Version: revised December 2011
1 PHLN Laboratory Definition
Diarrhoea caused by rotavirus
1) Definitive Criteria
Detection of rotavirus by antigen assay in an unvaccinated patient*
Detection of rotavirus by NADT
Detection of rotavirus by EM
Isolation of rotavirus
2) Suggestive Criteria
* Where the patient is known to have been vaccinated, or comes from a routinely vaccinated population (i.e. young children) false positve results are likely, therefore positive sample in these patients should be retested by a confirmatory assay or by PCR.
Rotavirus is one of nine genera amongst the family Reoviridae. It is, non-enveloped, 75 nm in diameter with a triple-layered icosahedral protein capsid with 11 segments of double stranded (ds) ribonucleic acid which code for six structural and six nonstructural proteins. The inner core contains VP1, VP2 and VP3, encoded by RNA segments 1-3; a middle capsid made up of VP6 encoded by segment 6, and an outer capsid of a VP7 shell encoded by segment 9 (or 7 or 8, depending on the strain) and a VP4 spike protein encoded by RNA segment 4 1. Rotaviruses are stable to heat, light and extremes of pH. The organism has a distinct ultrastructural appearance that resembles a wheel (rota, Latin). The rotavirus genus is divided into serological groups (A to E) based on the reactivity of the middle capsid protein VP6. All serogroups infect animals, but only group A commonly infects humans. While individual cases or clusters due to non-A serogroups have occurred, they are uncommon and the public health significance is uncertain. As nearly all rotavirus strains infecting humans belong to group A, the standard antigen detection and NADT in routine clinical use detect only group A. Groups B through G have been associated with human disease uncommonly and are variably called pararotavirus, atypical rotavirus, rotavirus-like rotavirus and adult diarrhoea rotavirus 2.
Rotavirus is a major cause of severe gastroenteritis in young children (with a peak incidence among children between 6 months and 2 years of age although younger children may be infected in developing countries) 2. Although discovered as a human pathogen only 32 years ago, the role of the virus in the burden of diarrhoeal disease in developed and developing countries was quickly accepted 3 and in 1985 it was estimated to be the cause of about 600,00 deaths annually in developing countries 1. It is rarely a cause of death in developed countries. It has been estimated that at least 30% of hospital admissions for acute gastroenteritis in young children are due to rotavirus infection and that it has a cyclical winter peak of disease in temperate climates. 2
Rotavirus infection may produce a spectrum of illness ranging from subclinical infection to severe and, on occasion fatal, dehydrating illness. Typically the clinical presentation is 3 days vomiting and 5 days of watery diarrhoea with moderate fever, following a 1-3 day incubation period.
Routine rotavirus vaccination of infants was introduced in Australia in 2007. Two vaccines are in use: a live attenuated vaccine containing a single serotype of serogroup A (Rotarix) and a live reassortant vaccine containing antigens of five serotypes within serogroup A (RotaTeq). 4 As serotype-specific immunity is important ongoing surveillance of serotypes is important in monitoring vaccine effectiveness. 1
Diagnosis was originally performed using electron microscopy, which is still occasionally used in centres where it is available. Routine diagnosis is now most commonly performed by antigen detection on faeces using commercially available, simple, rapid immunochromatographic dipstick style kits, latex particle agglutination and enzyme immunoassays. Reverse transcription polymerase chain reaction (RT-PCR) of faeces is available in a number of reference and research centres for diagnosis and strain characterisation, and is particularly useful for identification of outbreaks or cases occurring in recently vaccinated patients. Viral culture and serology are not routinely available and are used for research and for strain characterisation, and they do not have a role in diagnosis of acute disease.
Stool specimens collected from the first to fourth days of illness are optimal for rotavirus detection but virus may be shed for up to 3 weeks or longer depending on the severity of illness. Viral shedding usually coincides with the duration of diarrhoea but may stop prior to or after the cessation of diarrhoea.
Typing of rotavirus is based on the genetic and antigenic diversity of the 2 outer capsid proteins, VP4 and VP7. Surveillance of rotavirus G and P genotypes circulating in communities has become important with the recent introduction of vaccines for rotavirus. 1
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Rotavirus has been cultured in MA104 and primary African green monkey kidney cell cultures in roller bottles after trypsin pre-treatment. 5 This is not a practical method for routine diagnostic use as it is technically demanding, time consuming and expensive 6. The sensitivity of this technique is only 50% compared to antigen detection from stool specimens. This method will not be discussed further
3.2 Electron Microscopy
This was the original method used to detect rotavirus and it is a sensitive and specific diagnostic tool due to the high viral load in acute disease and characteristic morphology of the organism. EM has the advantage of being able to detect non-rotavirus causes of diarrhoea or infection by strains of rotavirus not detected in the antigen assays. The disadvantages of EM are the limited availability, and the cost and impracticality of screening large numbers of specimens. Titres of approximately 106/mL are required for detection by EM: shedding at these levels is typical of the first 48 hours of illness. Sensitivity may be increased 10-100 times by immune electron microscopy, which also increases specificity, but availability of reagents is relatively limited.
3.2.1 Suitable specimens
3.2.2 Test Sensitivity
80% to 90% compared to Immune Electron Microscopy. Fixation methods affect the results so proven methods must be used. Negative staining with 1% uranyl acetate at pH 4.3 or 2% phosphotungstic acid at pH 4.5 is recommended 6. EM is less sensitive than both antigen detection tests and PCR 7.
3.2.3 Test Specificity
Immune EM can be used to group or serotype the virions. Specificity approaches 100% compared to EM, EIA, Latex agglutination 6,8.
3.3 Antigen-detection methods
All commercially available kits are based on detection of the VP6 antigen of group A rotaviruses so this is the only rotavirus serogroup that is detected. Strict adherence to the manufacturers’ specifications are critical for optimal test performance 6 and, even then, it is recommended that laboratories carry out their own evaluation of the test prior to introduction as performance characteristics may be different in their patient population.
Recent experience in Australia has found a high false positive rate for antigen detection tests compared with PCR, especially in vaccinated children. This may reflect the lower pre-test probability in vaccinated patients, so positve antigen tests in these patients should be interpreted cautiously and referred for PCR testing if a false positive result is suspected. Therefore confirmation of positive results should be done where there is a low pretest probability that the person has rotavirus infection, including vaccinated patients or cases occurring outside the rotavirus “season”. This may be done by the use of an assay with confirmatory reagents 5,9 or referral of the sample to a reference laboratory for testing by PCR.
When the viral load is likely to be low e.g. late in the illness or in a rectal swab, retesting of negative samples by a more sensitive method such as PCR may be done if required.
3.3.1 Enzyme immunoassay (EIA)
126.96.36.199 Suitable specimens
188.8.131.52 Test sensitivity and specificity
The performance of each kit depends on the “gold” standard method used for comparison and is often based on carefully controlled clinical trials. In these situations sensitivities and specificities of >95% are possible. In actual practice these characteristics may be less than 90% and different assays may yield different results when used to test the same specimen.
184.108.40.206 Suitable test acceptance/validation criteria
Follow the manufacturers guidelines if using a commercial kit and use the “kit” confirmatory test for validation 10.
220.127.116.11 Suitable internal controls
Known positive (external) controls should be processed with each new kit
18.104.22.168 Suitable external QC programs
Need to be developed.
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3.3.2 Latex Agglutination
22.214.171.124 Suitable specimen
Fluid stool. (Rectal swabs are not suitable as they often have a low viral load)
126.96.36.199 Test sensitivity and specificity
This is a practical and cost effective way to test for group A rotavirus. The specificity is generally high so false positive results are unlikely unless the patient has a low pretest probability (see 3.3 above). Sensitivity is less than PCR, equivalent to or lower than EIA, and greater than EM 7,11. Non specific agglutination can be a problem.
188.8.131.52 Suitable test acceptance/validation criteria
Follow the manufacturers’ guidelines if using a commercial kit
184.108.40.206 Suitable internal controls
Known positive (external) controls should be processed with each new kit (batch)
220.127.116.11 Suitable external QC programs
Need to be developed
3.3.3 Immunochromatographic tests
These are relatively cheap, simple to use, dip-stick style assays. Each dipstick usually contains a strip of immobilised monoclonal antibody to VP6 and a separate strip of monoclonal antibody to the hexon antigen of adenovirus for simultaneous detection of both viruses.
3.3.4 Special considerations
Wilhelmi et al (2001)9 compared the performance characteristics of three different commercial rapid detection kits, resolving discordant results by RT-PCR. An EIA kit, latex agglutination kit and immunochromatographic kit were found to have, respectively, sensitivities of 96%, 68% and 99%; specificities of 99%, 99% and 96%; positive predictive values of 98%, 96% and 92%; and negative predictive values of 98%, 88% and 99%. However, these evaluations were performed in unvaccinated populations and may not be the same in contemporary Australia.
False positive results in rapid detection tests may be due to other viruses such as reovirus1 or other undetermined causes.
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3.4 Nucleic acid detection tests (NADT)
Reverse transcriptase polymerase chain reaction (RT-PCR) is the most sensitive method available to detect rotavirus and can be used. Most are designed to detect serogroup A rotaviruses only, though some assays have been designed for non-A rotavirus detection. One study found that real-time RT-PCR was 2-4 logs more sensitive than either conventional RT-PCR or nested PCR 12. PCRs for rotavirus detection are often directed at the gene encoding the VP6 inner capsid protein while genotyping PCR assays usually target VP7 and VP4, using consensus primers for each gene that are designed to detect the common human genotypes. PCRs directed at genotype-specific sequences in the VP7 and VP4 genes can then be performed to determine genotype.
3.4.1 Suitable sample
3.4.2 Test sensitivity
RT-PCR is the gold standard method for the detection of rotavirus. Sensitivity depends on the ability of the consensus primers to detect all possible genotypes circulating in the population and the RT- PCR method used. Most tests in use detect only serogroup A rotaviruses
3.4.3 Test specificity
Highly specific for rotavirus.
3.4.4 Predictive values and relevant populations
Positive and negative predictive values are high if primers are designed to detect currently circulating rotavirus subtypes.
3.4.5 Suitable test acceptance/validation criteria.
In house-assays should be validated according to the NPAAC Requirements for the Validation of In-house In Vitro Diagnostic Devices (IVDs).
3.4.6 Suitable internal controls
Internal and external controls should be included as described in the NPAAC Laboratory Accreditation Standards and Guidelines for Nucleic Acid Detection Techniques.
3.4.7 Suitable external QC programs
Need to be developed
3.4.8 Special issues
The NADT for rotavirus detection are highly sensitive and specific, but studies have shown that, compared with antigen detection tests, they are more likely to detect the virus in asymptomatic patients. 13 Therefore detection of rotavirus by PCR does not necessarily mean that it is causing disease, and a positive result must be interpreted taking into account the clinical and exposure history of the patient.
3.5 Typing Methods
Rotaviruses are classified according to the genetic and antigenic diversity of two outer capsid proteins, VP4 designating the P (protease sensitive) subtypes encoded on RNA segment 4, and VP7 encoded on segment 7, 8 or 9 designating G (glycoprotein) subtypes. The two genes segregate independently and various G and P combinations are seen in natural infections. 1 Both proteins are immunogenic and induce neutralizing antibodies. Epidemiological studies have shown that genotypically mixed infections can occur.
EIA using monoclonal antibodies to G serotypes (VP7) has been the most common method for serotyping rotavirus directly in faecal specimens. In 1990, a multiplexed, hemi-nested RT-PCR genotyping assay based on VP7 sequences was described and shown to correlate well with MAB-based G-serotyping 14. While a similar genotyping assay was directed towards identifying the VP4 genotypes.
In Australia, surveillance for rotavirus genotypes is performed by Enteric Virus Research Group, Murdoch Childrens Research Institute, Royal Children’s Hospital in Melbourne. They use 2 hemi-nested multiplex RT-PCR assays to characterize any specimens forwarded as rotavirus into the specific G and P genotypes. In instances where a sample is non-typeable for G and/or P type, the sample will be analysed by sequence analysis of the gene of interest. Annual reports of their typing results are published in Communicable Diseases Intelligence 15.
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3.6 Serological tests
3.6.1 Suitable specimens
Clotted blood 1 week after illness. Stool at the commencement and one week after illness.
3.6.2 Sensitivity and specificity
These tests have been used for epidemiologic studies of rotavirus but high seropositivity for rotavirus group A in the majority of older children and adults has precluded the use of serology as a diagnostic tool. Rotavirus IgM may be detected in serum about a week after illness commences but this is not useful where stool is available to be tested on presentation. Serum IgA has been the most widely used marker for rotavirus infection in vaccine trials, and is the best serologic indicator of reinfection. There are no commercial kits available to measure rotavirus antibodies. This method will not be discussed further.
- Fischer TK and Gentsch JR. Rotavirus typing methods and algorithms. Rev. Med. Virol. 2004; 14:71-82.
- Steele JC. Rotavirus. Clin Lab Med 1999; 19(3): 691-703
- Carlin JB, Chondros P, Masendycz P, Bugg H, Bishop RF, Barnes GL. Rotavirus infection and rates of hospitalisation for acute gastroenteritis in young children in Australia, 1993-1996. MJA 1998; 169: 252-256
- Generic protocol for monitoring impact of rotavirus vaccination on gastroenteritis disease burden and viral strains. World Health Organization 2008. Available at (www.who.int/vaccines-documents/)
- Sherlock CH, Brandt CJ, Middleton PJ et al. Laboratory diagnosis of viral infections producing enteritis. Washington DC Cumitech 26 American Society for Microbiology 1989
- Kapikian AZ, Chanock RM. Rotaviruses. In Fields BN, Knipe DM, Howley PM et al eds: Fields Virology ed 3, Philadelphia, Lippincott-Raven Publishers 1996
- Logan C, O’Leary JJ, O’Sullivan N. Real-time reverse transcription-PCR for detection of rotavirus and adenovirus as causative agents of acute viral gastroenteritis in children. J Clin Microbiol. 2006; 44:3189-3195.
- Doane FW, Anderson N. Retroviridae. In Electron Microscopy in diagnostic virology; a practical guide and atlas. New York, Cambridge University Press, 1987.
- Wilhelmi I, Colomina J, Martin-Rodrigo D, Roman E, Sanchez-Fauquier A. New immunochromatographic method for rapid detection of rotaviruses in stool samples compared with standard enzyme immunoassay and latex agglutination techniques. Eur J Clin Microbiol Infect Dis. 2001; 20:741-3
- LeBaron CW, Allen JR, Herbert M et al, Outbreaks of summer rotavirus linked to laboratory practices. Paediatric Inf Dis.J. 1992; 11:773
- Raboni SM, Nogueira MB, Hakim VM, Torrecilha, Lerner H, Tsuchiya LR. Comparison of latex agglutination with enzyme immunoassay for detection of rotavirus in fecal specimens. Am J Clin Pathol. 2002; 117:392-394
- Pang XL, Lee B, Boroumond N, Leblanc B, Preiksaitis JK, Yulp CC. Increased detection of rotavirus using reverse transcription-polymerase chain reaction (RT-PCR) assay in stools specimens from children with diarrhea. J Med Virol 2004 72:496-501.
- Amar CF, East CL, Gray J, Iturriza-Gomara M, Maclure EA, McLauchlin J. Detection by PCR of eight groups of enteric pathogens in 4,627 faecal samples: re-examination of the English case-control Infectious Intestinal Disease Study (1993-1996). Eur J Clin Microbiol Infect Dis. 2007;26:311-23.
- Gouvea JR, Glass RI, Woods K, Taniguchi K, Clark HF, Forrester B, Fung ZY. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J clin Microbiol 1990 28:276-282.
- Kirkwood CD, Bogdanovic-Sakran N, Cannan D, Bishop RF, Barnes GL. National Rotavirus Surveillance program Annual Report. 2006. Commun Dis Intell. 2006; 30: 133-136.