Enteric Viruses: Rapid Detection and Identification Methods

Key Characteristics

• Very significant time savings over

traditional methods

• Clear, easy to interpret results

• Simple, rapid operation allows use

at point-of-care

Intact rotavirus double-shelled

particles (image courtesy CDC)

Introduction
For this overview the term ‘enteric viruses’ refers to an important, but diverse, group of viruses found in the
intestinal tract of humans and animals. Some of the most important are listed in table 1 below. They are
mainly associated with diarrhoea and gastroenteritis, usually mild and self-limiting, although some members
of the group cause asymptomatic infections.

Enteric viruses are the commonest causes of gastroenteritis worldwide, they are most often transmitted via
the faecal-oral route, with transmission by direct human contact and via fomites being common. The
infective dose can be very low. For example, a single rotavirus is capable of causing human infection. Some
enteric viruses, notably noroviruses, are highly infectious and may be easily spread in aerosols and by
contact with contaminated surfaces, sometimes resulting in large outbreaks of illness.

Enteric viruses may also be present in contaminated water supplies and waterborne outbreaks of disease are
not uncommon. Foodborne transmission has been shown to occur in some outbreaks of viral gastroenteritis.

Table 1. Some Clinically Significant Enteric Viruses

Family Genus Types

Astroviridae Mamastrovirus human astroviruses

Adenoviridae Mastadenovirus human adenoviruses

Caliciviridae Norovirus noroviruses (Norwalk-like viruses)

Sapovirus human sapoviruses

Parvoviridae Parvovirus human parvoviruses

Picornaviridae Enterovirus Non-polio enteroviruses: cocksackievirus A & B, echoviruses,

human enteroviruses (types 68 to 71)

Reoviridae Rotavirus human rotaviruses

Detection and isolation techniques

Supplier reference Sampling and Preparation
for these items: Appropriate sampling procedures are critical for the successful isolation and detection of
enteric viruses. Materials collected may include clinical samples, such as stools, and
antibody non-clinical samples, such as water and foods.

antigen - strip
Water samples are tested both for specific viral contamination and for indicators of
antigen - EIA/ELISA faecal contamination, such as adenoviruses and enteroviruses. Since viruses are only
likely to be present in water at low levels, very large samples of many litres may need
antigen - latex agg. to be collected. Testing water samples for enteric viruses requires a specialised
concentration step prior to isolation and detection. Methods used with some success
IFA
include adsorption-elution techniques, precipitation and ultrafiltration.
molecular methods
Testing of food samples for enteric viruses is not routinely practiced, other than for
specific commodities, particularly bivalve molluscs, which concentrate viral particles
within their bodies when feeding in contaminated water and are a known hazard for
enteric virus infection. Specific sampling and virus concentration methods have been
developed for enteric virus detection in shellfish.

Clinical samples, especially stool samples, are the main application for routine testing
for enteric viruses. There are established procedures for taking clinical samples for
diagnostic virus testing and these should be closely followed to maximise the chances
of detecting the causative organism. Some enteric viruses are quite resistant to
freezing and samples can be stored at –20oC if longer term storage is required.

Immunological Detection Methods

The majority of immunological methods for enteric viruses are based on antigen
detection and employ enzyme immunoassay (EIA), latex agglutination, or
immunochromatography technologies.

A wide range of commercial products has been developed and many of these are
available as easy-to-use kits that can be applied at the point-of-care. For example, a
number of slide-based latex agglutination kits and lateral flow immunochromatography
test strips are available for the detection of rotavirus and adenoviruses in suspended
stool samples.

These tests offer simplicity in operation and can be performed outside of the laboratory
to provide a rapid diagnosis. Their main disadvantages are that they are usually only
qualitative tests, have limited sensitivity and cannot distinguish infective viruses.

Molecular Detection Methods

Most enteric viruses, apart from adenoviruses and parvoviruses, are RNA viruses, and
most molecular techniques therefore rely on detecting specific viral RNA sequences. By
focusing on the viral genome, molecular methods can improve specificity and sensitivity
significantly. A number of nucleic acid hybridisation methods have been developed for
enteric viruses, including dot-blot and sandwich hybridisation techniques, but their
sensitivity is often little better than that of conventional techniques.

Techniques that allow the amplification of target nucleic acid sequences have
revolutionised enteric virus detection in clinical and non-clinical samples. The principal
amplification technique used for RNA viruses is reverse-transcriptase polymerase-chain
reaction (RT-PCR). Theoretically, this technique is capable of detecting a single virus
within 24 hours. Detection of the amplified sequence may be done at the reaction
endpoint, or by continuous monitoring (real-time PCR). Real-time PCR has the
advantage of allowing the virus to be quantified. A number of commercial applications
are available for detection of enteric viruses, particularly enteroviruses. Multiplex PCR
methods have been developed that allow the detection of more than one nucleic acid
sequence, and therefore more than one virus type, in the same assay.

Other nucleic acid amplification methods have also been developed, notably nucleic acid
sequence base amplification (NASBA). This technique is also known as isothermal
amplification, as it does not require the repeated temperature cycling used in
conventional PCR methods. At least one commercial application of NASBA has been
developed for enterovirus detection. The advantages of PCR and related methods are
considerable. The main benefit is time saving – 24 hours to a result rather than several
 days or weeks for a cell culture assay. PCR also requires less operator skill and training
to carry out and can be automated to process large numbers of samples. It is also
extremely sensitive, although it can be vulnerable to contamination and cannot
distinguish infective viruses. Most PCR methods also require some costly equipment
and are not suitable for use outside the laboratory. Non-quantitative PCR results may
also be difficult to interpret, since low numbers of virus that do not signify an infection
may be detected, but this problem is largely overcome by real-time PCR.

This guide has been prepared by Food Safety Info, scientific and technical information providers
for the food industry. For more information, visit our web site at www.foodsafetywatch.com

Where do I get more information?

Suppliers by Sector:
Clinical
News items on rapidmicrobiology.com:
Oxoid Tests Allow Rapid Diagnosis of Gastroenteritis
Extensive Range for Enteric Pathogens Available from Oxoid
New Oxoid IDEIA Viral Gastroenteritis Panel Detects Norovirus in 2 Hours
Virocult® a Virus Transport Swab Suitable for Enteric Specimens!
Mast Launches New Eiken LoopAMP™ Norovirus Kit
Improved Rapid Tests for Rotavirus and Adenovirus

New Oxoid IDEIA Viral Gastroenteritis Panel Detects Norovirus in 2 Hours

Oxoid has launched the IDEIA range of products for the rapid diagnosis of viral
gastroenteritis.

These rapid enzyme immunoassays can be used to detect the four leading causes of viral
gastroenteritis: Norovirus, Rotavirus, Astrovirus and Adenovirus, in human stool
samples.

There has recently been an increase in norovirus activity throughout Europe [1,2] making the rapid detection of this virus of particular concern.
Timely identification of the etiological agent combined with appropriate infection control procedures can help to reduce the impact of norovirus
outbreaks on the institution involved [3]. IDEIA Norovirus allows norovirus to be detected in stool samples within just 2 hours.

Barbara Fallowfield, marketing manager, Oxoid (Ely) commented, "The sooner the cause of gastroenteritis outbreaks can be detected, the
sooner infection control procedures can be put in place to stop the spread of disease and limit the damage. IDEIA Norovirus provides rapid and
reliable results that are of enormous value to those involved in the investigation and prevention of norovirus outbreaks, including microbiology
laboratories, infection control teams, surveillance teams and cruise ship operators.

Each IDEIA viral gastroenteritis kit follows the same standardised protocol and is suitable for manual or automated testing. The assays are
 quick and easy to perform, either individually or in a panel using a single sample dilution. Sensitive and specific results are read
photometrically in less than 2 hours.

For further information about the IDEIA Viral Gastroenteritis Panel (IDEIA Norovirus, IDEIA Rotavirus, IDEIA Astrovirus (Amplified) or
IDEIA Adenovirus), please speak to your local Oxoid representative or contact Oxoid using the contact details at the top of this page.

References:
1. Food-borne Viruses in Europe Network (FBVE), (2006) Eurosurveillance Weekly 11(12).
2. Takkinen, J. (2006) Eurosurveillance Weekly 11(6).
3. Lopman, B.A., Reaches, M.H., Vipond, I.B. et al (2004) Emerg. Infect. Dis. 10(10):1827-1834

Mast Launches New Eiken LoopAMP™ Norovirus Kit

Mast is pleased to announce the launch of the new Eiken LoopAMP™ Norovirus kit at the
IBMS meeting in September.

This novel, rapid molecular test offers levels of sensitivity and specificity similar to RT-PCR
without the need for expensive specialist equipment.

The kit employs a simple 3-step methodology to amplify Norovirus RNA from as little as 200
viral copies of sample material.
Florescent Detection of Norovirus

Kits are available for both GI and GII using simple visual UV fluorescence to confirm detection within 60 minutes.

Mast's Technical Sales representatives will be "glowing with enthusiasm " to illustrate how this test can be easily incorporated into routine use in your
laboratory.

To request more information or book a demonstration, visit the Mast Booth (602) at IBMS meeting, or contact Paul Holmes, using the contact details at
the top of this page.

Extensive Range for Enteric Pathogens Available from Oxoid

The extensive ProSpecT® range of ELISA assays for the direct detection of enteric pathogens from faecal
specimens is now available from Oxoid Limited. This comprehensive range, which includes bacteriology,
parasitology and virology tests, is the method of choice in many laboratories, offering speed and accuracy in
situations where patients can deteriorate rapidly.

Diarrhoeal disease can be caused by a wide range of pathogens, including bacteria, parasites and viruses.

The ProSpecT® range is able to detect antigens produced by some of the most significant of these pathogens, such as:

• Campylobacter
• Clostridium difficile (Toxin A and B)
 • Shiga toxin-producing E. coli (STEC)
• Rotavirus
• Adenovirus
• Astrovirus
• Cryptosporidium
• Giardia
• Entamoeba histolytica

By allowing faecal specimens to be tested directly, without the need for culture or enrichment, the ProSpecT® range saves valuable time in identifying
the cause of enteric disease. The simple and convenient procedure can be completed within 2 hours and with almost 100% accuracy when compared to
routine stool culture methods. Furthermore, since the tests detect antigen or toxin produced by the organisms, they are not dependent on the shedding of
live organisms or on their growth.

The assays use the same procedure and share many common reagents (supplied in ready-to-use dropper bottles), allowing several strips for different
pathogens to be set up and run simultaneously. All incubations are at room temperature and results can be read easily - visually or
spectrophotometrically.

For further information about the ProSpecT® range please speak to your local Oxoid representative or contact Val Stroud, using the contact details at
the top of this page.

Rapid Detection of Respiratory Viruses

• Significant time savings over
traditional methods

• Screening kits allow detection of

several different viruses in a single
assay

• Simple, rapid operation allows use

at the point-of-care

Introduction
The respiratory viruses are a large and diverse group that includes some of the commonest causes of human
viral infection worldwide. They are capable of causing a wide range of diseases, from mild self-limiting upper
respiratory tract infections, such as sore throats and the common cold, to more serious infections like
influenza, bronchiolitis, potentially life threatening pneumonia and severe acute respiratory syndrome
(SARS). Some of the most important types from a clinical point of view are shown in table 1 below.

Historically, some respiratory viruses, notably the influenza viruses, have been associated with highly
destructive global pandemics causing millions of deaths. For example, the so-called ‘Spanish flu’ pandemic
of 1918-19 is estimated to have caused many more deaths than the Great War of the preceding four years –
probably at least 50 million worldwide. There are serious current concerns that the emergence of new
influenza virus types in the human population, through mutation or by antigenic shift between avian and
human viruses, could lead to further pandemics in the future.

The respiratory viruses are most often transmitted via the aerosol route by inhaling infected droplets,
although mechanical transmission through the nose or eye following direct contact with an infected person
may also play a significant role in some cases. Infected individuals usually produce large numbers of
infective particles in their respiratory secretions, with a peak often seen soon after the onset of symptoms.
Many respiratory virus infections, especially those caused by respiratory syncytial virus (RSV) and the
common cold viruses, are highly contagious and will spread rapidly through a population, especially in
crowded conditions.
Respiratory virus infections are extremely common worldwide. For example, acute viral nasopharyngitis –
more often known as the common cold – is the most common type of human infection and children may
have a many as six to eight episodes every year. RSV is the commonest cause of lower respiratory tract
infection in young children and influenza viruses are also major causes of human disease carrying significant
costs in terms of health care and lost economic productivity. Pandemic respiratory infections have the
potential to be among the most serious threats to global human health posed by any biological agent.

Table 1. Some Clinically Significant Respiratory Viruses

Family Genus Types Diseases caused

Adenoviridae Mastadenovirus Human adenoviruses Pharyngitis, pneumonia, gastroenteritis,

conjunctivitis

Coronaviridae Coronavirus Human coronaviruses, Common colds, severe acute respiratory

SARS coronavirus syndrome (SARS)

Orthomyxoviridae Influenzavirus A Influenza A, Avian Influenza, bronchiolitis, pneumonia,

influenza A severe lower respiratory tract infection

Influenzavirus B Influenza B Influenza

Paramyxoviridae Metapneumovirus Human Pharyngitis, bronchiolitis, pneumonia

metapneumovirus

Human respiratory Croup, bronchiolitis, pneumonia (in

Pneumovirus
syncytial virus (RSV) infants and young children)

Croup, non-specific upper respiratory

Human parainfluenza
Respirovirus tract infections, bronchopneumonia
virus types 1 & 3
(mainly in children)

Human parainfluenza Croup, pharyngitis and colds (mainly in

Rubulavirus
virus types 2 & 4 children)

Parvoviridae Bocavirus Human bocaviruses Thought to be associated with a variety

of respiratory infections, including
bronchiolitis and pneumonia

Picornaviridae Rhinovirus Human rhinovirus A & Common colds

B

Detection and isolation techniques

Supplier reference Diagnostic tests for viruses in general fall into one of three categories.
for these items:
1. Direct examination of the sample – electron microscopy, antigen
antibody detection detection, molecular detection of viral genomes
2. Indirect testing – cell culture techniques and animal tests
DFA/IFA
3. Serology – detection of specific serum antibodies
EIA and
Immunochromatography Methods for the isolation and detection of respiratory viruses in all three categories
have been developed, but the preferred method depends on the individual virus.
molecular methods However, sampling and specimen collection are always an important factor.

Sampling
Appropriate sampling procedures are critical for the successful isolation and
detection of respiratory viruses. Only clinical samples are routinely collected, and
these may include nasal and throat swabs, nasopharyngeal aspirates, tracheal
aspirates, broncho-alveolar lavage fluid and serum. There are established
procedures for taking clinical samples for diagnostic virus testing and these should
be closely followed to maximise the chances of detecting the causative organism.
For example, swabs should be transported in viral transport medium and all
samples should be refrigerated if there is a delay of more than 2 hours in reaching
the laboratory.

Traditional detection and isolation methods

The principal traditional or classical techniques for detecting and isolating
respiratory viruses are indirect cell culture methods and direct electron
microscopy. Most respiratory viruses can be detected by culture in common cell
lines specific to the virus concerned. This allows both quantification and isolation of
the virus. Cell lines used include green monkey kidney, Hep-2 and Madin-Darby
canine kidney (MDCK) cells. Traditionally, cell line cultures are inoculated with the
sample and then incubated for up to a week, before examination for any evidence
of cytopathogenic effects (CPE), such as damaged cells, or the sloughing off of the
cell monolayer. Positive and negative results are then confirmed by further
passages (subculturing) in the specific cell line. Unfortunately, some respiratory
viruses, notably the influenza viruses, produce little or no CPE and confirmation
and identification of positive cell cultures was normally done using
haemadsorption, or haemagglutination tests. More recently however,
immunofluorescence, or molecular tests have been used for confirmation and
identification. This improves the sensitivity and specificity of the test and gives a
significant time saving.

Modified culture methods allowing more rapid detection are commonly used for
isolation of respiratory viruses today. Foremost among these methods is shell vial
culture, in which the clinical specimen is inoculated onto a cell monolayer that has
been grown on a cover slip in a shell vial culture tube. After inoculation the tube is
centrifuged at low speed and then incubated. The centrifugation process is thought
to make the cells more susceptible to viral infection and thus reduce the detection
time. Shell vial culture is used to identify adenoviruses, influenza A & B,
parainfluenza viruses and RSV and can achieve time savings of several days,
especially when used with immunofluorescence, or molecular confirmation
methods. Shell vial cultures containing mixed cells lines, such as R-Mix cells, can
also be used as a rapid screening test for respiratory viruses. The technique can
reliably detect more than one virus type if combined with several different specific
fluorescent antibodies.

Cell culture methods have some major drawbacks. They are often time consuming
– although this problem has been overcome to some extent by modified culture
techniques – and require highly trained staff in specialised laboratories.
Interpretation of results can also be difficult and requires experienced staff. On the
other hand, cell culture is relatively sensitive, will detect virus infectivity and
allows the result to be quantified.

Electron microscopy (EM) is now little used for the detection of respiratory viruses
in samples or in culture. However, some types, such as parainfluenza viruses, the
SARS virus and adenoviruses, have a characteristic appearance and can be
presumptively identified by direct examination, using a negative staining
technique, or by immuno-electron microscopy, where a specific antibody is used to
agglutinate, or to capture virus particles in the sample. EM depends on there being
a large number of virus particles (at least 105-106 per gram) present in the
sample.

Rapid methods
In general, rapid methods for the detection of respiratory viruses are based either
on immunological, or on molecular techniques.

Immunological methods
The majority of immunological methods for respiratory viruses are based on
antigen detection and employ immunofluorescence, enzyme immunoassay (EIA),
 or immunochromatography technologies. Direct (DFA) and indirect
immunofluorescence (IFA) methods are widely used for detection of respiratory
viruses. DFA uses a specific labelled antibody to probe for a viral antigen, while IFA
uses an unlabelled antibody, followed by a labelled antibody specific to the first
probe – allowing some amplification and improved sensitivity. Detection is
performed using fluorescence microscopy. Both DFA and IFA methods are quite
labour intensive and need highly trained staff, but are very useful techniques for
confirming and identifying viruses in clinical samples and in positive cell cultures. A
number of commercial products are available, including screening kits for the
detection of up to seven different respiratory viruses in culture.

Commercial immunoassay-based products have also been developed as easy-to-

use kits that can be applied at the point-of-care. For example, a wide range of
lateral flow immunochromatography test strips is available for the detection of
influenza A & B viruses and RSV in clinical samples. These tests offer simplicity in
operation and can be performed outside of the laboratory to provide a rapid
diagnosis. Their main disadvantages are that they are usually only qualitative
tests, have limited sensitivity and cannot distinguish infective viruses.

Immunoassay methods based on antibody, rather than antigen, detection are also
available for the serological detection of respiratory virus infection. For example,
enzyme immunoassay kits are available for the detection of serum antibodies to
adenoviruses, influenza A & B viruses, parainfluenza 1, 2 & 3 viruses and RSV.

Molecular methods
Most respiratory viruses, apart from adenoviruses and parvoviruses, are RNA
viruses, and most molecular techniques therefore rely on detecting specific viral
RNA sequences. By focusing on the viral genome, molecular methods can improve
specificity and sensitivity significantly.

The principal amplification technique used for RNA viruses is reverse-transcriptase

polymerase-chain reaction (RT-PCR). Theoretically, this technique is capable of
detecting a single virus within 24 hours. Detection of the amplified sequence may
be done at the reaction endpoint, or by continuous monitoring (real-time PCR).
Real-time PCR has the advantage of allowing the virus to be quantified.

A number of commercial applications are available for detection of respiratory

viruses, particularly influenza A & B viruses and RSV. Multiplex PCR methods have
been developed that allow the detection of more than one nucleic acid sequence,
and therefore more than one virus type, in the same assay. Other nucleic acid
amplification methods have also been developed, notably nucleic acid sequence
base amplification (NASBA). This technique is also known as isothermal
amplification, as it does not require the repeated temperature cycling used in
conventional PCR methods. A commercial application of NASBA has been
developed for metapneumovirus detection.

The advantages of PCR and related methods are considerable. The main benefit is
time saving – 24 hours to a result rather than several days for a cell culture assay.
PCR also requires less operator skill and training to carry out and can be
automated to process large numbers of samples. It is also extremely sensitive,
although it can be vulnerable to contamination and cannot distinguish infective
viruses. Most PCR methods require some costly equipment and are not suitable for
use outside the laboratory. Non-quantitative PCR results may also be difficult to
interpret, since low numbers of virus that do not signify an infection may be
detected, but this problem is largely overcome by real-time PCR.

New Minitip Flocked Swabs Improve Respiratory Virus Diagnostics

 New Minitip Flocked Swabs Improve Respiratory Virus Diagnostics,
Collect and Release More Sample Material Reducing QNS Rejection

Copan have launched three new styles of plastic minitip swabs with heads that are manufactured using unique flocking technology. This new innovation
in sample collection swabs is patented by Copan worldwide. Copan minitip Flocked Swabs are very hydrophilic and absorb and release more liquid
sample volume compared to traditional fiber winded minitip swabs. The process of flocking sprays thousands of short nylon fibers onto an applicator tip
in a perpendicular fashion. When the swab is used liquid sample is rapidly absorbed between adjacent nylon strands by capillary action and is then
instantaneously and automatically eluted once the swab tip is placed in viral transport medium.

Unlike traditional fiber winded swabs Flocked Swabs release their entire sample rapidly and immediately. The Flocked Swab tips also have a distinctive
velvet brush-like texture which is extremely advantageous for respiratory virus diagnostics as this enables the swab to more effectively dislodge and
collect virus infected cells lining the nasopharynx.

Independent studies have demonstrated that Copan Flocked Swabs collect more respiratory epithelial cells compared to regular fiber winded swabs
which provides better quality samples, significantly reduces sample rejection due to Quantity Not Sufficient (QNS) and allows earlier diagnosis of
respiratory virus infections by rapid Direct Fluorescent Antibody microscopy.

Traditional Fiber Swab Flocked Swab Flocked Swab

Sample diffuses and becomes trapped Liquid sample stays close to the surface and elutes Velvet brush-like texture efficiently dislodges and collects
in the fiber mattress out rapidly and spontaneously infected respiratory epithelial cells.

Improved sample collection with Flocked Swabs provides a more convenient, less invasive and less
traumatic system for collecting nasophyarngeal samples compared to nasal washes and nasopharyngeal
aspirates.

Copan Minitip Flocked swabs are available in three different styles Regular Minitip, Flexible Shaft
Minitip and Ultra-Thin Minitip to suit different physician preferences. All three models comprise of
plastic shafts that feature molded breakpoints which allow the physician or nurse to simply break the
swab off into a transport medium tube. The product is available sterile in peel pouches or in dry pre-
labeled transport tubes.

More information