Identifying viruses can be child's play

Chris Waghorn discusses some of the most common enteric and respiratory viral infections in children and some rapid and convenient methods for detecting these viruses and distinguishing them from other infectious agents

Some common viral infections in children, such as chickenpox, measles and mumps, have very distinctive signs and symptoms. Other common childhood viral infections, such as those caused by enteric and respiratory viruses, are harder to differentiate from infections caused by other pathogens, such as bacteria and parasites, since their clinical presentation is often indistinguishable^1,2,3.

Viral gastroenteritis
Viral gastroenteritis can be caused by a number of different viruses. Rotavirus, for example, can infect any age group but it predominantly causes diarrhoea in infants and children under five. Enteric Adenovirus and Astrovirus infections are also most common in young children, whereas Norovirus is more likely to cause diarrhoea in older children and adults³.

Rotavirus and Astrovirus infections tend to be seasonal, occurring during the winter months in temperate regions and during periods of low humidity or the rainy season in tropical regions, whereas enteric Adenovirus infections occur throughout the year.

Most people will fully recover from viral gastroenteritis but, if fluids are not adequately replaced, there is a danger of dehydration. Few people die as a result of viral gastroenteritis in developed countries, however it is more frequently fatal in less developed countries. Around the world, diarrhoea accounts for 1.6-2.5 million deaths every year¹.

Rotavirus
Rotavirus is so named because of its characteristic wheel-like appearance when viewed by electron microscopy (Figure 1). There are 7 Rotavirus groups, A-G, with group A being the most common worldwide¹.

Figure 1. Rotovirus viewed by electron microscope

Rotavirus is the most common cause of infantile diarrhoea^4,5,6 and most children will have been infected in the first few years of life. In the United States, it is estimated that 3 million cases of diarrhoea are attributed to this virus every year, resulting in 55,000 cases being hospitalised due to dehydration⁵, and in England and Wales 18,000 children are hospitalised annually as a result of Rotavirus infection⁴. Around the world, severe Rotavirus infection results in over 600,000 deaths in children every year, particularly in developing countries⁶.

Rotavirus infection causes fever, nausea, vomiting, abdominal cramps and frequent watery diarrhoea, and may result in dehydration. The virus is extremely contagious and is transmitted via the faecal oral route. Furthermore, Rotavirus is very stable in the environment and so it can also be transmitted from contaminated food, water and surfaces6. Frequent hand washing may help to prevent the spread of disease and in hospitals isolation of infected patients and strict infection control procedures are required6 as vulnerable and immuno-compromised patients are more at risk of developing serious disease.

The incubation period for Rotavirus infection is 2-3 days and it is usually self-limiting, lasting for up to 9 days⁵. Treatment is supportive, with replacement of fluids and electrolytes.

Astrovirus
When viewed by electron microscope, Astroviruses often have a characteristic ‘star’ in the centre of each particle. There are 8 Astrovirus types, of which type 1 is the most prevalent¹.

Astrovirus is increasingly recognised as a common cause of paediatric diarrhoea1⁷. Symptoms of Astrovirus infection, including nausea, vomiting, abdominal pain, diarrhoea and fever, are less severe than Rotavirus infection and medical attention is not usually required⁷. The disease is usually self-limiting and a complete recovery normally occurs within 2-3 days.

Those most at risk of symptomatic infection include children, the immuno-compromised (e.g. HIV positive individuals, bone marrow recipients) and the elderly¹. If dehydration does occur, oral or intravenous rehydration may be required. A childhood infection is thought to provide some immunity⁷.

Adenovirus                                                             Figure 2. Adenovirus
Adenoviruses are a group of hardy, medium-sized icosohedral viruses (Figure 2) of which there are 49 serotypes known to cause human disease⁸. The viruses infect the membranous tissues of the respiratory tract, eyes, intestines and urinary tract⁹, causing illnesses, such as respiratory disease, gastroenteritis, conjunctivitis and cystitis⁸.

Serotypes 40 and 41 are common causes of viral gastroenteritis in children^8,10. Most enteric Adenovirus infections are mild and do not require therapy other than oral rehydration^8,9. Symptoms are similar to Rotavirus infection - watery diarrhoea, vomiting, headache, fever and abdominal cramps - but the incubation period is longer (up to 14 days⁹) and the symptoms can last for up to 2 weeks⁹.

Adenovirus particles are unusually stable to chemical and physical agents, allowing prolonged survival in the environment. The virus is highly infectious and spreads through contact with faecal contamination.

Infections in paediatric and neonatal wards can spread rapidly, resulting in prolonged hospitalisation of patients and, occasionally, ward closures for decontamination.  Strict infection control procedures are effective in stopping nosocomial outbreaks⁸.

Viral respiratory infections
Infections of the upper respiratory tract are common in children and may be caused by viruses, bacteria, fungi or parasites. Occasionally, infection will progress to the lower respiratory tract and can cause serious illness, such as bronchiolitis or pneumonia. Around 20% of all deaths in children under five are caused by acute lower respiratory tract infections and 90% of these deaths are due to pneumonia². Bacterial infections may be treated with antibiotics but it is not possible to differentiate between bacterial and viral infections based on clinical signs or radiology².

Bronchiolitis is when the tiny airways leading to the lungs (bronchioles) become inflamed, swollen and filled with mucous, making it hard for the child to breath. It typically occurs in infants and very young children (under two), as their bronchioles are more easily blocked¹¹. Pneumonia occurs when the lungs become infected.

Viral respiratory tract infections are often mild but occasionally they may require hospitalisation. Initially an infection may present with cold-like symptoms, which may progress to coughing and wheezing. In severe cases, the child may suffer from respiratory difficulties, including rapid, shallow breathing, rapid heart beat, abnormal retractions, flaring nostrils, irritability, difficulty sleeping, signs of fatigue and, in the worst cases, cyanosis (blue lips and fingernails), dehydration and respiratory failure.

Suggested treatments include rest, a cool-mist vaporiser (to sooth airways and relieves cough) and plenty of fluids to keep secretions watery and easy to clear. Hospitalised children may need humidified oxygen, medicines to open airways and, in extreme cases, mechanical ventilation^12,13.

Viruses that can cause respiratory tract infections in children include Respiratory Syncytial Virus (RSV) and Adenovirus¹¹.

Respiratory Syncytial Virus (RSV)
RSV particles are of variable shape and size and are relatively unstable in the environment¹³.

RSV infects the lungs and breathing passages. It is the most common cause of severe respiratory disease, such as bronchiolitis and pneumonia, in children under two^{13, 14} and is the most common cause of hospitalisation due to acute respiratory disease in young children¹⁴. Those most at risk of complications include premature babies and those with pre-existing lung, heart or immune problems. In the United States, this virus causes 125,000 hospitalisations and 2,500 deaths each year¹².

Infections are most prevalent in the winter months. The virus is highly contagious and is transmitted from person to person through contact with oral or nasal fluids, or contaminated surfaces. It spreads efficiently among children, particularly in child care centres and hospitals, with the highest rates of infection in infants of 2-6 months¹². The incubation period is 4-6 days and symptoms may last for up to three weeks.

Adenovirus
As discussed earlier, Adenovirus disease varies with the infecting serotype. It most commonly causes respiratory disease, usually in infants and young children, accounting for 10% of acute respiratory infections in children⁹.

Unlike enteric Adenovirus infection, which occurs throughout the year, Adenovirus respiratory infections are more common from late winter to early summer. Most infections are mild and do not require therapy other than symptomatic treatment. Symptoms include fever, pharyngitis, rhinitis, cough, swollen glands and infection may lead to acute otitis media or lower respiratory tract infections, such as bronchiolitis, croup or viral pneumonia.

The incubation period is 2-14 days and symptoms can last for up to 4 weeks⁹. Adenovirus is highly contagious and can spread rapidly through contact with respiratory secretions.

Detecting enteric and respiratory viruses
There are a number of reasons that clinicans and investigators may seek to identify the cause of infection, for example:
1. Since symptoms of respiratory and enteric viral infections are hard to distinguish from those caused by other pathogens (bacterial, fungal or parasite), identification of the infectious agent helps to direct treatment options.
2. These viral infections are highly infectious and can spread through hospital wards quickly and efficiently. Rapid identification of the infectious agent helps to identify those patients that should be isolated and allows infection control procedures to be initiated promptly.
3. Identification of the infectious agent is useful for epidemiological purposes and allows clinicians to track and predict problems.

In the past, identification of viral infections required samples to be sent to reference laboratories, since the methodology was not suitable for, or available in, routine microbiology laboratories. The techniques were time consuming and laborious, requiring specialised equipment and expertise.

Now, however, there are a variety of commercially available kits for the detection of specific viral antigen in clinical samples. These kits provide rapid, same-day results and are extremely easy to use, allowing even the smallest laboratories to offer a virus identification service.

Microplate assays
Microplate assays, such as the ProSpecT range (Oxoid Limited, Basingstoke, UK) provide a convenient and easy to use method for the detection of specific antigen directly from clinical faecal specimens (Figure 3). The ProSpecT tests include assays for the detection of Rotavirus (product code: 24920967), Adenovirus (product code: 2494596) and Astrovirus (product code: 2494096).

Figure 3. ProSpecT microplate assay

Diluted specimen is added to the wells of the microplate and, following appropriate room temperature incubations, are washed with conjugate and substrate prior to stopping the reaction. Within just 2 hours highly sensitive and specific results can be read easily - visually or spectrophotometrically.

These viral assays form part of a range of ProSpecT assays for enteric pathogens, which also includes assays for bacterial and parasite enteric pathogens. Each assay, irrespective of the micro-organism it detects, uses the same procedure and reagents, allowing several strips for different enteric pathogens (viral, bacterial or parasite) to be set up and run simultaneously. The results provide important information, quickly and reliably, for guiding therapeutic decisions.

Lateral flow tests
Lateral flow tests, such as the Xpect range (Oxoid Limited, Basingstoke, UK) offer incredible speed, with results in just 15 minutes. This range includes a rapid test for RSV (product code: 24601) and also includes a rapid test for the simultaneous detection and differentiation of Influenza types A and B (product code: 24600).

Diluted specimen is simply added to the sample window and within 15 minutes the test has developed and the result can be read visually (Figure 4). If a line appears in at the test and control points, the test is positive. If a line appears at the control point but not at the test point, the test is negative. The internal control ensures that the test is functioning correctly and if a line does not appear at the control point the test should be repeated.

The Xpect range also offers the flexibility of several specimen and transport media options.

Latex agglutination tests
A third rapid method for the direct detection of viral antigen from clinical specimens is latex agglutination, such as the Rotavirus Latex Agglutination Test (product code: 30950401) available from Oxoid. This test uses sensitised latex reagent to detect the presence of Rotavirus antigen in faecal specimens. It is extremely easy to perform and provides easily interpreted results within minutes.

In the presence of Rotavirus antigen, the latex particles agglutinate to form clearly visible clumps. In the absence of specific antigen the particles remain in smooth suspension and the test is negative.

Local virology testing
Childhood viral infections of the respiratory and digestive tracts are extremely contagious and have the potential to spread rapidly through groups of young children, for example in childcare facilities and in neonatal and paediatric wards.

In the hospital setting, such infections may result in longer stays and complications are more likely to develop because of underlying conditions and vulnerability of the patients. Therefore rapid diagnosis is extremely important to ensure patients receive correct treatment and to allow appropriate infection control measures to be taken as soon as possible. In severe outbreaks ward closures may be required for decontamination.

Rapid diagnosis is also extremely important for severe cases that are admitted from the community, ensuring that the true cause of infection is determined and the proper treatment is given.

The speed and simplicity of the tests described allow hospitals of any size to obtain same-day results. Furthermore, they are simple and convenient to perform and do not require specialised equipment or expertise.

By Chris Waghorn, product manager, Oxoid Ltd, Basingstoke, Hants. 

References
1. Hill-King, L. (2005) Biomedical Scientist, May 2005.
2. WHO Fact Sheet, Acute Respiratory Infections in Children.
3. Centers for Disease Control and Prevention (CDC), Respiratory and Enteric Viruses Branch.  Viral Gastroenteritis FAQ.
4. Health Protection Agency, Health Topics, Rotavirus.
5. KidsHealth.org, Rotavirus.
6. CDC, Respiratory and Enteric Viruses Branch.  Disease Information: Rotavirus.
7. Health Protection Agency, Health Topics, Astrovirus.
8. CDC, Respiratory and Enteric Viruses Branch.  Disease Information: Adenovirus.
9. KidsHealth.org, Adenovirus
10. Health Protection Agency, Health Topics, Adenovirus.
11. KidsHealth.org, Bronchiolitis.
12. KidsHealth.org, Respiratory Syncytial Virus.
13. CDC, NCID, Respiratory and Enteric Viruses Branch.  Disease Information: Respiratory Syncytial Virus.
14. Health Protection Agency, Health Topics, Respiratory Syncytial Virus (RSV).