Journal of Medical Virology 1:69-77 (1977)

A Study of the Prevalence of Rotavirus Infection in Children With Gastroenteritis Admitted to an Infectious Diseases Hospital C.J. Birch, F.A. Lewis, M.L. Kennett, M. Homola, H. Pritchard, and I.D. Gust Virology Department, Fair field Hospitai for CommunicabI e Diseases, Melbourne, Vietoria, Australia

In a 1 2 month survey of infants and children with gastroenteritis admitted to Fairfield Hospital, Melbourne, rotavirus was found in approximately 42% of patients. This virus was detected more often during the winter months, particularly in children aged between 12 months and 3 years. Detection of rotavirus by electron microscopy was found to be more sensitive than by counterimmunoelectrophoresis. Routine bacterial and viral studies revealed that bacterial pathogens and common enteric viruses were associated with relatively few cases of gastroenteritis. There is little doubt that rotavirus is the most important aetiological agent of acute gastroenteritis in young children in Melbourne. Key words: rotaviru., gastroenteritis,children, electron microscopy

INTRODUCTION

In 1973, Davidson et al. detected reovirus-like particles in the duodenal epithelium and faeces of six infants with acute nonbacterial gastroenteritis. Subsequent studies (Tan et al., 1974) have established that this agent, now commonly referred to as rotavirus, is a major cause of diarrhoea in children in many countries. At Fairfield Hospital, Melbourne, between 800 and 1,000 patients with gastroenteritis are admitted each year, a large proportion of whom are infants and young children. Until 1975, enteric pathogens were detected in less than 20% of these patients. In this report we present the results of a 12 month study carried out on children admitted to the gastroenteritis ward of this hospital in which, in addition to routine bacterial and viral studies, evidence of rotavirus infection was sought.

Received October 19, 1976 Address reprint requests to Dr. Ian Gust, Fairfield Hospital for Communicable Diseases, Melbourne, Victoria, Australia.

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@ 1977 Alan R. Liss, Inc., 150 Fifth Avenue, New York, NY 10011

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MATERIALS AND METHODS Patients and Specimens

Between March 1, 1975, and February 29, 1976,690 infants and young children with acute diarrhoea were available for study, and faecal specimens were obtained from 400 (57.9%). The specimens were obtained between 1 and 9 days after the onset of diarrhoea. Electron Microscopy (EM)

Faecal specimens were stored at 4°C until they could be processed. Solid specimens were prepared as a 20% suspension in Hank's balanced salt solution (using an MSE homogenizer, Model 7700-A). Liquid specimens were processed without homogenization or dilution. An equal volume of trifluorotrichloroethane (Arklone P, ICI) was added and the mixture was shaken for at least 30 sec with a vortex mixer (Lab. Line Instruments, Inc.) and then clarified by centrifugation at 10,000 g for 30 min in an International PR2 refrigerated centrifuge. Four ml of the resulting supernate was layered onto 1 ml of 45% sucrose (wlv) in 0.002 M Tris buffer (pH 7.4) and centrifuged at 100,000 g for 75 min in a Beckman L2 ultracentrifuge. The supernate was then discarded and the pellet resuspended in 0.1 ml of 0.002 M Tris. A drop of this suspension was placed on a Formvar-carbon-coated grid, which was then negatively stained with ammonium molybdate (4%, pH 7.0) and examined in a Philips 301 electron microscope. The number of particles in 4 intact grid squares of a 400 mesh grid were counted and ratings were given as follows: > 100 particles 4+, 51-99 particles 3+, 21-50 particles 2 t , and 1-20 particles 1t. Virus Isolation

A portion of each specimen was processed by a method previously described (Kennett et al., 1972) and then inoculated into duplicate phials of primary cynomolgus monkey kidney, MEK-3 (Ellis et al., 1975), HeLa, and Borrie (Lewis and Kennett, 1976) cells. The cell cultures were incubated at 34°C on roller drums rotating at 10 revolutions per hr and observed every 3 to 4 days for cytopathic effect (CPE). When a CPE developed, the supernatant fluid was collected and stored at 4OC whilst identifcation tests were in progress. Bacteriology

Separate specimens were transported to the laboratory in Carey-Blair transport medium (Blair et al., 1970), plated onto deoxycholate citrate agar (DCA) and MacConkey and thiosulphate-citrate-bile salts sucrose agar (TCBSA), and inoculated into selenite broth enrichment medium. After overnight incubation, differential biochemical testing was carried out on all nonlactose fermentors from the plates, and the selenite broth was subcultured onto DCA. Salmonella and Shigella strains were confirmed by slide agglutination tests and after grouping were sent to a reference laboratory for further identification. Counterimmunoelectrophoresis (CIEP)

Detection of rotavirus by CIEP was carried out by a method described by Nastasi et al., (1972), except that 0.8%agarose replaced 1% agarose. No staining techniques were utilized and the plates were read after 9 0 min. The rabbit antirotavirus serum used was kindly provided by Dr. H. Watanabe of the School of Microbiology, University of Melbourne.

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RESULTS Patients and Specimens

Over the 12 month period 679 infants and young children with diarrhoea were admitted to hospital and an additional 11 children developed diarrhoea whilst in hospital for other reasons. Acute phase faecal specimens were obtained from 400 (57.9%) of these 690 patients. The number of infants and children with diarrhoea treated each month is shown in Fig. 1 and the agents identified in their faeces in Fig. 2. Agents Identified Rotavirus. Human rotavirus was detected by EM in 167 (41.7%) of the 400 faecal specimens. Under the electron microscope the virus was spherical and had a characteristic double-shelled capsid structure. The diameter of the particles averaged 73 nm with an inner-core diameter of 44 nm. They usually occurred singly with very distinct edges, giving the impression that there was little or no surface antibody coating them (Fig. 3). Pathogenic bacteria. Salmonella and Shigella species were recovered from the faeces of 68 (17%) patients. The most common strains encountered were Salmonella typhimurium and Shigella sonnei. Three-quarters of the Shigella isolates were associated with an outbreak of gastroenteritis in a children’s institution. Other viruses. Viruses other than rotavirus were isolated in cell culture andlor identified by EM in 63 (15.8%) patients (see Table I). Of 22 adenoviruses isolated, 18 were types prevalent in Melbourne at the time (types 1 , 2 , 5 , and 7), 1 was an uncommon type (type IS), and 3 have not been identified. Adenoviruses were visualized by EM

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Fig. 1. Number of children admitted t o the gastroenteritis ward at Fairfield Hospital each month between March 1975 and February 1976.

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Fig. 2. Agents identified in the faeces of children with gastroenteritis from March 1975 to February 1976.

Fig. 5. Typical appearance of a rotavirus-positive specimen ( X 142,800).

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TABLE I. Results of Bacterial and V i a l Studies on 400 Children With Diarrhoea Number of admissions Number of specimens received and processed Rotavirus-positive(by EM) Other viruses - by EM and/or isolation Enteroviruses 24 Adenoviruses 35 Reoviruses 4 Salmonella Shigella No pathogens identified

690 400 167 (41.8%) 63

39 (9.8%) 29 (7.3%) 129 (32.2%)

in the faeces of another 13 patients, but isolation attempts were unsuccessful. Enteroviruses were isolated from 24 patients; 9 were polioviruses, presumably Sabin strains, and the remainder (Coxsackie type R 4 and Echovirus types 7, 16,21, and 22), were strains which were circulating in the community at the time. Keoviruses were isolated from 4 patients - 2 were type 1 and 2 were type 2. Mixed infections. In 13 of the 167 specimens containing rotavirus, one or more other potential pathogens were found. Rotavirus was associated with Salmonella species on 4 occasions, with Shigella on 4, with enteroviruses on 3, and with adenoviruses on one. One child had rotavirus, Shigella sonnei, and Echovirus type 16 in a single faecal specimen. Rotavirus Infection Seasonal distribution. The distribution of rotavirus infections showed a marked seasonal variation, with a peak in the cooler months of April to August and a low in the warmer months of November t o February (Table I1 and Fig. 4). Human rotavirus was the most common pathogen identified in all but three months, November, December, and January,

Age distribution. The age distribution of children with rotavirus-associated diarrhoea is shown in Fig. 5. The virus was detected in children as young as 7 days and as old as 10 years of age, but was most commonly found in children between 6 months and 3 years of age. It was detected in 31% of chldren with diarrhoea less than 6 months of age, in 50% between the ages of 6 months and 3 years, and in 12.5% of children aged 4 years and over. Detection of Virus by EM and CIEP

Faecal specimens from 384 of the 400 patients studied were tested for the presence of rotavirus by both EM and CIEP. The results are shown in Tables I11 and IV. Virus was detected by one or both methods in 162 (42.2%) specimens. Of the 160 specimens known to be positive by EM, 62 (38.8%) were also positive by CIEP. The success of CIEP appeared to be related to the number of virus particles. CIEP detected 54.4% of those specimens rated 4+ by EM, 41.6% with ratings of 2+ or 3+, and 15.2% of those rated 1+.

Number of patients admitted Number of specimens received and processed Percentage of ro tavirus-positive specimens

22 54.5

16 25.0

67.8

31

74

May

51

129 52

68

35.3

34

52 40.4

37

67

Aug. Sept. Oct.

60.3 51.0 48.1

63

126

June July

11.1

18

22

2 222

64 96

Detected by EM Detected by CIEP

42 23

4+

29 12

3+

43 18

2+

46 7

1+

160 60

Total

TABLE IV. Detection of Rotavirus by CIEP in the Stools of 160 Children in Whom the Virus Had Been Visualized by EM

CIEP positive CIEP negative

EM negative

EM positive

5.6

18

22

Nov. Dec.

TABLE 111. Comparison of the Detection of Rotavirus by EM and CIEP in the Stools of 384 Children With Diarrhoea

44

April

43

March

11.0

20

28

Jan.

13.0

23

30

41.8

400

690

Feb. Total

TABLE 11. Summary of the Number of Admissions, Specimens Processed and Percentage of Rotavirus-Positive Specimens for t h e 12 Month Period March 1975 to February 1976

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Rotavirus Infection in Hospitalized Children

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Fig. 4. Monthly distribution of rotavirus infections in children with gastroenteritis.

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Fig. 5. Age distribution of children with rotavirus-associated gastroenteritis.

DISCUSSION

Despite the importance of rotavirus diarrhoea, only three surveys (Bryden et al., 1975; Davidson et al., 1975; Kapikian et al., 1976) have been previously reported in which the pattern of infection is described over a 12 month period. The virus is clearly a major pathogen in infants and young children and in the current study it was detected in 41.7% of 400 patients. This compares with 50% o f 378 patients examined by Davidson et al. (1975) in Melbourne, and 42% of 143 examined by Kapikian et al. (1976) in the United States. In addition; each study probably underestimates the extent of infection, as some faecal specimens were obtained more than 6 days after the onset of symptoms, when the number of rotavirus particles is often below the threshold required for detection by EM (Davidson et al., 1975).

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Rotavirus is a common cause of diarrhoea in Melbourne in the cooler months (April t o August), whereas relatively few cases are detected in the warmer months. The monthly admissions of infants and young children with acute gastroenteritis each year follow a similar pattern, with a peak in May, June, and July and a low in November, December, and January, suggesting that rotavirus is the major cause of winter diarrhoea. Similar findings have been reported in Australia (Davidson et al., 1975) the United States (Kapikian et al., 19761, and England (Bryden et al., 1975). Whilst rotavirus infection was detected in children up to 10 years of age, those most commonly affected were aged from 6 months to 3 years. Serological studies carried out in this and other laboratories have confirmed that in developed countries most primary infections occur in this group (Gust et al., 1976; Kapikian et al., 1976). The evidence that enterovirus infection is an important cause of diarrhoea in children is unconvincing. Agents such as Echovirus type 18 and 19 have occasionally been associated with localized neonatal outbreaks (Ferris, 1965), but the role of other members of the group is uncertain (Ramos-Alvarez and Clarke, 1964). It is of interest that 9% of faecal specimens examined contained adenovirus, and that not all of these viruses could be isolated in cell culture. This phenomenon, which has been reported by other workers (Bryden et al., 1975; Kapikian et al., 1976), may be due to damage of the virus during processing, the presence of neutralizing antibody in the specimen, or perhaps to the existence of new adenovirus serotypes. The frequency of infection with Salmonella and Shigella species was low throughout the period of this study and only exceeded rotavirus infection in 3 months - November, December, and January. Most of the Shigella isolates (76%) were obtained from an outbreak in a large children’s institution. At present the electron microscope is commonly used for detecting rotavirus infection, but in view of the limited availability of these instruments, the expense involved in running them, and the daily demand on their use, alternative means of diagnosis are urgently required. Tufvesson et al. (1976) and Middleton et al. (1976) have reported encouraging results with a CIEP technique. However, in our hands CIEP was considerably less sensitive than EM, detecting only 37.5% of specimens in w h c h the virus had been visualized. The relatively low detection rate may have been due to failure to stain the gels, differences in the time at which specimens were obtained, or differences in the type of faeces examined. Recently, Banatvala et al. (1975) have reported detection of rotavirus in faeces by means of fluorescent antibody staining and it is possible that this technique could be adapted to provide a satisfactory rapid diagnostic test. However, at present, when a diagnosis is required urgently the electron microscope still offers advantages in sensitivity and speed. Although rotavirus has emerged as the major aetiological agent of winter diarrhoea in children, there remains a considerable proportion of both winter and summer cases in which an aetiological agent cannot be identified. The need for further work in this area is apparent. ACKNOWLEDGMENTS

This study was made possible by a grant from the Aboriginal Health Branch of the Australian Department of Health. The authors would like to thank the staff of Ward 2 at this hospital for their cooperation in obtaining specimens, Miss Jacqui Fuge for her expert technical assistance, and Mrs. Judy Westwood for typing the mansucript.

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REFERENCES Banatvala JE, Totterdell B, Chrystie IL, Woode GN: (1975). In-vitro detection of human rotavirus. Lancet October 25, p 821. Blair JE, Lennette EH, Truant JP: (1970). Manual of Clinical Microbiology. American Society for Microbiology, p 646. Bryden, AS, Davies HA, Hadley RE, Flewett TH, Morris CR, Oliver P: (1975). Rotavirus enteritis in the West Midlands during 1974. Lancet, August 9, pp 241-243. Davidson GP, Bishop RF, Townley RRW, Holmes IH, Ruck BJ: (1975). Importance of a new virus in acute sporadic enteritis in children. Lancet, February 1, pp 242-246. Ellis AW, Kennett ML, Lewis FA, Gust ID: (1975). Hand, foot, and mouth disease: An outbreak with interesting virological features. Pathology 5: 189-196. Ferris AA: (1965). The epidemiology of infectious diarrhoea. Australian Journal of Science (285): 187-1 92. Gust ID, Pringle RC, Barnes GL, Davidson GP, Bishop RF: CF antibody response t o rotavirus infection. (Submitted for publication, 1976.) Kapikian AZ, Kim HW, Wyatt RG, Cline WL, Arrobio JO, Brandt CD, Rodriguez WJ, Sack DA, Chanock RM, Parrott RH: (1976). Human reovirus-like agent as the major pathogen associated wi& “winter” gastroenteritis in hospitalized infants and young children. New England Journal of Medicine 294(18):965-972. Kennett ML, Ellls AW, Lewis FA, Gust ID: (1972). An epidemic associated with echovirus type 18. Journal of Hygiene (Cambridge) 70: 325-334. Lewis FA, Kennett ML.: (1976). Comparison o f rhinovirus-sensitive HeLa cells and human embryo fibroblasts for isolation of rhinoviruses from patients with respiratory disease. Journal of Clinical Microbiology 3( 5):5 28-532. Middleton, PF, Petric M, Hewitt CM, Szymanski MT, Tam JS: (1976). Counterimmunoelectroosmophoresis for the detection of infantile gastroenteritis virus (orbi group) antigen and antibody. Journal of Clinical Pathology 29:191-197. Nastasi MC, Pringle RC, Gust ID: (1972). The detection o f Australia antigen and antibody by crossover immunoelectrophoresis. Australian Journal of Medical Technology 3 : l l l - 115. Ratnos-Alvarez M, Clarke J: (1964). Diarrhoea1 diseases of children. American Journal of Diseases of Children 107:218-231. Tan GS, Townley RRW, Davidson GP, Bishop RF, Holmes IH, Ruck BJ: (1974). Virus in faecal extracts from children with gastroenteritis. Lancet June 1, p 1109. Tufvesson B, Johnsson T: (1976). Detection of rotavirus in faeces from gastroenteritis patients by immunoelectroosmophoresis. Accepted for publication in Acta Pathologica et Microbiologica Scandinavica.