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Laboratory case definitions

Diarrhoea Caused by Rotavirus Laboratory Case Definition (LCD) Summary

The Public Health Laboratory Network have developed a standard case definition for the diagnosis of diseases which are notifiable in Australia. This page contains the laboratory case definition for rotavirus.

Authorisation: PHLN

Consensus Date: 25 October 2006


1 PHLN Laboratory Definition

1.1 Condition

Diarrhoea caused by Rotavirus

1.1.1 Tests

    1) Definitive Criteria
    Detection of rotavirus by antigen assay
    Or
    Detection of rotavirus by NAA
    Or
    Detection of rotavirus by EM
    Or
    Isolation of rotavirus
    2) Suggestive Criteria
    Nil

2 Introcuction

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 4.4 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. Most rotavirus strains infecting humans belong to group A, and the standard antigen detection assays in routine clinical use detect only group A. Groups B through G have been associated with human disease less commonly and are variably called pararotavirus, atypical rotavirus, rotavirus-like rotavirus and adult diarrhoea rotavirus.14 All serogroups infect animals.

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).14 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 accepted2 and in 1985 it was estimated to be the cause of 870,000 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.14

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.

Diagnosis was originally performed using electron microscopy, which is still occasionally used in centres where it is available. Routine diagnosis is now routinely performed by antigen detection on faeces using commercially available, simple, rapid immunochromatographic dipstick style kits which have superseded the earlier latex agglutination and enzyme immunoassays. Reverse transcription polymerase chain reaction (RT-PCR) of faeces is available in some reference and research centres for diagnosis, and is particularly useful for identification of outbreaks due to serogroups other than group A. Viral culture and serology are available but 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 depending on the severity of illness. Viral shedding usually coincides with the duration of diarrhea but diarrhea can continue for an additional few days.

Subtyping is based on the antigenically diverse VP4 and VP7 proteins. Surveillance of rotavirus subtypes circulating in communities has become important with the recent development of vaccines for rotavirus.
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3 Tests

3.1 Culture

Rotavirus has been cultured in MA104 and primary African green monkey kidney cell cultures in roller bottles after trypsin pre-treatment.13 This is not a practical method for routine diagnostic use as it is technically demanding, time consuming and expensive.7 The sensitivity of this technique is only 75-90% 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 following 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

Fluid stool

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 7. EM is less sensitive than both antigen detection tests and PCR.10

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.3,7

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 detected. Other methods such as NAA or EM are needed when searching for non group A rotavirus in humans or animals.

3.3.1 Enzyme immunoassay (EIA)

3.3.1.1 Suitable specimens
Fluid stool

3.3.1.2 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. Strict adherence to the manufacturers’ specifications are critical for optimal test performance.7 An assay with confirmatory reagents should be selected and confirmation of unusual results undertaken; e.g. “summer” rotavirus positive tests in temperate climates.13,15

3.3.1.3 Suitable test acceptance/validation criteria
Follow the manufacturers guidelines if using a commercial kit and use the “kit” confirmatory test for validation.9

3.3.1.4 Suitable internal controls
Known positive (external) controls should be processed with each new kit

3.3.1.5 Suitable external QC programs
Need to be developed.
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3.3.2 Latex Agglutination

3.3.2.1 Suitable specimen
Fluid stool. (Rectal swabs are not suitable as they often have a low viral load)

3.3.2.2 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. Sensitivity is less than PCR, equivalent to or lower than EIA, and greater than EM.10,12 Non specific agglutination can be a problem. When the viral load is likely to be low e.g. late in the illness or in a rectal swab, negative results should be confirmed by another method.13

3.3.2.3 Suitable test acceptance/validation criteria
Follow the manufacturers’ guidelines if using a commercial kit

3.3.2.4 Suitable internal controls
Known positive (external) controls should be processed with each new kit (batch)

3.3.2.5 Suitable external QC programs
Need to be developed

3.3.3 Immunochromatographic tests

These relatively cheap, simple to use, dip-stick style assays are now used almost universally for the routine diagnosis of rotavirus in pathology laboratories. 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)15 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%, 99%, specificities of 99%, 99% 96%, positive predictive values of 98%, 96% 92% and negative predictive values of 98%, 88% and 99%.

False positive results in rapid detection tests may be due to other viruses such as reovirus.4

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3.4 Nucleic acid assays (NAA)

Reverse transcriptase polymerase chain reaction (RT-PCR) is the most sensitive method available to detect rotavirus and can be used, with the appropriate choice of primers, for nonA rotavirus detection. Of the NA assays, real-time RT-PCR has been shown to be 2-4 logs more sensitive than either conventional RT-PCR or nested PCR.11 PCR detection assays commonly target VP7 using consensus primers, designed to detect all subtypes. Subtyping of VP7 can then be performed using specific primers.

3.4.1 Suitable sample

Fluid stool

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.

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

3.5 Subtyping 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.4 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 subtyping 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.6

In Australia, surveillance for rotavirus subtypes is performed by Enteric Virus Research Group, Murdoch Children’s Research Institute, Royal Children’s Hospital in Melbourne. They use EIA to screen faecal specimens referred to them using a panel of MAb’s to G1, G2, G3, G4 and G9, followed by RT-PCR to characterize any specimens not reacting with the panel. Polyacrylamide gel electrophoresis (PAGE) is used to classify rotavirus strains into electrophoretypes. Annual reports of their typing results are published in Communicable Diseases Intelligence.8
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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. Coproconversion may be the best serologic indicator of reinfection. There are no commercial kits available to measure rotavirus antibodies. This method will not be discussed further.

References

1. Bern C, Glass RI. Impact of diarrhoeal diseases worldwide. In Kapikian AZ ed, Viral Infections of the gastrointestinal tract. New York: Marcel Dekker, 1994: 1-26

2. 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

3. Doane FW, Anderson N. Retroviridae. In Electron Microscopy in diagnostic virology; a practical guide and atlas. New York, Cambridge University Press, 1987.

4. Fischer TK and Gentsch JR. Rotavirus typing methods and algorithms. Rev. Med. Virol. 2004; 14:71-82.

5. Giordano MO, Martinez LC, Ferreyra LJ, Isa MB, Paez Rearte M, Pavan JV, Nates SV. J Clin Virol 2005; 32:71-72

6. 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.

7. Kapikian AZ, Chanock RM. Rotaviruses. In Fields BN, Knipe DM, Howley PM et al eds: Fields Virology ed 3, Philadelphia, Lippincott-Raven Publishers 1996

8. 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.

9. LeBaron CW, Allen JR, Herbert M et al, Outbreaks of summer rotavirus linked to laboratory practices. Paediatric Inf Dis.J. 1992; 11:773

10. 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;

11. 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.

12. 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

13. Sherlock CH, Brandt CJ, Middleton PJ et al. Laboratory diagnosis of viral infections producing enteritis. Washington DC Cumitech 26 American Society for Microbiology 1989

14. Steele JC. Rotavirus. Clin Lab Med 1999; 19(3): 691-703

15. 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