RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections

Journal of Clinical Virology 17 (2000) 151 – 158 www.elsevier.com/locate/jcv

RT-PCR identification and typing of astroviruses and Norwalk-like viruses in hospitalized patients with gastroenteritis: evidence of nosocomial infections O. Traore´ a,*, G. Belliot b,d, C. Mollat c, H. Piloquet c, C. Chamoux c, H. Laveran a, S.S. Monroe d, S. Billaudel c a

Ser6ice and Hygiene Hospitalie`re, Faculte´ de Me´decine, 28 place Henri Dunant, 63000 Clermont Fd Cedex, France b Atlanta Research and Education Foundation, Decatur, GA 30033, USA c Laboratoire de Virologie, Laboratoire de Bacte´riologie, Clinique Pe´diatrique, Centre Hospitalier Uni6ersitaire, 9 Quai Moncousu, 44 035 Nantes Cedex 01, France d Di6ision of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Pre6ention, Atlanta, GA, 30329, USA Received 23 March 2000; accepted 3 May 2000

Abstract Background: Astroviruses (HAstVs) and ‘Norwalk-like viruses’ (NLV) are frequent causes of gastroenteritis worldwide, though no data on the strains in circulation or their prevalence is available for France. Objecti6es: We applied molecular methods to detect HAstVs and NLVs by reverse transcription-polymerase chain reaction (RTPCR) in fecal samples collected during a 2-year period from children and adults hospitalized with gastroenteritis. Study design: All samples negative for rotavirus and adenovirus by latex agglutination which contained small (25 – 40 nm) viral particles observed by electron microscopy (EM) were examined by RT-PCR. RT-PCR products were sequenced to characterize the HAstV and NLV strains present. Results: A total of 75 samples were analyzed by RT-PCR, of which 15 were positive for HAstV and 24 for NLV. Several distinct strains of serotype 1 HAstV, the predominant serotype, circulated during the period. Nineteen of the 24 NLVs were of the G2 genogroup including Mexico-like (n= 10), Bristol-like (n =8), and Hawaii-like viruses (n= 1); two were genogroup 1. Overall, seven (47%) of the 15 HAstV infections and nine (37.5%) of the 24 NLV infections appeared to be nosocomially acquired based on the date of admission in hospital and the date of illness. Conclusion: This study provides additional evidence of the importance of nosocomial infections caused by NLV and HAstV. © 2000 Published by Elsevier Science B.V. Keywords: Astrovirus (HAstV); Gastroenteritis; Norwalk-like viruses (NLV); Nosocomial infections

* Corresponding author. Tel.: + 33-473-608007; fax: + 33-473-265432. E-mail address: [email protected] (O. Traore´). 1386-6532/00/$ - see front matter © 2000 Published by Elsevier Science B.V. PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 0 8 8 - 3

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1. Introduction Astroviruses (HAstVs) and human caliciviruses have been identified as important etiologic agents of gastroenteritis, particularly among young children. HAstVs were first associated with gastroenteritis in 1975 (Appleton and Higgins, 1975; Madeley and Cosgrove, 1975). Primers directed toward a highly conserved region of the HastV genome which encodes the non-structural proteins, allow for sensitive reverse transcriptionpolymerase chain reaction (RT-PCR) detection and genogrouping of HAstV strains (Belliot et al., 1997a). HAstV strains can be genotyped using primers directed toward the region that encodes the capsid protein (Noel et al., 1995). Human caliciviruses include the ‘Norwalk-like viruses’ (NLVs), and the morphologically ‘classic’ human caliciviruses, now known as ‘Sapporo-like viruses’. Since the initial description of Norwalk virus in 1972 (Kapikian et al., 1972), diagnosis of NLV infection has been difficult because these viruses cannot be cultured in vitro and no animal models are available, except human volunteers (Thornhill et al., 1975; Cubitt et al., 1998), to produce viral antigens and antisera for detection assays of antigen and antibody. Nucleotide sequence analysis of a conserved region of the NLV genome encoding the RNA polymerase, allowed the division of NLVs into two genetically distinct genogroups, G1 and G2 (Green et al., 1994; Ando et al., 1995). The nucleotide sequence variation in this region is usually sufficient to allow the dillerentiation of strains in circulation. Epidemiologic studies using these new assays have shown the high prevalence of HAstV and NLV among persons with gastroenteritis in several countries around the world (Moe et al., 1994; Green et al., 1995; Jiang et al., 1995a; Cubitt and Jiang, 1996; Levett et al., 1996; Pang et al., 1999a). Although the occurrence of HAstV-associated and NLV-associated gastroenteritis has recently been documented (Belliot et al., 1997b; Bon et al., 1999), the role of HAstV and NLV and the relative importance of each serotype and genotype in France are unknown. In our hospital, we routinely use electron microscopy (EM) to detect viral agents associated

with gastroenteritis. In this study, we applied RT-PCR to the detection of NLV and HAstV in fecal specimens from hospitalized patients with gastroenteritis associated with small viral particles observed by EM. The strains of HAstV and NLV detected during a 2-year period were characterized by RT-PCR and nucleotide sequencing. Comparison of the nucleotide sequence provided epidemiologic data on the circulation of HAstV and NLV strains in the hospital and the surrounding community.

2. Patients and methods

2.1. Study design The study was conducted in the University Hospital of Nantes, France, from January 1996 to January 1998. Stool specimens from children and adults submitted to the virological laboratory, for analysis, were included in the study. Diarrhea was defined as unusually loose stools at at least twice the usual daily frequency. The specimens were both from patients either with diarrhea at admission (community acquired) or with diarrhea which occured 72 h or more after hospitalisation (nosocomially acquired). All samples were screened for group A rotavirus and adenovirus by latex agglutination (Diarlex, Fumouze Diagnostics, Asnieres, France). All specimens negative for rotavirus or adenovirus were further examined by negativestain EM for other viral agents. Specimens, where small round viral particles were observed by EM, were examined by RT-PCR using primers specific for NLVs and HAstVs. During the last 2 months of the study due to increased rates of detection of NLVs in patients (December 1997 and January 1998), RT-PCR was performed on all rotavirusand adenovirus-negative samples, regardless of the EM results.

2.2. Negati6e-stain EM Ten percent suspensions in water of the stool specimen were clarified by extraction with 1,1,2trichloro-trifluoroethane (Freon) followed by centrifugation at 1500×g for 15 min. The

O. Traore´ et al. / Journal of Clinical Virology 17 (2000) 151–158

supernatant was further clarified by recentrifugation at 8000×g for 10 min. Nine hundred fifty microliter volume of the supernatant was mixed with 50 ml of standard gammaglobulin (Polygamma, Association Nationale pour la Distribution des Fractions Plasmatiques Humaines, Paris, France) and incubated at 37°C for 30 min. This preparation was centrifuged at 100 000×g for 30 min; the pellet was dropped onto a carbon-coated copper grid and negatively stained with 2% phosphotungstic acid. All the grids were examined by the same microscopist under a JEOL 100C electron microscope at a magnification of × 50 000.

2.3. RT-PCR Total RNA was purified from 200 ml of freonextracted 25% stool suspension prepared in sterile distilled water by using a RNA-PLUS purification kit (Bioprobe Systems, Montreuil, France) according to the manufacturer’s instructions. The RNA was suspended in a final volume of 25 ml of diethyl pyrocarbonate (DEPC)-treated water. RTPCR for the detection of HAstV was performed using primers Mon340 and Mon348 located within ORF 1a, yielding an amplicon of 289 bp (Belliot et al., 1997a; Traore´ et al., 1998). HAstV strains were genotyped using primer pairs Mon244/Mon245 located within ORF 2 yielding an amplicon of 413 bp (Noel et al., 1995) followed by nucleotide sequencing. For NLV RT-PCR, cDNA synthesis was primed using SR33, and the PCR was performed in combination with SR46 (G11) or SR48, SR50 and SR52 (G1) yielding amplicons of 123 bp (Ando et al., 1995). Briefly, 5–10 ml of RNA solution was added to 20 ml of a RT mix containing 10 units (HAstV) or 12 units (NLV) of avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI), 5 ml of 5× enzyme buffer (Promega), 2 ml of each of four 10 mM dNTP stocks (Boehringer Mannheim, Indianapolis, IN) and 25 pmole of reverse primer (Mon348 and Mon245 for HAstV or SR33 for NLV). The mixture was incubated for 1 h at 42°C. PCR was performed using 8 ml of cDNA with 2.5 units of Taq DNA polymerase (Appligene, Illkirch, France), 5 ml of 10 × Taq

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buffer (Appligene), 1 ml of each of four 10 mM dNTP stocks, and 25 pmol of each primer (Mon340/Mon348 and Mon244/245 for HAstV, SR33/SR48/SR50/SR52 for GI-NlV or SR33/ SR46 for GII-NLV), in a final volume of 50 ml. An initial denaturation was performed for 3 min at 94°C, followed by 30 cycles of amplification; denaturation for 30 s at 94°C, annealing for 20 s at 50°C and extension for 30 s at 72°C, for HAstVs or 40 cycles of denaturation for 1 min at 94°C, annealing for 1 min 30 s at 50°C, and extension for 2 min at 60°C, for NLVs. A final extension was performed for 5 min at 72°C. The amplification products were analyzed by electrophoresis using 1.5% (HAstV) or 2.5% (NLV) agarose gels. Amplified products were visualized by UV illumination following staining with ethidium bromide.

2.4. Sequencing of PCR products RT-PCR products were purified from 1.7% agarose gels, using a Qiaex gel extraction kit (Qiagen, Hilden, Germany), following the manufacturer’s instructions and sequenced using a Big Dye Reaction Dyedeoxy Terminator Cycle Sequencing kit (Perkin–Elmer Cetus) on a 377 automatic sequencer (Perkin–Elmer Cetus). Nucleotide sequences were aligned and compared by using the Sequencher program (Gene Codes Corporation, MI). Multiple sequence alignments were generated using the Pileup program of the Genetic Computer Group (GCG) sequence analysis package (Genetics Computer Group, 1994). Genetic analysis of sequences was performed using the Distances program of the GCG sequence analysis package and sequences of HAstV and NLV strains from GenBank.

3. Results From January 1996 to January 1998, 1103 fecal specimens from children and adults were submitted for laboratory diagnosis of the etiologic agents of gastroenteritis. Of these 377 (34%) fecal specimens were positive for rotavirus by either latex agglutination test (286) or EM (91) and 109

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(10%) were positive for adenovirus by latex agglutination test. Of the 617 remaining rotavirus- and adenovirus-negative samples, 75 samples from 69 patients had small viral particles observed by EM. Fifteen and nineteen of these were positive for HAstV and NLV, respectively. The remaining 41 EM-positive specimens were negative using both RT-PCR assays. Five additional NLV strains were detected by RT-PCR on the subset of EM negative samples (n =30) collected in December 1997 and January 1998. All samples positive for HAstV or NLV were negative for Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., Pseudomonas aeruginosa and Clostridium difficile. Of the 15 HAstV-positive specimens detected using the primers Mon340/Mon348, 13 were successfully adapted to culture in Caco-2 cells (Table 1). Nine of these positive specimens were detected during the first 3 months of the study (i.e. from January 1996 to March 1996). In the following two winter seasons, 1996 – 1997 and 1997 – 1998, HAstV infections were diagnosed only as sporadic cases. Seven of 15 HAstV strains were hospital-acquired (nosocomial infections). The eight remaining strains were community-acquired.

All patients, except patient 96001 (49 years old), were infants or children (1–144 months old, mean age: 26 months). All cultured strains were amplified using the primer set Mon244/Mon245. Genotyping results indicated genotype 1 was present in ten specimens, genotype 4 in two, and genotype 5 in one sample. All the HAstVs detected during the first 3-months of the study were genotype 1 (Fig. 1). Genetic analysis demonstrated that several of these genotype 1 strains (N98004, N96007, N96012, N96003 and N96002) showed up to 10% nucleotide sequence divergence with the reference strain Ox 1. The two genotype 4 strains detected in this study shared together 94% identity. Some community-acquired and nosocomial strains shared 100% nucleotide sequence identity (N96004/N96023, N96002/N96003 and N96014/N96008). Two hospital-acquired HAstV strains detected in the same pediatric unit had 100% nuclootide sequence identity (N96007 and N96012). For patient 96001, diagnosed with acute leukernia, and patient 96006, HAstVs were detected only using primers Mon 340 and 348 and could not be cultured. Both patients were hospitalized in the oncology unit.

Table 1 Characteristics of the patients with HAstV-associated diarrhea Patient no.

96004 96023 96003 96002 96007 96012 96014 96008 96001 96006 96015 97001c 97007b 97010 98004 a

Age (months)

16 1 8 6 1 1 12 12 49 years 144 121 12 12 40 2

Admission date Onset date

Nosocomial acquisition

Hospitalisation unita

HAstV typeb

19 Feb 1996 13 Apr 1996 1 Feb 1996 1 Aug 1995 22 Feb 1996 29 Feb 1996 3 Mar 1996 3 Mar 1996 31 Jan 1996 24 Feb 1996 24 Dec 1996 19 Jan 1997 29 Jan 1997 30 Jan 1997 15 Dec 1997

Yes No No Yes Yes Yes Yes No No No No Yes No No Yes

P P P PS P PS PS PS O O P P P P P

1 1 1 1 1 1 1 1 No typing No typing 4 5 4 1 1

23 Feb 1996 13 Apr 1996 2 Feb 1996 9 Feb 1996 27 Feb 1996 3 Mar 1996 9 Mar 1996 4 Mar 1996 1 Feb 1996 24 Feb 1996 25 Dec 1996 27 Jan 1997 29 Jan 1997 30 Jan 1997 5 Jan 1998

P, pediatric unit; PS, pediatric surgery, O, oncology unit. Framed data correspond to HAstV strains with 100% nucleotide sequence identity in the region analyzed. c Mixed infection NLV–HAstV. b

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4. Discussion

Fig. 1. Phylogenetic dendrogram based on nucleotide identity in the capsid region of HAstVs analyzed in this study and HAstVs Oxford reference strains (Ox 1–8). Accession nurnbers for the GenBank sequences are Ox 1 (L23513), Ox 2 (L13745), Ox 3 (L38505), Ox 4 (L38506), Ox 5 (U15136), Ox 6 (L38507), Ox 7 (L38508), and Ox 8 (Z66541).

A total of 24 (19 by EM and RT-PCR plus five by RT-PCR alone) NLV strains were detected in infants or children (1 – 156 months old, mean age: 21 months). Nine of these 24 NLV strains were attributed to nosocomial infections (Table 2). The sequence of 79 bp in the RNA-dependent RNA polymerase region was determined for 21 strains, and a dendrogram based on the nucleotide sequence was constructed (Fig. 2). Nineteen strams were characterized as GII strams and two were GI strains. Ten strains, of which eight were identical, were closely related to Mexico virus (90% and 95% identity). Eight strains were Bristol virus-like (91% to 94% identity), of the eight strains closely related to Bristol virus, six were detected during January 1998, and shared 95% to 100% identity. The strain N97027 could not be classified based on 79 base nucleotide sequence in the RNA polymerase. The two GI strains 97014 and 97030 were most closely related to GenBank reference strains JPNNV22 (95% identity) and SV7 (94% identity), which belong to genogroups 1B and 1A, respectively, based on the classification of Ando et al. (1995). Finally, mixed infections of HAstVs and NLVs were observed in two specimens, 97001 and 97007.

Our present study examined the circulation of HAstVs and NLVs among patients admitted to a large French hospital during a 2-year period. We used RT-PCR and nucleotide sequence data to compare the strains in circulation. Our data probably underestimate the true prevalence of HAstVs and NLVs because of the relative insensitivity of EM as a screening method (Lew et al., 1990; Cubitt et al., 1999) and because we did not look for mixed infections of HAstV or NLV with rotavirus or adenovirus (Riepenhoff-Talty et al., 1983; Wolfaardt et al., 1997). Knowledge of the admission date to the hospital and the date of onset of gastroenteritis has allowed us to assess the importance of nosocomial infections in the hospital. We assume that the extent of nosocomial transmission of HAstVs and NLVs is still greater because of the high number of asymptomatic children (Madeley and Cosgrove, 1975; Cubitt et al., 1999). HAstV and NLV infections were found to occur more frequently during the first year of life. Of note, children as young as 1 month of age were infected with HAstV or NLV These results are in agreement with seroprevalence studies that observed an antibody response in the first year of life suggesting an early natural exposure to NLV (Smit et al., 1997) and HAstV (Kurtz and Lee, 1978; Kriston et al., 1996). In the hospitalized population, we observed predominantly HAstV infections during the first winter (1996–1997) of the study period and NLV infections during the winter of 1997–1998. In our study, all HAstV infections were diagnosed during the winter months (December–April), showing a winter seasonality as previously described by Monroe et al. (1991). HAstV serotype 1 was predominant among the HAstV strains detected, an observation seen globally (Lee and Kurtz, 1994; Mitchell et al., 1999; Pang and Vesikari, 1999b) although HAstV serotype 2 predominated in a recent study (Guerrero et al., 1998). Two HastVs strains could not be adapted on culture on Caco-2 cells. These two strains were detected in patients with hematologic malignancies who are a population at risk of HAstV infections (Cox et al., 1994; Bjorkholm et al., 1995; Cubitt et al.,

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1999). Sequence analysis of the genotype 1 amplicons, circulating during 1995 – 1996, showed a close relationship between the HAstV strains circulating in the community and in the hospitalized population. It also showed that several HAstV strains from nosocomially infected patients were identical. Once introduced into the hospital from the community, HAstV strains may spread within the hospital if appropriate infection control measures are not implemented. Moreover, the efficiency of enteric precautions may be reduced because of subclinical infections and prolonged virus shedding (Bjorkholm et al., 1995; Cubitt et al., 1999). Several NLV strains circulated in the community during the study period, the most prevalent were Mexico-like NLVs. A common Mexico-like strain was found during the winter of 1996 – 1997

and 1997–1998. As described in other countries, NLVs belonging to group GI were rarely detected during this period (Caul, 1996; Smit et al., 1997; Pang et al., 1999a). The second most prevalent NLV strains were Bristol-like viruses. However, none of these strains was identical to the world-distributed common strains described by Noel et al. (1995). During the winter of 1997– 1998, Bristol-like strains were more prevalent than Mexico-like viruses. Four of these Bristol-like strains (98020, 98022, 98029 and 98031) were detected in 2 weeks and were found to be identical to each other. These observations suggest the possibility that these strains could be outbreak-related. Eleven strains were detected between December 1997 and January 1998. Five of these strains were from EM negative samples. Poor excretion of the virus in the stool could be the

Table 2 Characteristics of the patients with NLV-associated diarrhea Patient no.

96016 96024 96029 96042 97001c 97004 97009 98023 98028 96032 98014 98020 98022 98029 98031 96041 96038 96013 97007c 97014 97030 97027 98034 98011 a

Age (months)

1 7 12 8 12 24 6 1 1 40 30 156 12 12 6 18 18 10 12 42 60 6 6 10

Admission date Onset date

Nosocomial acquisition

Hospitalisation unita

NLV typeb

11 Dec 1996 26 Sep 1996 3 Oct 1996 16 Sep 1996 19 Jan 1997 12 Jan 1997 1 Feb 1997 29 Dec 1997 4 Jan 1998 30 Dec 1996 7 Jan 1998 12 Nov 1997 17 Jan 1998 7 Jan 1998 9 Jan 1998 21 May 1996 3 Sep 1996 8 Dec 1996 29 Jan 1997 15 Jan 1997 30 Dec 1997 23 Dec 1997 6 Jan 1998 8 Jan 1998

Yes Yes No Yes Yes No No Yes No Yes No Yes No No Yes No No No No No No Yes No No

P P P P P P P Pr P P P P P P P P PS P P P P P PS P

GII MV-like GII MV-like GII MV-like GII MV-like GII MV-like GII MV-like GII MV-like GII MV-like GII MV-like GII BV-like GII BV-like GII BV-like GII BV-like GII BV-like GII BV-like GII BV-like No typing No typing No typing GI GI NV-like GII HV-like GII BV-like GII MV-like

18 Dec 1996 7 Oct 1996 4 Oct 1996 24 Dec 1996 27 Jan 1997 13 Jan 1997 1 Feb 1997 8 Jan 1998 5 Jan 1998 13 Jan 1997 8 Jan 1998 20 Jan 1998 18 Jan 1998 7 Jan 1998 18 Jan 1998 21 May 1996 4 Sep 1996 9 Dec 1996 29 Jan 1997 15 Jan 1997 31 Dec 1997 28 Dec 1997 6 Jan 1998 9 Jan 1998

P, pediatric unit; PS, pediatric surgery; Pr, premature. Framed data correspond to NLV strams with 100% nucleotide sequence identity in the region analyzed. c Mixed infection NLV–HAstV. b

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This study provides an epidemiologic insight on the circulation of HAstV and NLV strains in France and on their similarity with reference and international strains. Our study shows the epidemiological importance of the nosocomial infections by NLVs and HAstVs and that reinforced hygiene control will be probably useful to reduce the risks of infection in hospital wards with frequent passage, such as pediatric unit. Further investigations are necessary to assess the incidence of diarrheal morbidity due to HAstV and NLV in France.

Acknowledgements

Fig. 2. Phylogenetic dendrogram based on nucleotide identity in the RNA polymerase region of NLVs from this study and reference strains Southampton virus (SOV), 1283, Kyoto89 (KY89), Norwalk virus (NV), SV7, JPNNV22, Toronto virus (TV), Mexico virus (MV), OTH, Snow Mountain virus (SMV), Hawaii agent (HV), and Bristol virus (BV). Accession numbers for the GenBank sequences are L07418 (SOV), L23832 (1283), L23828 (Ky89), M87661 (NV), J07614 (SV7), D82830 (JPNNV22), U02030 (TV), U22498 (MV), L23830 (OTH), L23831 (SMV), U07611 (HV) and X76716 (BV).

cause of the EM negative results. Conversely, in five samples originating from children and containing viruses with NLV-like morphology by EM, we observed, like other authors, that RTPCR could be negative (Smit et al., 1997; Wolfaardt et al., 1997). These viruses could be Sapporo-like viruses or this could be due to the large genetic diversity within NLVs strains (Ando et al., 1995), the presence of RT-PCR inhibitors in the samples, or the deterioration of viruses during specimen storage. Ninety-one of the samples, negative by latex agglutination, were rotavirus-positive by using EM. Since these stool specimens were unavailable for further testing, these EM-positive rotavirus strains were not characterized. Further investigations need to be done to determine whether these rotaviruses might belong to group C, an observation found in studies in the US (Jiang et al., 1995b).

We would like to thank John O’Connor for editorial assistance, Jacqueline Noel and Roger Glass for their skilful assistance and comments.

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