Surveillance of human calicivirus in Spain.

To the Editor: Human caliciviruses (HuCVs) are an important cause of acute viral gastroenteritis in young children worldwide (1,2). In Spain, norovirus infections are not subject to specific surveillance; few data exist about sporadic cases (3,4) and none about outbreaks across the country.

We have conducted a surveillance study of acute gastroenteritis epidemics to determine the prevalence of HuCV infections. Our goal was to gain insight into the epidemiology of these infections in Spain and consider new directions to prevent them and control improvements in food and water quality and sanitary practices.

From October 1998 to October 1999, a pilot prospective program was designed to study viral strains causing annual epidemics of severe diarrhea in children. A total of 822 stool specimens were obtained from children <4 years of age with sporadic gastroenteritis, who visited the emergency room of a hospital in Madrid. A gastroenteritis episode was defined as [less than or equal to] 3 looser-than-normal stools within a 24-h period. Clinical and epidemiologic information was collected. No pathogens were detected in fecal specimens from 292 children. A subset of 201 of these samples was tested for HuCVs.

Additionally, 741 fecal samples were collected from 135 outbreaks that occurred throughout Spain (13 of 17 geographic areas) from 2000 to 2002. An outbreak was defined with Kaplan criteria (5). Epidemiologic data (mode of transmission, setting and size of the outbreak, persons affected, persons at risk, attack rate, and age of affected persons) were recorded on a standardized form submitted to the National Microbiology Center.

Enteropathogenic bacteria in fecal specimens were examined by conventional culture procedures. Viruses (group A rotavirus, adenovirus, and astrovirus) were also examined by commercial enzyme immunoassay (Dako Diagnostics, Cambridgeshire, UK). In negative samples (1,033 of 1,563), viral RNA was extracted as described (3) and analyzed by reverse transcription-polymerase chain reaction (RT-PCR) for HuCVs by using JV12/JV13 primers (6). RTPCR-negative samples were also reanalyzed with p289/290 (7) and NVpl10-Nvp69 (8) primers pair, which detect sapovirus. HuCV-positive specimens were confirmed and genetically characterized by the reverse line blot hybridization method (9) and sequencing assays by using High Pure PCR Product Purification kit (Boehringer, Mannheim, Germany) and ABI PRISM BigDye Terminator Cycle Sequencing Reaction Kit (Perkin Elmer Biosystems, Foster City, CA, USA) on an automated sequencer (Applied Biosystems model 3700). For HuCV phylogenetic analyses, a multiple-sequence alignment was generated from the consensus sequence of each of the isolates and the reference strains by using ClustalX 1.8 methods. The nucleotide sequences were analyzed with the MEGA 2.1 analytical package by using neighbor-joining methods and Kimura 2-parameters algorithm. GenBank accession numbers for sequences described in this study are AY207341-AY207365.

In pediatric gastroenteritis cases, HuCVs were detected by RT-PCR in 63 specimens (31%). Twenty-nine of these were genotyped, and results of phylogenetic analyses are shown in the Table. The median age of children with HuCV infection was 15 months (range 1-47). In HuCV-positive cases, 83% were associated with vomiting, 32% with fever, 24% with mild dehydration, and 2% with severe dehydration. Hospitalization was required in 13% of the cases.

Additionally, noroviruses were detected in 85 (63%) of 135 outbreaks. Seventy-seven (91%) of them were segregated into genogroup II (Table). Detection rates within outbreaks ranged from 13% to 100%. The setting was provided for 82 (97%) of 85 norovirus outbreaks. Nursing homes (57%) were the most common setting, followed by schools (10%), camping and vacation destinations (7%), hospitals (6%), and restaurants and hotels (4% each). The attack rate was provided for 33 (39%) norovirus outbreaks, and the median number of persons affected was 35. The mode of transmission was provided for 30 (35%) of the norovirus-positive outbreaks; the most common mode of transmission was person-to-person contact (n = 15), followed by contaminated food (n = 10) and contaminated water (n = 5).

This report shows the importance and diversity of HuCVs circulating throughout Spain. Noroviruses particularly have been found as a main causative agent of sporadic pediatric cases and outbreaks. The lower prevalence of sapovirus is similar to that shown by other authors (2), perhaps because sapovirus causes milder symptoms than norovirus. Analysis showed the predominance of norovirus genogroup II and Lordsdale cluster as the main genotypes both in sporadic cases and outbreaks, also shown in other reports (1,2,6).

Our study confirms that noroviruses are the main cause of nonbacterial gastroenteritis outbreaks throughout Spain, as in other European countries (1,10). However, we consider that HuCV infections could be underdiagnosed because a substantial number of nonbacterial outbreaks are labeled of unknown etiology. The systematic application of sensitive techniques to detect these viruses, as well as a more systematic surveillance system for viral diarrhea, would provide broader knowledge of norovirus infection in Spain.

Table. Human caliciviruses (HuCVs) and phylogenetic clusters found
in sporadic pediatric cases and gastroenteritis outbreak

                                          Pediatric       Outbreak
                                            cases         incident
                                        (N = 201) (%)   (N = 135) (%)

HuCVs found by reverse transcription-      63 (31)         85 (63)
  polymerase chain reaction
HuCVs analyzed phylogenetically              29              83
  Sapovirus                                3 (10)             0
  Norovirus *                              26 (90)        85 (100)
    GI-Desert Shield cluster                  0               5
    GI-Queens Arms cluster                    1               1
    GII-Hawaii cluster                        0               8
    GII-Leeds cluster                         0               1
    GII-Lordsdale cluster                    23              63
    GII-Meklsham cluster                      0               5
    GII-Mexico cluster                        2               0

* GI, genogroup I; GII, genogroup II.

Acknowledgments

We thank A. Alonso and S. Moreno for their technical assistance and R. Glass, C. Domingo, and E. Bouza for continual support and advice.

This study was supported in part by a grant (08.2/0003/2003.1) from Madrid Community (Spain) and, in part, financed by ISCIII (MPY 1176/04).

References

(1.) Bon F, Fascia P, Dauvergne M, Tenenbaum D, Planson H, Petion AM, et al. Prevalence of group A rotavirus, human calicivirus, astrovirus, and adenovirus type 40 and 41 infections among children with acute gastroenteritis in Dijon, France. J Clin Microbiol. 1999;37:3055-8.

(2.) Kirkwood CD, Bishop R. Molecular detection of human calicivirus in young children hospitalized with acute gastroenteritis in Melbourne, Australia, during 1999. J Clin Microbiol. 2001:39:2722-4.

(3.) Roman E, Negredo A, Dalton RM, Wilhelmi I, Sanchez-Fauquier A. Molecular detection of human calicivirus among Spanish children with acute gastroenteritis. J Clin Microbiol. 2002;40:3857-9.

(4.) Buesa J, Collado B, Lopez-Andujar P, Abu-Mallouh R, Rodriguez-Diaz J, Garcia-Diaz A, et al. Molecular epidemiology of caliciviruses causing outbreaks and sporadic cases of acute gastroenteritis in Spain. J Clin Microbiol. 2002;40:2854-9.

(5.) Kaplan JE, Feldman R, Campbell DS, Lookabaugh C, Gary GW. The frequency of a Norwalk-like pattern of illness in outbreaks of acute gastroenteritis. Am J Public Health. 1982;72:1329-32.

(6.) Vinje J, Koopmans MPG. Molecular detection and epidemiology of small round structured viruses in outbreaks of gastroenteritis in the Netherlands. J Infect Dis. 1996;174:610-5.

(7.) Jiang X, Huang PW, Zhong WM, Farkas T, Cubitt DW, Matson DO. Design and evaluation of a primer pair that detects both Norwalk- and Sapporo-like caliciviruses by RT-PCR. J Virol Methods. 1999;83:145-54.

(8.) Le Guyader F, Estes MK, Hardy ME, Neill FH, Green J, Brown DW, et al. Evaluation of a degenerate primer for the PCR detection of human caliciviruses. Arch Virol. 1996;141:2225-35.

(9.) Vinje J, Koopmans MP. Simultaneous detection and genotyping of polymerase chain reaction 'Norwalk-like viruses' by oligonucleotide array in a Reverse Line Blot hybridization format. J Clin Microbiol. 2000;38:2595-601.

(10.) Vainio K, Stene-Johansen K, Oystein-Jonassen T, Bruu AL, Grinde B. Molecular epidemiology of calicivirus infections in Norway. J Med Virol. 2001;65:309-14.

Alicia Sanchez-Fauquier, * Isabel Wilhelmi, ([dagger]) Enriqueta Roman, ([double dagger]) Javier Colomina, ([section]) Vanessa Montero, * and Ana Negredo *

* Instituto De Salud Carlos III, Madrid, Spain; ([dagger]) Hospital Severo Ochoa, Madrid, Spain; ([double dagger]) Hospital De Fuenlabrada, Madrid, Spain; and ([section]) Hospital De La Ribera, Valencia, Spain

Address for correspondence: Alicia Sanchez Fauquier, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Carretera Majadahonda-Pozuelo Km. 2, 28220-Madrid, Spain; fax: [phone]; email: asanchez @isciii.es