FTA filter paper and were stable for 24h at room temperature for Infectious bursal disease virus [28] (Table 2).

Table 2. Inactivation of different viruses by various transport medium^a.

Transport media	Viruses inactivated^a	Duration of exposure	Preservation of nucleic acid^b	References
PrimeStore® MTM	Influenza A (H3N2)	Seconds	ND^c	[22]
	Influenza A (A/Vietnam/1203/04) H5N1		ND
	Adenovirus type 5		ND
	Influenza A A/California/04/2009 H1N1	ND	30 days
SupraSens microbial transport medium (SSTM)	SARS-CoV-2	15 min	72 h	[24]
FTA® filter paper	Newcastle disease virus	In contact	<14 days	[26]
	Infectious Bronchitis Virus	1–5 days	ND	[29]
	Avian influenza virus A/turkey/Germany/R617/2007 (H6N2)	<60 min	5 months (difference of < 1 Ct from day 0)	[25]
	Avian influenza virus A/chicken/Egypt/0879/2008 (H5N1)	<60 min	3 days
CyMol (COPAN diagnostics)	H3N2 (A/Victoria/3/75)	< 5 min	21 days (1 log10 reduction from day 0)	[30]
	Pandemic H1N1 (A/California/04/09-like virus)		ND
	H1N1 patient isolate		ND
	Influenza A virus subtypes (H1N1, H6N5, H8N4, H10N8, and H15N8)	30 min	ND

^aThe inactivation was determined by the failure of the virus to show CPE in the cell culture.

^bThe stability of the nucleic acid was determined by reduction in the virus titre as analysed by real-time PCR and quantitative PCR.

^cND-No data.

2.3. Transport medium with cell lines

The recovery of viruses after long-distance transport, mainly the enveloped virus is improved with the immediate inoculation of the sample into the virus transport medium containing cell culture compared to the traditional virus transport medium [16]. The Transporter (Bartels Immunodiagnostics, Bellevue, Wash) was compared against sucrose-phosphate-glutamate (SPG) buffer for recovery of herpes simplex virus. The samples were mainly taken from obstetrical patients and consisted of respiratory swabs, tissues, faeces, skin and urine. The samples in the transporter tube were transported at ambient temperature. The recovery rate of the virus in the transporter tube (92 of 101 samples) was better than SPG buffer (82 of 101 samples). The SPG had more false negatives than the transporter tube [31].

2.4. Transport medium with additional substrates

The incorporation of additional substrates like foam pad has been shown to improve the recovery of the virus [32] and substrates including filter papers [27, 29] and high-density polyethene preserve the nucleic acid at room temperature [33]. The use of additional substrates enhances the preservation and recovery of viruses. Virocult transport tubes were evaluated for the transport of herpes simplex virus at 22 °C. The half-life of herpes simplex virus at 22 °C in the medium was 2.75 days. The titre of the sample was 0.5 log lower compared to the sample held at 2 °C [32]. The samples in the Virocult transport tube were refrigerated before transporting at ambient temperature and were refrigerated again between receipt and inoculation. Among 448 positive samples out of 2000 (22.4%), 85% showed a cytopathic effect on day 3. And all the 448 were positive by day 7. Statistical analysis showed that the transport of the virus for 12 days didn’t cause a significant loss in viability.

ViveST (ViveBio, Norcross, GA) is a device utilized for the preservation of nucleic acids at ambient temperature [33]. ViveST with foetal bovine serum and ViveST with MEM were compared with MEM, universal transport medium (M4RT), and FBS alone. At ambient temperature (∼22 °C) adenovirus, HSV, human rhinoviruses, echovirus, and coxsackie B virus were stable for 7 days in ViveST with FBS [33]. The stability of the echovirus was better than other viruses [33]. There was an average reduction in two orders of magnitude of infection units in the viruses when compared with the starting concentration of respective viruses. Echovirus was preserved at the highest rate compared to the other viruses (p-0.00006) [33]. Adenovirus was preserved better in ViveST with FBS and MEM than in other media. ViveST with FBS preserved CVB and HSV better than other media compared [33] (Table 3).

Table 3. Composition of transport medium with cell lines and additional substrates.

Transport media	Composition	References
Transporter	The transport medium tubes containing 2 ml of EMEM, 20 mM HEPES buffer and 2% foetal bovine serum with gentamicin sulphate (10 µg/ml) amphotericin B (4 µg/ml), and human diploid fibroblast monolayer.	[31]
Virocult	Plastic tube containing a swab and a foam pad saturated with a balanced salt solution, lactalbumin hydrolysate (3.0 g/ litre), glucose (1.0 g/litre), cycloheximide (10 µg/ml), and chloramphenicol (2.5 µg/ml)	[32]
FTA® filter paper	A cotton-based cellulose paper soaked with anionic detergent and buffer	[29]
ViveST^TM	A clear polypropylene tube with a clear high-density polyethylene (HDPE) containing a proprietary polyolefin fibre-based synthetic matrix and a colour indicating DriCap Desiccator	[33]

2.5. Other microbial transport media evaluated for viruses

Transport media for chlamydia (ChlamydiaPort, Scott Laboratories) was evaluated as a viral transport medium for the HSV. It was compared with ViraPort (Scott Laboratories) and cell culture medium [34] for the stability of HSV type 1 (strain McIntyre) and HSV type 2 (strain 333) and clinical isolates (HSV type 1 strain A0218 and HSV type 2 strain A0301). The viruses were diluted 1:10 in all ChlamydiaPort and ViraPort and held at 22 °C and 2 °C. At 22 °C the half-life of A0301, 333, and McIntyre strains in the ChlamydiaPort was 5-6 days and 2- 3.75 days in ViraPort. ViraPort could stabilise strain AO218 better ChlamydiaPort at 22 °C. Clinical samples were stable for 5 days in the ChlamydiaPort than Virocult transport system [34].

Stuart’s medium used for many bacteria was utilized for determining the stability of herpes virus in comparison with VTM containing Eagle’s tissue culture medium containing 10% foetal calf serum, streptomycin (100 µg/ml), mycostatin (50 µg/ml) and penicillin (200 µg/ml). The VTM functioned better as a transport medium than Stuart’s transport medium. The advantage of Stuart’s medium over the viral transport medium was that it didn’t have any antibiotics and hence could be a better choice detection of other microbial respiratory pathogens [35] (Table 4).

Table 4. Stability of viruses in different transport mediums.

Virus	Transport media	Half-life of virus in days^1,2	References
Adenovirus type 4	Leibovitz Charcoal viral transport medium (CVTM)	21*	[8]
Adenovirus type 5	Bentonite transport medium	2.5*	[16]
Adenovirus type 7	CVTM	21*	[8]
Herpes simplex virus	CVTM	21*	[8]
Herpes Simplex virus 1	Leibovitz-Emory medium	8*	[9]
	Richards viral transport medium (ChlamydiaPort)	15	[10]
	Sucrose phosphate- glutamate (SPG)	3.6	[10]
	Virocult	2.7	[10]
	HH medium	4.2	[10]
	Tryptose phosphate broth	4.2	[10]
	M4	2.9	[12]
	CVM	1.9	[12]
	MPT	1.5	[12]
	Transystem	1.7	[12]
	BBL	2.0	[12]
	Bentonite transport medium	3*	[16]
Herpes simplex virus 2	Leibovitz-Emory medium	8*	[9]
	M4	2.8	[12]
	CVM	3.8	[12]
	MPT	1.9	[12]
	Transystem	2.0	[12]
	BBL	2.3	[12]
Coxsackie virus A9	Bentonite transport medium	21*	[16]
Coxsackie virus B5	CVTM	21*	[8]
	M4	20.3	[12]
	CVM	38.2	[12]
	MPT	4.3	[12]
	Transystem	8.4	[12]
	BBL	5.2	[12]
	Bentonite transport medium	14*	[16]
Coxsackie virus A21	CVTM	21 *	[8]
Echovirus type 5	M4	16.7	[12]
	CVM	8.2	[12]
	MPT	3.5	[12]
	Transystem	2.3	[12]
	BBL	8.2	[12]
Echovirus type 11	Bentonite transport medium	21*	[16]
Influenza A PR8	CVTM	21*	[8]
	Universal transport medium	1.9*	[36]
Respiratory syncytial virus	Universal transport medium	4*	[36]
	M4-RT	2 *	[36]
Parainfluenza virus 3	M4	1.9	[12]
	CVM	1.8	[12]
	MPT	1.1	[12]
	Transystem	1.1	[12]
	BBL	1.1	[12]
	Bentonite transport medium	7*	[16]
Rubella virus	Bentonite transport medium	4.5*	[16]

Data represents the half-life of the virus when stored at ambient temperature (22-25˚ C) as determined by the regression analysis of the viral decay curve.

No data of the half-life of the virus for data marked ‘*’.

*The viability of the virus in the sample was determined by inoculation in cell culture and the occurrence of the cytopathic effect.

2.6. Extraction reagents for preserving the nucleic acid

Nucleic acid extraction reagents are known to stabilize nucleic acid. The capability of the lysis buffer AVL (Qiagen, Valencia, CA) was evaluated for suspensions of dengue virus serotype 4 (DENV 4), Rift Valley fever virus (RVFV) and Venezuelan equine encephalitis virus (VEEV), at 32 °C, 20 °C, 4 °C, and −20 °C temperatures and the preservation of nucleic acid was determined by quantitative real-time PCR [37]. The RVFV was detected on day 0 at 32 °C and on day 7 at 20 °C. DENV 4 was detected in replicate samples until day 7 at 20 °C, and in one sample on days 14, 21, and 35; only one sample was detected on day 7 at 32 °C. At 20 °C VEEV was not detected until 7 days; at 32 °C all the samples were inactivated for 0–7 days [37].

3. Stability of viruses in various transport media

There has been extensive research on the evaluation of the virus in the transport medium maintained at ambient temperature. In the transport media reviewed in this review, Leibovitz Charcoal viral transport medium tested by Leibovitz et al. maintained the viability of the viruses both RNA and DNA, though the half-life of the virus was not recorded, the virus was recovered by cell culture after 21 days of maintaining at 22 °C. Among the transport media for which the half-life of the virus was analysed by studying the decay curve, Dunn et al. found that the half-life of coxsakie virus B5 and echovirus type 5 was 38.2 days and 20.3 respectively in the CVM transport medium and the half-life of these viruses in the M4 transport medium was 8.2 days and 16.7 days respectively suggesting that non-enveloped RNA viruses were best preserved in these media compared to others. The enveloped DNA viruses had the highest half-life of 4.2 days in the HH medium and tryptose phosphate medium suggesting the rapid degradation of the enveloped viruses at ambient temperature in comparison to non-enveloped viruses. The decay of the virus in the transport medium at ambient temperature results in a false diagnosis with samples subjected to delayed transportation. The challenges faced with the logistical expense involved in the cold chain transport of the virus diagnostic samples and decay of the virus at ambient temperature have resulted in the development of transport media like CyMol and PrimeStore® MTM that can inactivate the virus and preserve the nucleic acid of influenza A virus for 21 days and 30 days respectively with less than a log reduction from the day 0 virus titre as determined by real-time PCR [22, 30]. FTA filter paper has not been evaluated for the transport of human diagnostic samples but has the potential to be investigated [25, 27]. This approach of transport of virus samples that are inactivated at the time of collection and can be transported at ambient temperature is economically and logistically beneficial.

4. Conclusion

An ideal transport media helps to preserve the stability of the samples under fluctuating temperature conditions, helps in long-term storage of specimens and has a long shelf life. With the advancement in the field of research and diagnostics in virology the need for the transport of viral specimens in cold conditions could be gradually eliminated. The transport of viral specimens at ambient temperature is cost-effective and beneficial in areas with poor public health settings. These media are particularly beneficial in remote areas which require long-term storage of samples before a diagnostic test is performed. This eliminates the false diagnosis of an etiology, improving treatment and public health measures. Similarly, the change towards molecular techniques for diagnosis and research in virology makes it less necessary to retain the viability of the virus. Rather the focus should be more towards the preservation of viral nucleic acid. Molecular diagnostic methods offer better specificity, which is crucial in accurate diagnosis, mainly during public health emergencies. The preservation of the nucleic acid has been achieved by commercially available media which can preserve the stability of the nucleic acid for a month at ambient temperature. Additionally, the rapid inactivation of the virus renders it incapable of transmission of infection and reduces the risk involved in the transport of the virus specimens. This is particularly beneficial during the public health emergencies. There is always a risk of laboratory-acquired infection during the handling of infectious specimens. The inactivation of the infectious specimen at the first step of laboratory diagnosis can be beneficial in the rest of the process essentially eliminating the risk of laboratory-acquired infections. Therefore, the focus is on improving the research to develop a virus transport medium which can transport the virus specimens at ambient temperature that can inactivate the virus with minimum contact time and preserve the nucleic acid during the transport.

Author contribution

Oliver Christy Dsa outlined the paper structure performed the comprehensive literature review, conducted the data analysis and drafted the manuscript. Trupti Sathish Kadni coordinated the literature review processes and edited the manuscript. Sudheesh N provided expert guidance throughout the review process, reviewed and revised the manuscript and edited the final version of the manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability

Data sharing is not applicable to this article as no new data were created or analysed in this study.

Additional information

Funding

No funding was received for this review.