Human Torovirus: a New Nosocomial Gastrointestinal Pathogen

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Human Torovirus: A New Nosocomial Gastrointestinal Pathogen

Frances B. Jamieson, Elaine E. L. Wang, Cindy Bain, Departments of Microbiology and Pediatrics and the Clinical Jennifer Good, Lynn Duckmanton, and Martin Petric Epidemiology and Health Care Research Program, Hospital for Sick Children and University of Toronto, Toronto, Canada

Studies were undertaken to determine if human torovirus is associated with gastroenteritis and to examine the clinical features of torovirus illness in children. The fecal excretion of torovirus in patients with gastroenteritis was compared with that in matched asymptomatic controls in a case-control study. Toroviruses were identiﬁed in 72 (35.0%) of 206 gastroenteritis cases compared with 30 (14.5%) of 206 controls (P ! .001 ). Clinical features of torovirus gastroenteritis in 172 patients positive for torovirus were compared with those of 115 patients infected with rotavirus or astrovirus. Persons infected with torovirus were more frequently immunocompromised (43.0% vs. 15.7%) and nosocomially infected (57.6% vs. 31.3%). They also experienced less vomiting (46.4% vs. 66.7%) but had more bloody diarrhea (11.2% vs. 1.8%). An antibody response to torovirus developed mainly in older, nonimmunocompromised children (P ! .01 ). These studies demonstrate an association between torovirus excretion and gastroenteritis in the pediatric population among immunocompromised hospitalized patients and in previously healthy patients.

Acute viral gastroenteritis in family studies was second in long. Toroviruses were classified on the basis of the Berne virus prevalence only to the common cold and accounted for 16% genome sequence as members of the family Coronaviridae, of illnesses or 1.52 cases per person per year [1]. Despite ad- which together with the family Arteriviridae are now classified vances in management that have reduced mortality, viral gas- in the order Nidovirales [12–14]. troenteritis continues to be a major cause of hospitalization, Torovirus-like particles were first documented in 1984 by elec- resulting in admissions of more than 200,000 children and al- tron microscopy of fecal specimens of persons with gastroen- most 900,000 in-patient days per year in the United States [2]. teritis [15]. Evidence that these were toroviruses came from their In our pediatric setting, nosocomial diarrhea occurs in up to immunospecific cross-reactions with the Breda virus and the 10.3% of room contacts [3]. Viral agents known to cause gas- ability of nucleic acid probes based on the Berne virus sequence troenteritis include rotaviruses, astroviruses, caliciviruses, and to hybridize with RNA extracted from stool specimens positive enteric adenoviruses [4–6]. However, the etiologic agent can re- for toroviruses by electron microscopy [16, 17]. Studies of these main undiagnosed in over half the cases of suspected infectious viruses were limited because they cannot be grown in cell cul- gastroenteritis despite advances in diagnostic technology [7]. ture, they lack the distinct icosahedral structure of other gas- Studies on animal gastroenteritis viruses have provided val- troenteritis viruses, and their peplomers are less pronounced uable insights into the discovery of new human pathogens, such than those of coronaviruses [16]. as rotavirus [8]. Among the established agents of gastroenteritis In our diagnostic electron microscopy laboratory, pleomor- in animals are the toroviruses, which include the Breda virus phic fringed particles that resembled particles previously des- of cattle and Berne virus of horses [9–11]. These enveloped ignated as toroviruses were observed in a substantial number RNA viruses measure 100–140 nm at their largest diameter and of fecal specimens from children with gastroenteritis. These contain a tightly coiled tubular nucleocapsid that generally as- were shown to be toroviruses because of a high concordance sumes a donut or torus shape in the virion [9]. The particles between the identification of torovirus-like particles by electron have a fringe of peplomers on their surface that are 10 nm ∼ microscopy and a positive enzyme immunoassay with Breda virus reference antiserum [18]. Torovirus-like particles purified from stool specimens were further shown to be toroviruses on Received 14 January 1998; revised 18 June 1998. The investigation was approved by the Hospital for Sick Children Re- the basis of their morphology, immunospecific interactions with search Ethics Board. Informed consent was obtained from patients or their Breda virus antiserum, ability to elicit an immune response parents or guardians. following infection, and the high degree of homology of the 30 Grant support: National Health Research and Development Program of Health and Welfare Canada. end of their genome with that of the Berne and Breda viruses Reprints or correspondence: Dr. Martin Petric, Division of Microbiology, [19]. These data support the hypothesis that these toroviruses Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada are infectious agents, but better evidence is needed to establish M5G 1X8 ([email protected]). that toroviruses actually cause gastroenteritis. We therefore The Journal of Infectious Diseases 1998;178:1263±9 conducted two epidemiologic studies to determine the role of ᭧ 1998 by the Infectious Diseases Society of America. All rights reserved. 0022-1899/98/7805-0005$02.00 these virus particles in gastroenteritis. The first study was a 1264 Jamieson et al. JID 1998;178 (November)

Figure 1. Electron micrograph of torovirus particles from patient fecal specimen. Bar ϭ 100 nm. case-control comparison of torovirus shedding in patients with Study 1 gastroenteritis and in asymptomatic controls. The second com- Stool specimens from patients with gastroenteritis were examined pared the clinical features of symptomatic patients shedding for viruses by negative contrast electron microscopy [19, 20]. Case- torovirus and those shedding rotavirus or astrovirus. patients were selected from among this symptomatic group using a table of random numbers. For each case, a control patient was selected and matched by ward and date of admission within 1 week of the case. These matching criteria were used to ensure that the Methods two populations were comparable with respect to underlying illness and season and duration of hospitalization. Controls were not Patients symptomatic for gastroenteritis and had experienced no changes in stool frequency or consistency on the day of selection. Patients The studies were conducted between 1 October 1993 and 30 were excluded from enrollment if they had an underlying gastro- September 1995 in patients admitted to the Hospital for Sick Chil- dren, a 410-bed primary, secondary, and tertiary care center. In Table 1. Characteristics of case-control study patients. this setting, fecal specimens are submitted for diagnostic virology Diarrhea patients Asymptomatic controls for all patients admitted with symptoms of gastroenteritis or who Characteristic (n ϭ 206) (n ϭ 206) develop these symptoms while hospitalized. Specimens from 1500 ∼ patients are examined each year. In the ﬁrst study, the frequency Age in years (mean, range) 3.3 (0.02–18.2) 3.2 (0.01–17.6) Gender of torovirus identiﬁcation in patients with gastrointestinal symp- Male 133 122 toms (cases) was compared with that of asymptomatic controls. Female 73 84 Once this was accomplished, a second study was undertaken to % immunocompromised (no./total) 19.9 (41/206) 16.0 (33/206) a establish a more complete microbiologic diagnosis, examine the % torovirus-positive (no./total) 35.0 (72/206) 14.5 (30/206) a serologic response, and describe the progress of the infection. P ! .001, x2 test. JID 1998;178 (November) Human Torovirus Gastroenteritis 1265

Table 2. Torovirus-positive stool specimens in immunocompromised an electron microscope (Phillips EM300) at ϫ50,000 magnification. and nonimmunocompromised patients (study 1). Specimens were considered positive for torovirus if they contained Cases Controls spherical or kidney-shaped particles (100–140 nm in diameter) with Patient group (n ϭ 206) (n ϭ 206) a characteristic fringe of peplomers 10 nm long as shown in figure ∼ Immunocompromised 19/41 (46.3)a 3/33 (9.0)a 1 [19]. Other viral pathogens, including rotaviruses, adenoviruses, Nonimmunocompromised 53/165 (32.1)b 27/173 (15.6)b astroviruses, and Norwalk virus-like agents, were identified by their NOTE. Data are no./total (%). morphologic features [20, 21]. All specimens were examined by an a P ! .005, x2 test. experienced electron microscopist blinded to specimen source. For b P ! .001, x2 test. patients participating in study 2, stool samples were also submitted for bacterial isolation [22] and tested for Escherichia coli O157:H7 [23], examined for ova and parasites [24], and tested for Clostridium intestinal disorder (e.g., Crohn’s disease). Stool specimens from difficile cytotoxin [25]. controls were collected within 72 h of enrollment of the case spec- Serology. Antibody to torovirus was quantitated in acute and imen and examined for viruses by electron microscopy; the electron convalescent sera by HAI assay as described previously [19] by use microscopist was blinded to patient clinical status. All stool spec- of a purified torovirus preparation from a single patient. The test imens were collected in a standard stool specimen container (25 was done without knowledge of the clinical and demographic fea- mL) and processed within 48 h. Control patients were reassessed tures of the cases. The HAI assay used a 0.5% suspension of rabbit on day 4 after enrollment to determine if they developed gastro- erythrocytes and 4 hemagglutination units of virus per well. In the intestinal symptoms subsequent to enrollment. Case patients were analysis of the results of these tests, age and immune status (im- excluded from the analysis when no matching control was available munocompromised vs. immunocompetent) were included in a lo- or when a stool specimen was not obtained from the matched gistic regression model to predict for seroconversion. asymptomatic control. Statistical analysis. We used McNemar’s x2 test to compare concordance of matched pairs of gastroenteritis patients and Study 2 asymptomatic controls described in study 1. Relative odds were calculated by dividing the number of cases with torovirus times At a different time from that of study 1, patients whose stool the number of controls without torovirus by the number of controls specimens were positive for torovirus, rotavirus, or astrovirus by with torovirus times the number of cases without torovirus. Un- electron microscopy were enrolled in study 2. Information on clin- paired Student’s t tests for continuous measures and x2 analysis or ical presentation and course throughout hospitalization was col- Fisher’s exact tests were used to compare clinical findings between lected from patients or parents by questionnaire. After discharge, patients positive for torovirus and patients positive for astrovirus each patient was followed with a visit from the study nurse. In or rotavirus. Descriptive statistics were used to express duration torovirus-positive patients, stool specimens were examined daily by of symptoms and shedding of virus. Geometric means and 95% electron microscopy during the hospitalization and at follow-up. confidence intervals were used to describe serologic responses [26]. Acute sera from these patients were collected at the time of diagnosis, and convalescent sera were obtained 2–6 weeks after enrollment. Results Study 1: torovirus excretion in gastroenteritis patients and Definitions asymptomatic controls. Two hundred and six case-control

Diarrhea was defined as 3 loose stools in a 24-h period. Nos- ≥ ocomial infection was defined as onset of gastrointestinal symptoms on or after day 3 of admission [3]. Immunosuppressed patients included those with the following conditions: severe combined immunodeficiency, leukemia, other malignancies treated by chemo- therapy, solid organ or bone marrow transplant recipients, human immunodeficiency virus infection or AIDS, and those receiving large doses of immunosuppressive therapy for any cause. Sero- conversion was defined as a rise in antibody titer to torovirus of 4-fold by hemagglutination-inhibition (HAI) assay. ≥

Laboratory Tests

Stool specimens. These were prepared for diagnostic electron microscopy as described previously [19, 20]. In brief, a 10%–20% suspension of specimen was prepared in a 1% solution of ammo- nium acetate, applied to a polyvinyl carbon–coated 400-mesh electron microscope grid, and stained with a 2% solution of sodium Figure 2. Rotavirus and torovirus diagnoses at Hospital for Sick phosphotungstate at pH 7.0. The specimen was examined under Children in 1993. 1266 Jamieson et al. JID 1998;178 (November)

Table 3. Demographic features of torovirus and rotavirus/astrovirus virus-, and 13 astrovirus-infected gastroenteritis patients. The cases (study 2). monthly frequency distribution of viruses identified by electron Torovirus Rotavirus/ microscopy in 1993 is shown in figure 2. While rotavirus in- cases astrovirus cases Demographic feature (n ϭ 172) (n ϭ 115) P fections were most frequent during the winter and spring, the incidence of torovirus infections remained relatively constant Age in years Mean 4.0 2.0 .006 throughout the year. As shown in table 3, torovirus-positive Range 0.02–18.6 0.05–16.5 patients were older, developed nosocomial infections more of- !2 91 81 ten, and were more frequently immunocompromised than were 2 81 35 ≥ Gender patients infected with rotavirus or astrovirus. Male 102 76 NS The clinical manifestations of torovirus were similar to those Female 70 39 of rotavirus or astrovirus except that children infected with % nosocomial infections 57.6 (99/172) 31.3 (36/115) !.001 Mean days in hospital before illness 19.4 10.7 torovirus had less vomiting and more bloody diarrhea (table % immunosuppressed (no./total) 43.0 (74/172) 15.7 (18/115) !.001 4). Of the 19 torovirus-positive patients presenting with bloody NOTE. NS ϭ not significant. diarrhea, 9 were immunocompetent, and 1 had a concomitant C. jejuni infection. Nine additional torovirus-positive patients pairs were included in this analysis. Of the initial cases selected, developed bloody diarrhea within 2–15 days after study en- 176 were excluded for one or more of the following reasons: rollment and had no other pathogen in the stool sample. underlying gastrointestinal disorder (5), lack of a control patient Among those with rotavirus or astrovirus, only 2 presented with hospitalized at a comparable time (106), or lack of a stool bloody diarrhea; another 6 developed bloody diarrhea after specimen from the control patient (65). enrollment, 2 of whom were positive for C. jejuni. Demographic characteristics were similar for cases and con- Bacterial pathogens were detected in stool specimens from trols as summarized in table 1. Among the 206 cases, a virus both groups of patients. Among those infected with torovirus, was identified in 88: torovirus, 72; rotavirus, 8; and other vi- Yersinia enterocolitica was isolated from 1 and C. jejuni from ruses, including astrovirus, Norwalk-like viruses, and adeno- 2. C. difficile cytotoxin was detected in 7 patients in a subset virus, 8. In contrast, a virus was detected in 33 of the 206 of 81 tested in the rotavirus-astrovirus group and in 8 of a matched asymptomatic patients: 30 had torovirus, 1 had ro- subset of 128 torovirus-positive patients. One torovirus-positive tavirus, and 2 had other viruses. Significantly more cases patient (of 64 tested) had Giardia lamblia cysts, and 1 rotavirus- (35.0%) than controls (14.5%) were positive for torovirus positive patient (of 61 tested) had Dientamoeba fragilis present (P ! .001). Since a substantial number of the patients were im- in stool specimens. munocompromised, cases and controls of this subset were in- The average duration of symptoms was similar, although dependently analyzed for excretion of torovirus (table 2). In torovirus-positive patients required parenteral hydration for a this subset, significantly more cases than controls were again longer period (6.4 vs. 4.8 days). Stool specimens collected on positive for torovirus. This was also true among the immu- a follow-up visit 2–6 months after study enrollment from 168 nocompetent patients. When controls were reassessed, 23 con- patients infected with torovirus showed that 14 were shedding trols had developed gastroenteritis within 4 days of study en- the virus. rollment. Of these, 10 were positive for torovirus. When these When immunocompromised patients were compared with 10 controls were excluded, only 20 (10.2%) of 196 controls were previously healthy torovirus-positive patients, they were sig- positive for torovirus. nificantly older and more likely to have acquired the infection This study was designed to determine whether there was an in the hospital (78% vs. 42%) (table 5). Three immunocom- association between torovirus excretion and gastroenteritis. Ac- cordingly, the patient and control specimens were examined only for viruses with the understanding that other microbial Table 4. Clinical features of torovirus cases and rotavirus/astrovirus pathogens would be tested for in study 2. To obtain an insight cases. as to whether the symptomatic patients may have had bacterial Rotavirus/ infections not diagnosed in study 1, a retrospective analysis was astrovirus Torovirus cases cases performed on a subset of symptomatic patients whose stool Clinical feature (n ϭ 172) (n ϭ 115) P specimens were submitted for bacteriologic testing as part of Vomiting 46.4 (78/168) 66.7 (76/114) !.001 the routine investigation outside the study protocol. This anal- Diarrhea 87.6 (149/170) 80.0 (91/114) NS ysis revealed no bacterial pathogens in stool specimens from Blood in stool 11.2 (19/170) 1.8 (2/112) .006 28 cases for whom bacteriologic testing was done. Abdominal pain/colic 51.3 (79/154) 53.9 (56/104) NS Temperature 138.5ЊC 36.1 (52/144) 33.8 (22/65) NS Study 2: clinical features of torovirus-positive gastroenteritis Duration of symptoms (days) 7.8 (n ϭ 144) 7.9 (n ϭ 99) NS patients. Between 1 October 1993 and 30 September 1995, Duration intravenous hydration (days) 6.3 (n ϭ 139) 4.8 (n ϭ 102) !.005 clinical features were analyzed for 172 torovirus-, 102 rota- NOTE. Data are % (no./total), except low duration. NS ϭ not significant. JID 1998;178 (November) Human Torovirus Gastroenteritis 1267

Table 5. Comparison of immunocompromised and nonimmuno- sion in older children in our study is similar to findings in compromised torovirus cases. animals reinfected with Breda virus and suggests a booster re- Nonimmuno- Immuno- sponse to repeated infections [28, 29]. Finally, other known compromised compromised gastroenteritis pathogens were only sporadically identified in (n ϭ 98) (n ϭ 74) P torovirus-positive patients. These were also found in children Age (years) 0–2 72 19 !.001 infected by rotavirus or astrovirus, established gastroenteritis 2 26 55 viruses. The association of torovirus with acute and persistent ≥ Gender (no.) diarrhea was recently reported in a smaller cohort of children Male 62 40 NS Female 36 34 in an urban Brazilian slum [30]. In that study, 33 children had % nosocomial infections (no./total) 41.8 (41/98) 78.3 (58/74) !.001 acute diarrhea and 41 had persistent diarrhea. There were 17 % shedding torovirus in stool 18 days controls. (no./total) 43.9 (18/41) 62.5 (35/56) NS Torovirus appears to be a common agent of gastroenteritis NOTE. NS ϭ not significant. at our institution. Particles resembling toroviruses have been reported in stools of gastroenteritis patients, although in some promised patients required admission to the intensive care unit cases they were designated “coronavirus-like particles” [15, after developing severe diarrhea and vomiting. 31–33]. During a 2-year period at our hospital, 20% of stool Seroconversion. In paired sera from 88 patients, 51 dem- samples from gastroenteritis patients were positive for torovi- onstrated seroconversion with a 4-fold increase in antibody rus, more than double those positive for rotavirus (7%) and for ≥ titer (table 6). Of the 44 patients 2 years old, half (22) ex- astrovirus and Norwalk-like viruses (2%). Toroviruses were pre- ≤ perienced seroconversion, whereas of the 44 patients 2 years sent all year and had seasonal distribution similar to that de- ≥ old, 29 (66%) seroconverted. Of the 30 immunocompromised scribed for corona-like viruses [33]. These findings compare patients, 11 seroconverted, and of the latter 11, 10 (91%) were favorably with our recent report on the role of bovine torovirus older than 2 years. Of the 58 immunocompetent patients, 40 in diarrhea of calves [34]. In that study, toroviruses were the seroconverted and 19 (47.5%) were older than 2 years. After predominant viral agent associated with diarrhea. controlling for age in the analysis, immunocompromised pa- Torovirus is the principal identifiable cause of nosocomial tients were significantly less likely to seroconvert than were diarrhea in immunocompromised and older children in our in- immunocompetent patients (P ! .001). The geometric mean ti- stitution. Although no deaths in the immunocompromised pa- ter of convalescent sera was also significantly lower for im- tients studied could be directly attributable to torovirus infec- munocompromised patients (17.61) than for immunocompetent tion, enteric viral infections cause severe morbidity and patients (33.97). mortality among such patients [35]. Among asymptomatic controls, torovirus was detected at a lower rate in immunocompromised patients than in immunocompetent patients (9.0% vs. Discussion 15.6%; table 2), whereas among children with gastroenteritis, These studies provide strong evidence for a causative role for it was more common among immunocompromised than in im- torovirus in gastroenteritis. First, the relative odds of torovirus munocompetent patients. This further suggests that the recov- among gastroenteritis cases was 3.1-fold that for controls. How- ery of torovirus by the immunocompromised is related to the ever, if the 10 control patients who became symptomatic within illness rather than to a proclivity of immunocompromised pa- 4 days of sampling are excluded, the relative odds become 4.7, tients to carry this organism. Furthermore, this virus was a which approaches those described with enteric adenovirus and common cause of diarrhea in hospitalized patients who were astrovirus [4, 27]. Secondly, over half of the torovirus-positive previously healthy. An outbreak of gastrointestinal illness in a gastroenteritis patients developed an immune response to the neonatal intensive care unit due to particles similar to torovi- virus. This suggests that the virus caused the infection rather ruses (coronavirus-like particles) has been described [36]. than being a simple bystander or an electron microscopy artifact Two clinical features that distinguish torovirus-infected pain patients with diarrhea. The higher frequency of seroconver- tients are the lower frequency of vomiting and high frequency

Table 6. Geometric mean titers of torovirus antibodies in acute and convalescent sera and occurrence of seroconversion in immunocompromised and nonimmunocompromised patients (study 2). Titer (95% conﬁdence interval) Patient group Seroconversion Acute Convalescent

Immunocompromised (n ϭ 30) 11 6.5 (5.66–7.46) 17.61a (13.31–23.28) Nonimmunocompromised (n ϭ 58) 40 7.02 (6.48–7.60) 33.97a (26.54–43.48)

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