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Identification of viral agents causing gastroenteritis among children in Buenos Aires, Argentina
Journal of Clinical Virology 25 (2002) 197– 203 www.elsevier.com/locate/jcv
Identification of viral agents causing gastroenteritis among children in Buenos Aires, Argentina Alesio Bereciartu, Karin Bok, Jorge Go´mez * Viral Gastroenteritis Laboratory, National Institute of Infectious Diseases, ANLIS Dr. Carlos G. Malbra´n, Buenos Aires, Argentina Received 20 August 2001; received in revised form 16 November 2001; accepted 1 February 2002
Abstract Background: Gastroenteritis is one of the most common diseases affecting children and rotavirus is the major etiological agent worldwide. Although the role of rotaviruses as a causal agent of gastroenteritis has been previously established in Argentina, little is known about the frequency of other gastrointestinal viruses. Objecti6es: The present study was designed to determine the frequency of calicivirus in comparison with rotavirus, astrovirus and adenovirus type 40/41 in our country. Study design: Sixty six diarrhea patients (10%) = 3 years of age, were randomly selected from a previous series (9/1997–8/1998) and virus frequency determined using RT-PCR and/or commercial EIAs. In addition, we sequenced the RNA polymerase region of calicivirus-positive samples to characterize Argentine strains. Results: We detected at least one viral agent in 48.5% (32/66) of the patients. Rotavirus, calicivirus, astrovirus, and enteric adenovirus were found in 17, 16, 5, and 2 children, respectively. Sequence analysis of 4 calicivirus positive samples allowed us to identify a strain clustered to Lorsdale virus, one strain clustered with Manchester virus and two strains clustered with London/92 virus. Conclusions: Our data suggest that calicivirus and rotavirus are major agents of gastroenteritis in children younger than 3 years of age in Buenos Aires, Argentina. Unfortunately, viral diagnosis is seldom available in Argentine hospitals and coproculture results are mostly negative. Our data supports the need to develop rapid and sensitive tests for calicivirus diagnosis. The introduction of affordable viral diagnosis in our pediatric hospitals will improve patient care by reducing the unnecessary use of antibiotics. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Infantile diarrhea; Calicivirus; Rotavirus; Astrovirus; Enteric adenovirus
Abbre6iations: RT-PCR, reverse transcriptase polymerase chain reaction; EIA, enzyme immunoassay; RNA, ribonucleic acid; NLV, Norwalk-like virus; SLV, Sapporo-like virus. * Corresponding author. Present address: Av. Velez Sarsfield 563, (1281) Buenos Aires, Argentina. Tel./fax: + 5411-4302-5064. E-mail address: [email protected] (J. Go´mez).
1. Introduction Gastrointestinal diseases are one of the most frequent and preventable health problems worldwide, presenting high rates of morbidity and mortality especially in developing countries. In
1386-6532/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 0 1 0 - 0
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A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
addition, acute diarrheas cause an estimated onethird of all hospitalizations per year in children younger than 5 years of age (Bern et al., 1992; Murray and Lopez, 1997), which carry a heavy cost. During 1997 (National Surveillance System, Ministry of Health), 431,000 diarrhea cases were reported in the Argentine public health sector in children younger than 5 years of age, with virtually one death per day. While the role of rotaviruses as a causal agent of gastroenteritis has been previously established in the country (Bok et al., 2001, 1999; Gomez et al., 1986; Gonzalez et al., 1999; O’Ryan et al., 2001), little is known about the frequency of other gastrointestinal viruses such as calicivirus, astrovirus and adenovirus type 40/41. An accurate understanding of the relative prevalence of these agents would help design strategies to control the disease. The present study was performed to determine the relative frequency of these viruses in a pediatric hospital of the country, using improved diagnostic methods to broaden our knowledge about the epidemiology of diarrheal disease. In this report, 66 children with watery diarrhea randomly selected from a previous series (Gonzalez et al., 1999; O’Ryan et al., 2001) were analyzed to determine the frequency of calicivirus, rotavirus, astrovirus, and enteric adenovirus.
2.2. Rota6irus diagnosis Stool samples were tested for rotavirus within a week of collection using Pathfinder™ Rotavirus Direct Antigen Detection System (Kallestad Diagnostics, Austin, TX, USA) following the manufacturer’s instructions.
2.3. Enteric adeno6irus detection Stool samples were tested using a commercial EIA kit (Premier Adenoclone® Type 40/41, Cambridge Biotech, Worcester, MA, USA) following the manufacturer’s recommendations.
2.4. Viral RNA extraction Viral RNA from calicivirus and astrovirus was extracted from 150 ml of 10% stool suspension using TRIzol (Gibco-BRL, Life Technologies™, New York, NY, USA), following manufacturer’s recommendations.
2.5. Astro6irus detection
2. Materials and methods
Astrovirus coproantigen was detected using a commercial EIA kit (IDEIATM Astrovirus Dako Diagnostics Ltd., Denmark) and by RT-PCR with the MON 340 and MON 348 primers as previously described (Belliot et al., 1997). RTPCR products were analyzed by agarose gel electrophoresis (2%) followed by visualization under ultraviolet light after ethidium bromide staining.
2.1. Specimen collection
2.6. Calici6irus detection
Stool samples from 659 children under 3 years of age attending the Out-patient Division of Ricardo Gutie´ rrez Children’s Hospital of Buenos Aires, with diagnosis of acute watery diarrheas, were collected from September 1997 to August 1998. Watery diarrhea was defined as three or more semiliquid or liquid stools per day of B 5 days duration. Sixty-six randomly selected cases (10%) were tested for rotavirus and adenovirus type 40/41 by EIA, calicivirus by RT-PCR, and astrovirus by RT-PCR and EIA. All samples were stored at −20 °C until testing.
Two different RT-PCR methods were used to amplify the RNA polymerase region of the virus (Ando et al., 1995; Jiang et al., 1999). The first method uses a generic pair of primers (P289 and P290), which are designed to detect NLVs (319 bp) and SLVs (331 bp) genus of calicivirus. The second technique uses two sets of primers (GI and GII) to amplify (123 bp) genogroups I and II separately, both part of NLVs genus. Amplification products were analyzed by electrophoresis in 2% agarose gels and visualized under ultraviolet light after ethidium bromide staining.
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
199
2.7. Nucleotide sequencing
3. Results
Calicivirus DNA from 4 samples (BA1026, BA1160, BA1322 and BA1443) was purified using a commercial kit (Wizard® PCR Preps, Promega, Madison, WI, USA) and then sequenced in both senses (ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer, Applied Biosystems, Foster city, CA, USA). Each sample was cluster related after performing a phylogenetic analysis. Raw sequence data were first analyzed by CHROMAS software (version 1.3, Mc Carthy 1996, Griffith University, Queensland, Australia) and forward and reverse sequence data of each sample were aligned using the EditSeq and MegAlign programs (DNASTAR Inc. Software, Madison, WI, USA) to obtain the final consensus sequence. Complete alignment was performed with Clustal X and alignment was analyzed using Kimura 2 parameters as a method of substitution and Neighbor-Joining to reconstruct the phylogenetic tree using MEGA version 2.0 (Kumar et al. 2001, Molecular Evolutionary Genetics Analysis, Pennsylvania State University, University Park, and Arizona State University, Tempe, Arizona). The statistical significance of phylogenies constructed was estimated by bootstrap analysis with 1000 pseudoreplicate data sets.
The 66 randomly selected diarrhea cases presented a monthly distribution similar to the total number of diarrhea cases analyzed during the study period (Spearman Rank Correlation, r= 0.9788, PB0.0001) (Fig. 1). One or more viral agents were identified in 32 out of 66 (48.5%) diarrhea cases tested. Dual infections combining calicivirus with other agents were detected in 8 out of 16 calicivirus cases (50%) (Fig. 2). The frequency of each individual agent was as follows: rotavirus 17 (25.8%), calicivirus 16 (24.2%), astrovirus 5 (7.6%) and enteric adenovirus 2 (3.0%). Comparison between RT-PCR and EIA for astrovirus detection showed 5 positive samples by RT-PCR and 3 by EIA. The seasonal trends of rotavirus and calicivirus detection reflected a similar pattern, with peaks during the cold months of the year (April to August), while most virus-negative cases (probably bacterial diarrheas) were observed in summer months (January and February) (Fig. 3). Thirteen out of 16 caliciviruses identified were detected using P289/290 primers while five were amplified using GI/GII primers. Therefore, only two samples were amplified with both RT-PCR methods.
Fig. 1. Monthly distribution of the number of total diarrhea cases collected at the Buenos Aires Children’s Hospital and the randomly selected diarrhea cases analyzed in this report.
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Fig. 2. Dual and mono infections detected at the Buenos Aires Children’s Hospital.
Nucleotide sequence analysis of 274 bp region of RNA polymerase gene showed that three of four strains (BA1160, BA1322 and BA1443) clustered with SLV genus and only one strain clustered with NLV genus (BA1026). In SLV genus, BA1443 showed 99.9% amino acid identity with Manchester virus, while BA1322 and BA1160 showed 97.8% and 95.6% identity with London/92 virus, respectively. Finally, BA1026 showed 85.7% amino acid identity with Lorsdale virus (Table 1). Surprisingly, BA1322 which was amplified with the GII set of primers (originally designed for the detection of genogroup II, NLV genus) was identified as a member of SLV genus by sequencing analysis.
4. Discussion This report attempts to complete a comprehensive study of the viral etiology of diarrhea in a randomly selected subset of cases attended for diarrhea in the Buenos Aires Children’s Hospital. The study was based on a low socio-economic status urban population of the Greater Buenos Aires, consulting at this reference hospital for diarrheal disease. The original study was designed to evaluate the rotavirus disease burden in the hospital. While rotavirus was extensively studied in our country, only one single report on enteric adenovirus performed from 1983 to 1987
(Mistchenko et al., 1992) was published, and little is known about the frequency of other viral agents of diarrhea. In this study, a viral pathogen was identified in 48.5% of the diarrhea cases analyzed. Both rotavirus (25.8%) and calicivirus (24.2%) presented high frequency in this population, while astrovirus was detected in 7.6% and enteric adenovirus in 3.0% of cases. Commercial ELISA tests are considered the ‘gold standard’ and there is no clear evidence to show that PCR tests are better diagnosis methods for rotavirus. The ELISA test used in this study only detects 5× 105 pfu/ml and RT-PCR was shown to detect 5× 103 pfu/ml (Buesa et al., 1996). However, rotavirus disease usually eliminates so many viral particles (1010 pfu/ml) in feces that molecular methods have not improved viral diagnosis. Although enteric adenovirus had been previously detected at similar rates, this is the first report of the identification of calicivirus and astrovirus in the country. Other recent studies described calicivirus identification at a similar frequencies (Pang et al., 2000, 1999). The four calicivirus samples sequenced confirmed RT-PCR results, suggesting that the PCR methods used are suitable for diagnosis. Although a small number of cases were randomly selected and analyzed, calicivirus and rotavirus appeared to be frequent agents of diarrhea in the population under study. Interestingly, a high frequency (8/32 positive samples; 25%) of mixed infections was
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
also observed, to all of which calicivirus was associated. Seasonality for viral pathogens was observed in autumn and winter months (March to August), when both rotavirus and calicivirus presented peaks. Diarrheal disease is known to present a higher prevalence from January to June in Argentina (Bok et al., 2001, 1999; Gonzalez et al., 1999; O’Ryan et al., 2001), with peaks of rotavirus-negative cases (most probably bacterial
201
diarrheas) from January to March and rotaviruspositive cases from April to June. The seasonal pattern herein described for caliciviruses provides further support for the higher prevalence of viral pathogens during the winter. Similarly, although a reduced number of cases were analyzed, we did not find any significant difference among the age distribution of viral agents. These results encourage us to extend the analysis of calicivirus epidemiology to the whole set of
Fig. 3. Calicivirus and rotavirus seasonality.
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
202
Table 1 Calicivirus detected in Greater Buenos Aires, Argentina from September 1997 to August 1998 Sample identification RT-PCR Ia
BA1026 BA1090 BA1160 BA1261 BA1314 BA1322 BA1354 BA1389 BA1399 BA1409 BA1443 BA1465 BA1473 BA1480 BA1765 BA1797
+ + + − + + + + − + + + − + + +
RT-PCR IIb
GI
GII
− − − − − − − − − − − − − − − −
+ − − + − + − − + − − − + − − −
Amino acidd identity (%)
Nucleotided identity (%)
Genetic identityc
85.7 nd 95.6 nd nd 97.8 nd nd nd nd 99.9 nd nd nd nd nd
84.7 nd 85.0 nd nd 94.9 nd nd nd nd 95.6 nd nd nd nd nd
Lorsdale virus London/92 virus
London/92 virus
Manchester virus
a
Jiang et al., 1999; Ando et al., 1995; c The sequences used were available from GenBank (Manchester virus [accession no. X86559]; London virus [accession no. U95645]; Lorsdale virus [accession no. 86557]; BA1026 [accession no. AY048118]; BA1160 [accession no. AY048117]; BA1322 [accession no. AY048116]; BA1443 [accession no. AY048115]); d Nucleotide and amino acid sequence identities of a 274 bp RNA polymerase region. Bold letters indicate sequenced samples. nd, not done. b
samples of the study. Viral diagnosis is seldom available in Argentine hospitals and coproculture results are mostly negative. This report provides clear evidence on the frequency of viral pathogens as agents of infantile diarrhea. Rotavirus diagnosis in Argentine pediatric hospital laboratories should be implemented, given its repeatedly observed high frequency. Our data also supports the need to develop rapid and sensitive tests for calicivirus diagnosis. The introduction of affordable viral diagnosis in our pediatric hospitals will improve patient care by reducing the unnecessary use of antibiotics. Finally, this study shows a high frequency of viral agents, mainly calicivirus and rotavirus, in children attended for diarrhea in Buenos Aires Children’s Hospital. Rotavirus diarrhea may be prevented with vaccines, and the same has been recently proposed for caliciviruses (Ball et al., 1999; Tacket et al., 2000). Our data suggest that a
significant proportion of the diarrheal disease burden in a developing country like Argentina might be prevented in the near future.
Acknowledgements We are grateful to Fernanda Gonzalez for her assistance in the sample collection. This study was partially supported by a grant from Alberto Roemmers Foundation to JAG.
References Ando T, Monroe SS, Gentsch JR, Jin Q, Lewis DC, Glass RI. Detection and differentiation of antigenically distinct small round-structured viruses (Norwalk-like viruses) by reverse transcription-PCR and southern hybridization. J Clin Microbiol 1995;33(1):64 – 71.
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203 Ball JM, Graham DY, Opekun AR, Gilger MA, Guerrero RA, Estes MK. Recombinant Norwalk virus-like particles given orally to volunteers: phase I study. Gastroenterology 1999;117(1):40 – 8 [see comments]. Belliot G, Laveran H, Monroe SS. Detection and genetic differentiation of human astroviruses: phylogenetic grouping varies by coding region. Arch Virol 1997;142(7):1323 – 34. Bern C, Martines J, de Zoysa I, Glass RI. The magnitude of the global problem of diarrhoeal disease: a 10-year update. Bull World Health Organ 1992;70(6):705 – 14. Bok K, Castagnaro N, Borsa A, Nates S, Espul C, Fay O, Fabri A, Grinstein S, Miceli I, Matson D and Gomez J. Surveillance for rotavirus in Argentina, J Med Virol 2001; 65(1):190– 8.. Bok K, Castagnaro NC, Diaz NE, Borsa A, Cagnoli MR, Nates S, Yudowsky S, Espul C, Cuello H, Fay O, Brunet B, Ues OC, Santoro R, Grinstein S, Gonzalez F, Miceli I, Gomez JA. Rotavirus laboratory network: results after 1 year of observation. Rev Argent Microbiol 1999;31(1):1 – 12. Buesa J, Colomina J, Raga J, Villanueva A, Prat J. Evaluation of reverse transcription and polymerase chain reaction (RT/PCR) for the detection of rotaviruses: applications of the assay. Res Virol 1996;147:353 –61. Gomez JA, Biscotti EL, Bercovich JA, Grinstein S. Epidemiology of human rotaviruses in Argentina as determined by RNA genome electrophoresis. Intervirology 1986;26(3):174 – 80. Gonzalez F, Sordo M, Rowensztien G, Sabbag L, Roussos A, De Petre E, Garello M, Medei A, Bok K, Grinstien S,
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Gomez J. Diarrea por Rotavirus, Impacto en un Hospital de Nin˜ os de Buenos Aires. Medicine (Buenos Aires) 1999;59:321 – 6. 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(1 – 2):145 – 54. Mistchenko AS, Huberman KH, Gomez JA, Grinstein S. Epidemiology of enteric adenovirus infection in prospectively monitored Argentine families. Epidemiol Infect 1992;109(3):539 – 46. Murray CJ, Lopez AD. Global mortality, disability, and the contribution of risk factors: global burden of disease study. Lancet 1997;349(9063):1436 –42. O’Ryan M, Pe´ rez-Schael I, Mamani N, Pen˜ a A, Salinas B, Gonza´ lez G, Gonza´ lez F, Go´ mez J. Rotavirus associated outpatient visits and hospitalizations in South America: a prospective study in three large sentinel hospitals. Pediatr Infect Dis J 2001;20(7):685 – 93. Pang XL, Honma S, Nakata S, Vesikari T. Human caliciviruses in acute gastroenteritis of young children in the community. J Infect Dis 2000;181(2):S288 – 94. Pang XL, Joensuu J, Vesikari T. Human calicivirus-associated sporadic gastroenteritis in Finnish children less than 2 years of age followed prospectively during a rotavirus vaccine trial. Pediatr Infect Dis J 1999;18(5):420 – 6. Tacket CO, Mason HS, Losonsky G, Estes MK, Levine MM, Arntzen CJ. Human immune responses to a novel norwalk virus vaccine delivered in transgenic potatoes. J Infect Dis 2000;182(1):302 – 5.
Identification of viral agents causing gastroenteritis among children in Buenos Aires, Argentina Alesio Bereciartu, Karin Bok, Jorge Go´mez * Viral Gastroenteritis Laboratory, National Institute of Infectious Diseases, ANLIS Dr. Carlos G. Malbra´n, Buenos Aires, Argentina Received 20 August 2001; received in revised form 16 November 2001; accepted 1 February 2002
Abstract Background: Gastroenteritis is one of the most common diseases affecting children and rotavirus is the major etiological agent worldwide. Although the role of rotaviruses as a causal agent of gastroenteritis has been previously established in Argentina, little is known about the frequency of other gastrointestinal viruses. Objecti6es: The present study was designed to determine the frequency of calicivirus in comparison with rotavirus, astrovirus and adenovirus type 40/41 in our country. Study design: Sixty six diarrhea patients (10%) = 3 years of age, were randomly selected from a previous series (9/1997–8/1998) and virus frequency determined using RT-PCR and/or commercial EIAs. In addition, we sequenced the RNA polymerase region of calicivirus-positive samples to characterize Argentine strains. Results: We detected at least one viral agent in 48.5% (32/66) of the patients. Rotavirus, calicivirus, astrovirus, and enteric adenovirus were found in 17, 16, 5, and 2 children, respectively. Sequence analysis of 4 calicivirus positive samples allowed us to identify a strain clustered to Lorsdale virus, one strain clustered with Manchester virus and two strains clustered with London/92 virus. Conclusions: Our data suggest that calicivirus and rotavirus are major agents of gastroenteritis in children younger than 3 years of age in Buenos Aires, Argentina. Unfortunately, viral diagnosis is seldom available in Argentine hospitals and coproculture results are mostly negative. Our data supports the need to develop rapid and sensitive tests for calicivirus diagnosis. The introduction of affordable viral diagnosis in our pediatric hospitals will improve patient care by reducing the unnecessary use of antibiotics. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Infantile diarrhea; Calicivirus; Rotavirus; Astrovirus; Enteric adenovirus
Abbre6iations: RT-PCR, reverse transcriptase polymerase chain reaction; EIA, enzyme immunoassay; RNA, ribonucleic acid; NLV, Norwalk-like virus; SLV, Sapporo-like virus. * Corresponding author. Present address: Av. Velez Sarsfield 563, (1281) Buenos Aires, Argentina. Tel./fax: + 5411-4302-5064. E-mail address: [email protected] (J. Go´mez).
1. Introduction Gastrointestinal diseases are one of the most frequent and preventable health problems worldwide, presenting high rates of morbidity and mortality especially in developing countries. In
1386-6532/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 2 ) 0 0 0 1 0 - 0
198
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
addition, acute diarrheas cause an estimated onethird of all hospitalizations per year in children younger than 5 years of age (Bern et al., 1992; Murray and Lopez, 1997), which carry a heavy cost. During 1997 (National Surveillance System, Ministry of Health), 431,000 diarrhea cases were reported in the Argentine public health sector in children younger than 5 years of age, with virtually one death per day. While the role of rotaviruses as a causal agent of gastroenteritis has been previously established in the country (Bok et al., 2001, 1999; Gomez et al., 1986; Gonzalez et al., 1999; O’Ryan et al., 2001), little is known about the frequency of other gastrointestinal viruses such as calicivirus, astrovirus and adenovirus type 40/41. An accurate understanding of the relative prevalence of these agents would help design strategies to control the disease. The present study was performed to determine the relative frequency of these viruses in a pediatric hospital of the country, using improved diagnostic methods to broaden our knowledge about the epidemiology of diarrheal disease. In this report, 66 children with watery diarrhea randomly selected from a previous series (Gonzalez et al., 1999; O’Ryan et al., 2001) were analyzed to determine the frequency of calicivirus, rotavirus, astrovirus, and enteric adenovirus.
2.2. Rota6irus diagnosis Stool samples were tested for rotavirus within a week of collection using Pathfinder™ Rotavirus Direct Antigen Detection System (Kallestad Diagnostics, Austin, TX, USA) following the manufacturer’s instructions.
2.3. Enteric adeno6irus detection Stool samples were tested using a commercial EIA kit (Premier Adenoclone® Type 40/41, Cambridge Biotech, Worcester, MA, USA) following the manufacturer’s recommendations.
2.4. Viral RNA extraction Viral RNA from calicivirus and astrovirus was extracted from 150 ml of 10% stool suspension using TRIzol (Gibco-BRL, Life Technologies™, New York, NY, USA), following manufacturer’s recommendations.
2.5. Astro6irus detection
2. Materials and methods
Astrovirus coproantigen was detected using a commercial EIA kit (IDEIATM Astrovirus Dako Diagnostics Ltd., Denmark) and by RT-PCR with the MON 340 and MON 348 primers as previously described (Belliot et al., 1997). RTPCR products were analyzed by agarose gel electrophoresis (2%) followed by visualization under ultraviolet light after ethidium bromide staining.
2.1. Specimen collection
2.6. Calici6irus detection
Stool samples from 659 children under 3 years of age attending the Out-patient Division of Ricardo Gutie´ rrez Children’s Hospital of Buenos Aires, with diagnosis of acute watery diarrheas, were collected from September 1997 to August 1998. Watery diarrhea was defined as three or more semiliquid or liquid stools per day of B 5 days duration. Sixty-six randomly selected cases (10%) were tested for rotavirus and adenovirus type 40/41 by EIA, calicivirus by RT-PCR, and astrovirus by RT-PCR and EIA. All samples were stored at −20 °C until testing.
Two different RT-PCR methods were used to amplify the RNA polymerase region of the virus (Ando et al., 1995; Jiang et al., 1999). The first method uses a generic pair of primers (P289 and P290), which are designed to detect NLVs (319 bp) and SLVs (331 bp) genus of calicivirus. The second technique uses two sets of primers (GI and GII) to amplify (123 bp) genogroups I and II separately, both part of NLVs genus. Amplification products were analyzed by electrophoresis in 2% agarose gels and visualized under ultraviolet light after ethidium bromide staining.
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
199
2.7. Nucleotide sequencing
3. Results
Calicivirus DNA from 4 samples (BA1026, BA1160, BA1322 and BA1443) was purified using a commercial kit (Wizard® PCR Preps, Promega, Madison, WI, USA) and then sequenced in both senses (ABI PRISM Big Dye Terminator Cycle Sequencing Ready Reaction Kit, Perkin Elmer, Applied Biosystems, Foster city, CA, USA). Each sample was cluster related after performing a phylogenetic analysis. Raw sequence data were first analyzed by CHROMAS software (version 1.3, Mc Carthy 1996, Griffith University, Queensland, Australia) and forward and reverse sequence data of each sample were aligned using the EditSeq and MegAlign programs (DNASTAR Inc. Software, Madison, WI, USA) to obtain the final consensus sequence. Complete alignment was performed with Clustal X and alignment was analyzed using Kimura 2 parameters as a method of substitution and Neighbor-Joining to reconstruct the phylogenetic tree using MEGA version 2.0 (Kumar et al. 2001, Molecular Evolutionary Genetics Analysis, Pennsylvania State University, University Park, and Arizona State University, Tempe, Arizona). The statistical significance of phylogenies constructed was estimated by bootstrap analysis with 1000 pseudoreplicate data sets.
The 66 randomly selected diarrhea cases presented a monthly distribution similar to the total number of diarrhea cases analyzed during the study period (Spearman Rank Correlation, r= 0.9788, PB0.0001) (Fig. 1). One or more viral agents were identified in 32 out of 66 (48.5%) diarrhea cases tested. Dual infections combining calicivirus with other agents were detected in 8 out of 16 calicivirus cases (50%) (Fig. 2). The frequency of each individual agent was as follows: rotavirus 17 (25.8%), calicivirus 16 (24.2%), astrovirus 5 (7.6%) and enteric adenovirus 2 (3.0%). Comparison between RT-PCR and EIA for astrovirus detection showed 5 positive samples by RT-PCR and 3 by EIA. The seasonal trends of rotavirus and calicivirus detection reflected a similar pattern, with peaks during the cold months of the year (April to August), while most virus-negative cases (probably bacterial diarrheas) were observed in summer months (January and February) (Fig. 3). Thirteen out of 16 caliciviruses identified were detected using P289/290 primers while five were amplified using GI/GII primers. Therefore, only two samples were amplified with both RT-PCR methods.
Fig. 1. Monthly distribution of the number of total diarrhea cases collected at the Buenos Aires Children’s Hospital and the randomly selected diarrhea cases analyzed in this report.
200
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
Fig. 2. Dual and mono infections detected at the Buenos Aires Children’s Hospital.
Nucleotide sequence analysis of 274 bp region of RNA polymerase gene showed that three of four strains (BA1160, BA1322 and BA1443) clustered with SLV genus and only one strain clustered with NLV genus (BA1026). In SLV genus, BA1443 showed 99.9% amino acid identity with Manchester virus, while BA1322 and BA1160 showed 97.8% and 95.6% identity with London/92 virus, respectively. Finally, BA1026 showed 85.7% amino acid identity with Lorsdale virus (Table 1). Surprisingly, BA1322 which was amplified with the GII set of primers (originally designed for the detection of genogroup II, NLV genus) was identified as a member of SLV genus by sequencing analysis.
4. Discussion This report attempts to complete a comprehensive study of the viral etiology of diarrhea in a randomly selected subset of cases attended for diarrhea in the Buenos Aires Children’s Hospital. The study was based on a low socio-economic status urban population of the Greater Buenos Aires, consulting at this reference hospital for diarrheal disease. The original study was designed to evaluate the rotavirus disease burden in the hospital. While rotavirus was extensively studied in our country, only one single report on enteric adenovirus performed from 1983 to 1987
(Mistchenko et al., 1992) was published, and little is known about the frequency of other viral agents of diarrhea. In this study, a viral pathogen was identified in 48.5% of the diarrhea cases analyzed. Both rotavirus (25.8%) and calicivirus (24.2%) presented high frequency in this population, while astrovirus was detected in 7.6% and enteric adenovirus in 3.0% of cases. Commercial ELISA tests are considered the ‘gold standard’ and there is no clear evidence to show that PCR tests are better diagnosis methods for rotavirus. The ELISA test used in this study only detects 5× 105 pfu/ml and RT-PCR was shown to detect 5× 103 pfu/ml (Buesa et al., 1996). However, rotavirus disease usually eliminates so many viral particles (1010 pfu/ml) in feces that molecular methods have not improved viral diagnosis. Although enteric adenovirus had been previously detected at similar rates, this is the first report of the identification of calicivirus and astrovirus in the country. Other recent studies described calicivirus identification at a similar frequencies (Pang et al., 2000, 1999). The four calicivirus samples sequenced confirmed RT-PCR results, suggesting that the PCR methods used are suitable for diagnosis. Although a small number of cases were randomly selected and analyzed, calicivirus and rotavirus appeared to be frequent agents of diarrhea in the population under study. Interestingly, a high frequency (8/32 positive samples; 25%) of mixed infections was
A. Bereciartu et al. / Journal of Clinical Virology 25 (2002) 197–203
also observed, to all of which calicivirus was associated. Seasonality for viral pathogens was observed in autumn and winter months (March to August), when both rotavirus and calicivirus presented peaks. Diarrheal disease is known to present a higher prevalence from January to June in Argentina (Bok et al., 2001, 1999; Gonzalez et al., 1999; O’Ryan et al., 2001), with peaks of rotavirus-negative cases (most probably bacterial
201
diarrheas) from January to March and rotaviruspositive cases from April to June. The seasonal pattern herein described for caliciviruses provides further support for the higher prevalence of viral pathogens during the winter. Similarly, although a reduced number of cases were analyzed, we did not find any significant difference among the age distribution of viral agents. These results encourage us to extend the analysis of calicivirus epidemiology to the whole set of
Fig. 3. Calicivirus and rotavirus seasonality.
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202
Table 1 Calicivirus detected in Greater Buenos Aires, Argentina from September 1997 to August 1998 Sample identification RT-PCR Ia
BA1026 BA1090 BA1160 BA1261 BA1314 BA1322 BA1354 BA1389 BA1399 BA1409 BA1443 BA1465 BA1473 BA1480 BA1765 BA1797
+ + + − + + + + − + + + − + + +
RT-PCR IIb
GI
GII
− − − − − − − − − − − − − − − −
+ − − + − + − − + − − − + − − −
Amino acidd identity (%)
Nucleotided identity (%)
Genetic identityc
85.7 nd 95.6 nd nd 97.8 nd nd nd nd 99.9 nd nd nd nd nd
84.7 nd 85.0 nd nd 94.9 nd nd nd nd 95.6 nd nd nd nd nd
Lorsdale virus London/92 virus
London/92 virus
Manchester virus
a
Jiang et al., 1999; Ando et al., 1995; c The sequences used were available from GenBank (Manchester virus [accession no. X86559]; London virus [accession no. U95645]; Lorsdale virus [accession no. 86557]; BA1026 [accession no. AY048118]; BA1160 [accession no. AY048117]; BA1322 [accession no. AY048116]; BA1443 [accession no. AY048115]); d Nucleotide and amino acid sequence identities of a 274 bp RNA polymerase region. Bold letters indicate sequenced samples. nd, not done. b
samples of the study. Viral diagnosis is seldom available in Argentine hospitals and coproculture results are mostly negative. This report provides clear evidence on the frequency of viral pathogens as agents of infantile diarrhea. Rotavirus diagnosis in Argentine pediatric hospital laboratories should be implemented, given its repeatedly observed high frequency. Our data also supports the need to develop rapid and sensitive tests for calicivirus diagnosis. The introduction of affordable viral diagnosis in our pediatric hospitals will improve patient care by reducing the unnecessary use of antibiotics. Finally, this study shows a high frequency of viral agents, mainly calicivirus and rotavirus, in children attended for diarrhea in Buenos Aires Children’s Hospital. Rotavirus diarrhea may be prevented with vaccines, and the same has been recently proposed for caliciviruses (Ball et al., 1999; Tacket et al., 2000). Our data suggest that a
significant proportion of the diarrheal disease burden in a developing country like Argentina might be prevented in the near future.
Acknowledgements We are grateful to Fernanda Gonzalez for her assistance in the sample collection. This study was partially supported by a grant from Alberto Roemmers Foundation to JAG.
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