- Email: [email protected]
An outbreak of norovirus infection in an Italian residential-care facility for the elderly
Research Notes
CMI
9. Kane CT, Ndiaye HD, Diallo S et al. Quantitation of HIV-1 RNA in dried blood spots by the real-time NucliSENS EasyQ HIV-1 assay in Senegal. J Virol Methods 2008; 148: 291–295. 10. O’Shea S, Mullen J, Corbett K, Chrystie I, Newell ML, Banatvala JE. Use of dried whole blood spots for quantification of HIV-1 RNA. AIDS 1999; 13: 630–631. 11. Volberding PA. HIV quantification: clinical applications. Lancet 1996; 347: 71–73. 12. Tang N, Huang S, Salituro J et al. A RealTime HIV-1 viral load assay for automated quantitation of HIV-1 RNA in genetically diverse group M subtypes A–H, group O and group N samples. J Virol Methods 2007; 146: 236–245. 13. Schumacher W, Frick E, Kauselmann M, Maier-Hoyle V, Van der Vliet R, Babiel R. Fully automated quantification of human immunodeficiency virus (HIV) type 1 RNA in human plasma by the COBAS AmpliPrep/COBAS TaqMan system. J Clin Virol 2007; 38: 304–312. 14. Pasloske BL, Walkerpeach CR, Obermoeller RD, Winkler M, DuBois DB. Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards. J Clin Microbiol 1998; 36: 3590–3594. 15. Agresti A, Coull BA. Approximate is better than ‘exact’ for interval estimation of binomial proportions. Am Stat 1998; 52: 119–126. 16. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1996; 1: 307– 310. 17. Gueudin M, Plantier JC, Lemee V et al. Evaluation of the Roche Cobas TaqMan and Abbott RealTime extraction–quantification systems for HIV-1 subtypes. J Acquir Immune Defic Syndr 2007; 44: 500– 505. 18. Thomson MM, Na´jera R. Molecular epidemiology of HIV-1 variants in the global AIDS pandemic: an update. AIDS Rev 2005; 7: 210–224. 19. Romano L, Venturi G, Catucci M, De Milito A, Valensin PE, Zazzi M. Evaluation of cell-free and cell-associated peripheral blood human immunodeficiency virus type 1 RNA response to antiretroviral therapy. J Infect Dis 1999; 179: 361–366. 20. Zhang J, Crumpacker CS. Human immunodeficiency virus type 1 RNA in peripheral blood mononuclear cells of patients receiving prolonged highly active antiretroviral therapy. J Infect Dis 2001; 184: 1341–1344. 21. Zhang L, Ramratnam B, Tenner-Racz K et al. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med 1999; 340: 1605–1613. 22. Fransen K, Mortier D, Heyndrickx L, Verhofstede C, Janssens W, Van der Groen G. Isolation of HIV-1 RNA from plasma: evaluation of seven different methods for extraction (part two). J Virol Methods 1998; 76: 153–157.
An outbreak of norovirus infection in an Italian residential-care facility for the elderly M. C. Medici1, A. Morelli2, M. C. Arcangeletti1, A. Calderaro1, F. De Conto1, M. Martinelli 1, L. A. Abelli1, G. Dettori1 and C. Chezzi1 1) Section of Microbiology, Department of Pathology and Laboratory Medicine, University Medical School, Parma and 2) Public Hygiene Service, Department of Public Health, Local Health Unit, Langhirano, Parma, Italy
97
Abstract On December 2006, an outbreak of gastroenteritis occurred at a residential-care facility for the elderly in northern Italy. Thirtyfive of 61 individuals interviewed (attack rate, 57.4%) fell ill. In 94.3% of cases, the onset of illness was within 48 h of a Christmas party at the facility. Norovirus (NoV) was detected by RT-PCR in 24 of 31 individuals examined, including three asymptomatic food-handlers, in whom there was evidence of long-lasting excretion of viral particles. The identification of a sequence referring to the ‘2006a GII.4 NoV variant’ in all examined strains supported the hypothesis of a common point source. This retrospective cohort study is the first report on an outbreak of NoV gastroenteritis in an Italian residential-care facility for the elderly.
Keywords: Asymptomatic infection, norovirus outbreak, residential-care facility, sequence analysis, virus shedding Original Submission: 22 May 2008; 23 June 2008;
Revised Submission:
Accepted: 5 July 2008
Editor: J.-C. Desenclos Clin Microbiol Infect 2009; 15: 97–100 10.1111/j.1469-0691.2008.02117.x
Corresponding author and reprint requests: M. C. Medici, Section of Microbiology, Department of Pathology and Laboratory Medicine, University Medical School of Parma, Viale Antonio Gramsci, 14, 43100 Parma, Italy E-mail: [email protected]
Outbreaks of gastroenteritis due to norovirus (NoV) often occur during the winter months, and take place in closed and semi-closed settings. This retrospective cohort study describes an outbreak of gastroenteritis caused by NoV at a residential-care facility for the elderly in northern Italy. On 22 December 2006, the manager of the facility reported the occurrence of several cases of acute gastroenteritis among residents. All individuals had attended a Christmas party on 20 December at the facility. A case was defined as an illness in any resident, staff member or guest who took part in the event and experienced diarrhoea (three or more loose stools within 24 h) and/or vomiting. The food served on that occasion by individual serving (different food for residents and for staff members and guests) was suspected to be
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 93–102
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Clinical Microbiology and Infection, Volume 15 Number 1, January 2009
contaminated and thus the source of the infection. Face-toface interviews with 61 of the 126 individuals who attended the Christmas party (25 of 79 residents, 19 of 23 staff members, 17 of 24 guests) were undertaken by the local public health officer. These data were analysed by Epi Info 3.4 software (CDC, Atlanta, GA, USA), and data comparisons were performed by using chi-squared, Fisher’s exact and Mann– Whitney U- or G-tests, as appropriate. Thirty-five of the 61 (57.4%) interviewees had gastroenteritis. The median age of these 35 individuals was 74 years (range 19–97 years); this median age was significantly different (p <0.05) from that of the healthy individuals (45 years, range 27–86 years). In 94.3% (33 of 35 cases), the onset of illness was within 48 h of the Christmas party (mean 43 h, range 29– 74 h) (Fig. 1). The median duration of illness was 24 h (range 2–84 h). Illness was significantly associated with residency in the facility (relative risk 2.5) (Table 1). According to univariate analysis, the unique significant association between food consumed and illness was found to be thin soup (relative risk 1.9; p <0.05). Faecal samples collected from 31 individuals (residents and staff members only: 12 ill, eight healthy, and 11 with no information available) were examined at the Section of Microbiology of the University Hospital of Parma for Salmonella spp., Shigella spp., Yersinia spp., Vibrio spp., Campylobacter spp., Staphylococcus aureus and intestinal parasites (helminths and protozoa), according to standard protocols. In 11 cases, the presence of Clostridium perfringens was also investigated [1–3]. Virological investigations were performed on stool suspensions by electron microscopy, conventional cell culture, and RT-PCR targeting the NoV ORF1 gene [4]. NoV-positive results were confirmed by testing a second specimen aliquot. Kitchen environmental samples were subjected to culture tests for Salmonella spp., Bacillus cereus and
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TABLE 1. Attack rates and relative risks for interviewed individuals according to category, residential-care facility, Italy, December 2006 Category
No. of persons No. ill Attack Relative (n = 61) (n = 35) rate (% %) risk 95% % CI p-value
Guests 18 Staff members 18 Residents 25
6 8 21
33.3 44.4 84
Referent – – 1.3 0.6–3.0 NS 2.5 1.3–4.9 <0.001
CI, confidence interval; NS, not statistically significant.
C. perfringens [5–7], and yielded negative results. No virological investigation was performed on environmental samples. All faecal specimens except one (containing Blastocystis hominis) were negative for bacterial enteropathogens and intestinal parasites. When NoV RT-PCR was used, 24 specimens yielded positive results: 19 (79.2%) from residents and five (20.8%) from staff members, four of whom were asymptomatic, with no history of gastroenteritis before the outbreak. Three of the four NoV-positive asymptomatic staff members (75%) were food-handlers. All of the 12 individuals who were ill examined were NoV-positive. Sequencing of 11 NoV ORF1 amplicons (from specimens collected from either symptomatic or asymptomatic individuals) indicated a single outbreak virus strain and revealed the best fit (nucleotide identity 98.4%) with the new ‘2006a GII.4 variant’, namely GII.4/Terneuzen 70/2006/NL (accession number EF126964) [8]. This finding was confirmed by sequencing of ORF2 [9] and long ORF1–ORF2 fragments [10] in three cases. Phylogenetic analysis was conducted by using the neighbour-joining method and Kimura-2 correction parameter with MEGA software [11]. When follow-up samples of ten NoV-positive individuals were examined, the mean duration of NoV excretion was found to be 13 days (range 5–24 days) for six symptomatic
20 18
Guests (6)
16
Staff members (8) Residents (21)
No. of cases
14 12 10 8 6
Christmas party
4 2 0
FIG. 1. Epidemic curve for norovirus gastroam pm 20 December
am pm 21 December
am pm 22 December Date of onset
pm am 23 December
pm am 24 December
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 93–102
enteritis (n = 35) in the residential-care facility according to clinical onset (December 2006).
Research Notes
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individuals, 15 days (range 10–20 days) for two healthy individuals, and 17 days (range 10–24 days) for two individuals for whom no information was available. Semiquantitative analysis of serially diluted RNAs from initial and follow-up specimens (at a 20-day distance) from one asymptomatic food-handler and one symptomatic resident revealed a consistent decrease in the viral load (3 log reduction: from 10)3 to 100, last positive dilution) for the asymptomatic individual and a constant viral load for the symptomatic resident (10)2, last positive dilution). This study yielded an outbreak epidemic curve consistent with a common point source. This hypothesis was further supported by the identical nucleotide sequence detected in specimens collected from residents and staff members, both symptomatic and asymptomatic, i.e. that of the ‘2006a GII.4 NoV variant’ ciculating in The Netherlands [8] during the same period. The suspicion of a foodborne outbreak motivated the investigation. Several limitations, e.g. participation bias, the unreliability of most interviews due to dementia among the resident population and fear of suspension among kitchen staff members, and the absence of bacteriological and virological investigations of tap water and food, prevented confirmation of this suspicion. Thin soup eaten only by residents was associated with an increased risk of illness. However, residency was determined to be a risk factor in itself, probably due to the particular susceptibility of this population, on the basis of physiological and immunological conditions. Also, environmental contamination to which residents had been previously exposed, e.g. an episode of vomiting in a common area of the facility, or the presence of an infected staff member who had attended the six individuals eating thin soup, and who may have contaminated other food items served, in particular to the residents, cannot be excluded. This environmental contamination might explain, in addition to host factors, the high attack rate among the residents (84%). Insufficient hygiene, as well as the long-lasting high level of virus excretion among asymptomatic infected food-handlers, could also have played an important role in the spread of the pathogen and the contamination of food. Indeed, once an outbreak was suspected, enforcement of hygiene measures made possible the containment of the suspected outbreak and the avoidance of secondary transmission (person-to-person) of NoV. In conclusion, this event highlights the fact that in countries such as Italy, where NoV outbreaks are increasing in frequency [12–14], the lack of a surveillance system for nonbacterial gastroenteritis and the low level of awareness on the part of general practitioners and public health officers concerning NoV as a food-borne agent or a highly contaminating virus in the environment may allow this pathogen to go widely unrecognized.
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Acknowledgements The authors gratefully acknowledge F. Casula, H. P. Esteban Villanueva, S. Larini, M. G. Menozzi, F. Pinardi, P. Portincasa, S. Preti, S. Rossi and P. Somenzi (Section of Microbiology, Department of Pathology and Laboratory Medicine, University Medical School of Parma, Italy) for their expert assistance and contribution to the bacteriological, parasitic or virological investigations. CC and GD contributed equally to the coordination of the study.
Transparency Declaration This study was partly supported by the grant ‘Ricerca Scientifica FIL (ex 60%) 2006’, prot. 60A06-0979, from the University of Parma, Italy. The authors have no conflicting interests to declare.
References 1. Engelkirk PG, Duben-Engelkirk J, Dowell VR Jr. Principles and practice of clinical anaerobic bacteriology. Belmont, CA: Star Publishing Company, 1992. 2. Holdemon LV, Cato EP, Moore WEC (eds). Anaerobe laboratory manual, 4th edn. Blacksburg, VA: VPI Laboratory, Virginia Polytechnic Institute and State University, 1977. 3. Summanen P, Baron EJ, Citron DM, Strong CA, Wexler HM, Finegold SM. Wadsworth anaerobic bacteriology manual. Belmont, CA: Star Publishing Company, 1993. 4. Medici MC, Martinelli M, Ruggeri FM et al. Broadly reactive nested reverse transcription-PCR using an internal RNA standard control for detection of noroviruses in stool samples. J Clin Microbiol 2005; 43: 3772–3778. 5. Anonymous. Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella (EN ISO 6579:2002/Cor 1:2004). Geneva, Switzerland: International Organization for Standardization, 2004. 6. Anonymous. Microbiology of food and animal feeding stuffs—horizontal method for the enumeration of presumptive Bacillus cereus—colony count technique at 30 degrees C (EN ISO 7932:2004). Geneva, Switzerland: International Organization for Standardization, 2004. 7. Anonymous. Microbiology of food and animal feeding stuffs—horizontal method for the enumeration of Clostridium perfringens—colony-count technique (EN ISO 7937: 2005). Geneva, Switzerland: International Organization for Standardization, 2005. 8. Siebenga JJ, Vennema H, Renckens B et al. Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006. J Virol 2007; 81: 9932–9941. 9. Kojima S, Kageyama T, Fukushi S et al. Genogroup-specific PCR primers for detection of Norwalk-like viruses. J Virol Methods 2002; 100: 107–114. 10. Vennema H, de Bruin E, Koopmans M. Rational optimization of generic primers used for Norwalk-like virus detection by reverse transcriptase polymerase chain reaction. J Clin Virol 2002; 25: 233–235.
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11. Kumar S, Tamura K, Jakobsen IB, Nei M. MEGA2: molecular evolutionary genetics analysis software. Tempe, AZ: Arizona State University, 2001. 12. Boccia D, Tozzi AE, Cotter B et al. Waterborne outbreak of Norwalk-like virus gastroenteritis at a tourist resort, Italy. Emerg Infect Dis 2002; 8: 563–568. 13. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in south Italy. BMC Infect Dis 2004; 4: 37. 14. Le Guyader FS, Bon F, DeMedici D et al. Detection of multiple noroviruses associated with an international gastroenteritis outbreak linked to oyster consumption. J Clin Microbiol 2006; 44: 3878–3882.
Prevalence of infection with high-risk human papillomavirus in women in Colombia S. C. Soto-De Leon1, M. Camargo1, R. Sanchez1,2, S. Leon1, M. Urquiza1, J. Acosta1, D. Monsalve1, L. E. Rodriguez1, M. E. Patarroyo1,2 and M. A. Patarroyo1 1) Fundacio´n Instituto de Inmunologia de Colombia (FIDIC) and 2) Universidad Nacional de Colombia, Bogota, Colombia
Abstract The prevalence of human papillomavirus (HPV) infections in 2109 females inhabiting five cities of Colombia was determined. Of the 49.2% with an HPV infection, 59.8% were infected with more than one viral type. Species 7 (of the the genus Alphapapillomavirus) was associated with multiple infections. Analysis of the socio-demographic data revealed a statistically significant protective effect associated with the status of civil union (civil recognition of cohabitation without marriage), and indigenous ethnicity proved to be a risk factor for HPV infection. This is the first study comparing HPV infection among women from geographical regions of Colombia with different socio-cultural structures.
Keywords: Colombia, genotypes, high-risk HPV, human papillomavirus, multiple infections Original Submission: 13 February 2008; Submission: 22 July 2008;
Revised
Accepted: 23 July 2008
Editor: M. Clementi Clin Microbiol Infect 2009; 15: 100–102 10.1111/j.1469-0691.2008.02120.x
Corresponding author and reprint requests: M. A. Patarroyo, Cra 50 # 26-20, Bogota, Colombia E-mail: [email protected]
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Cervical cancer (CC) has been primarily associated with infection with high-risk human papillomavirus (HPV) (HR-HPV) types, mainly with Alphapapillomavirus species 7 (HPV-18, HPV39, HPV-45, HPV-59 and HPV-68) and species 9 (HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, HPV-58 and HPV-67) [1]. Although prior HPV infection is a necessary step for the development of CC, there are several other factors that can increase the risk of developing CC, including age, number of sexual partners, age at first intercourse, pregnancies, smoking habits, contraceptive methods, and co-infection with other pathogens or different HPV types [2]. Co-infection with other pathogens or different HPV types (which ranges in frequency between 30% and 40% around the world [3,4]) especially contributes to the severity of the lesions. Colombia is one of the countries with the highest incidence of CC in the world, the disease affecting 23–48 individuals per 100 000 females, mainly those aged 30–39 years [5]. The present study involved females from five geographical regions, where CC mortality rates ranged between 3.12 and 5.67 [6]: Leticia (Amazon jungle); Chaparral, mainly inhabited by a mestizo population; Tumaco, where most of the black females enrolled in this study live; Bogota, the country’s capital; and Girardot, a tourist destination. The study involved 2109 women (mean age 38.8 years), attending a local CC prevention programme during 2007, from the different regions as follows: 173 women from the League Against Cancer (Leticia, south-east Colombia); 174 women from the Hospital San Juan Bautista (Chaparral region); 921 women from the Hospital de Engativa (Bogota); 334 women from the Nuevo Hospital San Rafael de Girardot-Cundinamarca (central Colombia); and 508 women from the Hospital San Andres de Tumaco-Narin˜o (south-west Colombia). Each patient filled out a questionnaire regarding risk factors and gave her signed consent. The study was supervised and approved by each institution’s ethics committee. All cervical epithelium samples were preserved in 95% ethanol [7]. Samples were washed with phosphate-buffered saline and digested in lysis buffer [8]. To assess cervical sample integrity, the human b-globin gene was amplified using the GH20/PC04 primers, as previously described [9]. Samples yielding DNA of adequate quality were amplified following two PCR protocols using consensus primers GP5+/ GP6+ and MY09/MY11 [10]. HPV DNA-positive samples, according to one or both generic amplification reactions, were assessed by DNA amplification with HR-HPV-16, HR-HPV-31, HR-HPV-33, HR-HPV-45, HR-HPV-58 and HR-HPV-18 typespecific primers [11–13]. The viral types HPV-16, HPV-31, HPV-33 and HPV-58 were grouped into species 9, and viral types HPV-18 and HPV-45 were grouped into species 7 [14].
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 93–102
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9. Kane CT, Ndiaye HD, Diallo S et al. Quantitation of HIV-1 RNA in dried blood spots by the real-time NucliSENS EasyQ HIV-1 assay in Senegal. J Virol Methods 2008; 148: 291–295. 10. O’Shea S, Mullen J, Corbett K, Chrystie I, Newell ML, Banatvala JE. Use of dried whole blood spots for quantification of HIV-1 RNA. AIDS 1999; 13: 630–631. 11. Volberding PA. HIV quantification: clinical applications. Lancet 1996; 347: 71–73. 12. Tang N, Huang S, Salituro J et al. A RealTime HIV-1 viral load assay for automated quantitation of HIV-1 RNA in genetically diverse group M subtypes A–H, group O and group N samples. J Virol Methods 2007; 146: 236–245. 13. Schumacher W, Frick E, Kauselmann M, Maier-Hoyle V, Van der Vliet R, Babiel R. Fully automated quantification of human immunodeficiency virus (HIV) type 1 RNA in human plasma by the COBAS AmpliPrep/COBAS TaqMan system. J Clin Virol 2007; 38: 304–312. 14. Pasloske BL, Walkerpeach CR, Obermoeller RD, Winkler M, DuBois DB. Armored RNA technology for production of ribonuclease-resistant viral RNA controls and standards. J Clin Microbiol 1998; 36: 3590–3594. 15. Agresti A, Coull BA. Approximate is better than ‘exact’ for interval estimation of binomial proportions. Am Stat 1998; 52: 119–126. 16. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1996; 1: 307– 310. 17. Gueudin M, Plantier JC, Lemee V et al. Evaluation of the Roche Cobas TaqMan and Abbott RealTime extraction–quantification systems for HIV-1 subtypes. J Acquir Immune Defic Syndr 2007; 44: 500– 505. 18. Thomson MM, Na´jera R. Molecular epidemiology of HIV-1 variants in the global AIDS pandemic: an update. AIDS Rev 2005; 7: 210–224. 19. Romano L, Venturi G, Catucci M, De Milito A, Valensin PE, Zazzi M. Evaluation of cell-free and cell-associated peripheral blood human immunodeficiency virus type 1 RNA response to antiretroviral therapy. J Infect Dis 1999; 179: 361–366. 20. Zhang J, Crumpacker CS. Human immunodeficiency virus type 1 RNA in peripheral blood mononuclear cells of patients receiving prolonged highly active antiretroviral therapy. J Infect Dis 2001; 184: 1341–1344. 21. Zhang L, Ramratnam B, Tenner-Racz K et al. Quantifying residual HIV-1 replication in patients receiving combination antiretroviral therapy. N Engl J Med 1999; 340: 1605–1613. 22. Fransen K, Mortier D, Heyndrickx L, Verhofstede C, Janssens W, Van der Groen G. Isolation of HIV-1 RNA from plasma: evaluation of seven different methods for extraction (part two). J Virol Methods 1998; 76: 153–157.
An outbreak of norovirus infection in an Italian residential-care facility for the elderly M. C. Medici1, A. Morelli2, M. C. Arcangeletti1, A. Calderaro1, F. De Conto1, M. Martinelli 1, L. A. Abelli1, G. Dettori1 and C. Chezzi1 1) Section of Microbiology, Department of Pathology and Laboratory Medicine, University Medical School, Parma and 2) Public Hygiene Service, Department of Public Health, Local Health Unit, Langhirano, Parma, Italy
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Abstract On December 2006, an outbreak of gastroenteritis occurred at a residential-care facility for the elderly in northern Italy. Thirtyfive of 61 individuals interviewed (attack rate, 57.4%) fell ill. In 94.3% of cases, the onset of illness was within 48 h of a Christmas party at the facility. Norovirus (NoV) was detected by RT-PCR in 24 of 31 individuals examined, including three asymptomatic food-handlers, in whom there was evidence of long-lasting excretion of viral particles. The identification of a sequence referring to the ‘2006a GII.4 NoV variant’ in all examined strains supported the hypothesis of a common point source. This retrospective cohort study is the first report on an outbreak of NoV gastroenteritis in an Italian residential-care facility for the elderly.
Keywords: Asymptomatic infection, norovirus outbreak, residential-care facility, sequence analysis, virus shedding Original Submission: 22 May 2008; 23 June 2008;
Revised Submission:
Accepted: 5 July 2008
Editor: J.-C. Desenclos Clin Microbiol Infect 2009; 15: 97–100 10.1111/j.1469-0691.2008.02117.x
Corresponding author and reprint requests: M. C. Medici, Section of Microbiology, Department of Pathology and Laboratory Medicine, University Medical School of Parma, Viale Antonio Gramsci, 14, 43100 Parma, Italy E-mail: [email protected]
Outbreaks of gastroenteritis due to norovirus (NoV) often occur during the winter months, and take place in closed and semi-closed settings. This retrospective cohort study describes an outbreak of gastroenteritis caused by NoV at a residential-care facility for the elderly in northern Italy. On 22 December 2006, the manager of the facility reported the occurrence of several cases of acute gastroenteritis among residents. All individuals had attended a Christmas party on 20 December at the facility. A case was defined as an illness in any resident, staff member or guest who took part in the event and experienced diarrhoea (three or more loose stools within 24 h) and/or vomiting. The food served on that occasion by individual serving (different food for residents and for staff members and guests) was suspected to be
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 93–102
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Clinical Microbiology and Infection, Volume 15 Number 1, January 2009
contaminated and thus the source of the infection. Face-toface interviews with 61 of the 126 individuals who attended the Christmas party (25 of 79 residents, 19 of 23 staff members, 17 of 24 guests) were undertaken by the local public health officer. These data were analysed by Epi Info 3.4 software (CDC, Atlanta, GA, USA), and data comparisons were performed by using chi-squared, Fisher’s exact and Mann– Whitney U- or G-tests, as appropriate. Thirty-five of the 61 (57.4%) interviewees had gastroenteritis. The median age of these 35 individuals was 74 years (range 19–97 years); this median age was significantly different (p <0.05) from that of the healthy individuals (45 years, range 27–86 years). In 94.3% (33 of 35 cases), the onset of illness was within 48 h of the Christmas party (mean 43 h, range 29– 74 h) (Fig. 1). The median duration of illness was 24 h (range 2–84 h). Illness was significantly associated with residency in the facility (relative risk 2.5) (Table 1). According to univariate analysis, the unique significant association between food consumed and illness was found to be thin soup (relative risk 1.9; p <0.05). Faecal samples collected from 31 individuals (residents and staff members only: 12 ill, eight healthy, and 11 with no information available) were examined at the Section of Microbiology of the University Hospital of Parma for Salmonella spp., Shigella spp., Yersinia spp., Vibrio spp., Campylobacter spp., Staphylococcus aureus and intestinal parasites (helminths and protozoa), according to standard protocols. In 11 cases, the presence of Clostridium perfringens was also investigated [1–3]. Virological investigations were performed on stool suspensions by electron microscopy, conventional cell culture, and RT-PCR targeting the NoV ORF1 gene [4]. NoV-positive results were confirmed by testing a second specimen aliquot. Kitchen environmental samples were subjected to culture tests for Salmonella spp., Bacillus cereus and
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TABLE 1. Attack rates and relative risks for interviewed individuals according to category, residential-care facility, Italy, December 2006 Category
No. of persons No. ill Attack Relative (n = 61) (n = 35) rate (% %) risk 95% % CI p-value
Guests 18 Staff members 18 Residents 25
6 8 21
33.3 44.4 84
Referent – – 1.3 0.6–3.0 NS 2.5 1.3–4.9 <0.001
CI, confidence interval; NS, not statistically significant.
C. perfringens [5–7], and yielded negative results. No virological investigation was performed on environmental samples. All faecal specimens except one (containing Blastocystis hominis) were negative for bacterial enteropathogens and intestinal parasites. When NoV RT-PCR was used, 24 specimens yielded positive results: 19 (79.2%) from residents and five (20.8%) from staff members, four of whom were asymptomatic, with no history of gastroenteritis before the outbreak. Three of the four NoV-positive asymptomatic staff members (75%) were food-handlers. All of the 12 individuals who were ill examined were NoV-positive. Sequencing of 11 NoV ORF1 amplicons (from specimens collected from either symptomatic or asymptomatic individuals) indicated a single outbreak virus strain and revealed the best fit (nucleotide identity 98.4%) with the new ‘2006a GII.4 variant’, namely GII.4/Terneuzen 70/2006/NL (accession number EF126964) [8]. This finding was confirmed by sequencing of ORF2 [9] and long ORF1–ORF2 fragments [10] in three cases. Phylogenetic analysis was conducted by using the neighbour-joining method and Kimura-2 correction parameter with MEGA software [11]. When follow-up samples of ten NoV-positive individuals were examined, the mean duration of NoV excretion was found to be 13 days (range 5–24 days) for six symptomatic
20 18
Guests (6)
16
Staff members (8) Residents (21)
No. of cases
14 12 10 8 6
Christmas party
4 2 0
FIG. 1. Epidemic curve for norovirus gastroam pm 20 December
am pm 21 December
am pm 22 December Date of onset
pm am 23 December
pm am 24 December
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 93–102
enteritis (n = 35) in the residential-care facility according to clinical onset (December 2006).
Research Notes
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individuals, 15 days (range 10–20 days) for two healthy individuals, and 17 days (range 10–24 days) for two individuals for whom no information was available. Semiquantitative analysis of serially diluted RNAs from initial and follow-up specimens (at a 20-day distance) from one asymptomatic food-handler and one symptomatic resident revealed a consistent decrease in the viral load (3 log reduction: from 10)3 to 100, last positive dilution) for the asymptomatic individual and a constant viral load for the symptomatic resident (10)2, last positive dilution). This study yielded an outbreak epidemic curve consistent with a common point source. This hypothesis was further supported by the identical nucleotide sequence detected in specimens collected from residents and staff members, both symptomatic and asymptomatic, i.e. that of the ‘2006a GII.4 NoV variant’ ciculating in The Netherlands [8] during the same period. The suspicion of a foodborne outbreak motivated the investigation. Several limitations, e.g. participation bias, the unreliability of most interviews due to dementia among the resident population and fear of suspension among kitchen staff members, and the absence of bacteriological and virological investigations of tap water and food, prevented confirmation of this suspicion. Thin soup eaten only by residents was associated with an increased risk of illness. However, residency was determined to be a risk factor in itself, probably due to the particular susceptibility of this population, on the basis of physiological and immunological conditions. Also, environmental contamination to which residents had been previously exposed, e.g. an episode of vomiting in a common area of the facility, or the presence of an infected staff member who had attended the six individuals eating thin soup, and who may have contaminated other food items served, in particular to the residents, cannot be excluded. This environmental contamination might explain, in addition to host factors, the high attack rate among the residents (84%). Insufficient hygiene, as well as the long-lasting high level of virus excretion among asymptomatic infected food-handlers, could also have played an important role in the spread of the pathogen and the contamination of food. Indeed, once an outbreak was suspected, enforcement of hygiene measures made possible the containment of the suspected outbreak and the avoidance of secondary transmission (person-to-person) of NoV. In conclusion, this event highlights the fact that in countries such as Italy, where NoV outbreaks are increasing in frequency [12–14], the lack of a surveillance system for nonbacterial gastroenteritis and the low level of awareness on the part of general practitioners and public health officers concerning NoV as a food-borne agent or a highly contaminating virus in the environment may allow this pathogen to go widely unrecognized.
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Acknowledgements The authors gratefully acknowledge F. Casula, H. P. Esteban Villanueva, S. Larini, M. G. Menozzi, F. Pinardi, P. Portincasa, S. Preti, S. Rossi and P. Somenzi (Section of Microbiology, Department of Pathology and Laboratory Medicine, University Medical School of Parma, Italy) for their expert assistance and contribution to the bacteriological, parasitic or virological investigations. CC and GD contributed equally to the coordination of the study.
Transparency Declaration This study was partly supported by the grant ‘Ricerca Scientifica FIL (ex 60%) 2006’, prot. 60A06-0979, from the University of Parma, Italy. The authors have no conflicting interests to declare.
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11. Kumar S, Tamura K, Jakobsen IB, Nei M. MEGA2: molecular evolutionary genetics analysis software. Tempe, AZ: Arizona State University, 2001. 12. Boccia D, Tozzi AE, Cotter B et al. Waterborne outbreak of Norwalk-like virus gastroenteritis at a tourist resort, Italy. Emerg Infect Dis 2002; 8: 563–568. 13. Prato R, Lopalco PL, Chironna M, Barbuti G, Germinario C, Quarto M. Norovirus gastroenteritis general outbreak associated with raw shellfish consumption in south Italy. BMC Infect Dis 2004; 4: 37. 14. Le Guyader FS, Bon F, DeMedici D et al. Detection of multiple noroviruses associated with an international gastroenteritis outbreak linked to oyster consumption. J Clin Microbiol 2006; 44: 3878–3882.
Prevalence of infection with high-risk human papillomavirus in women in Colombia S. C. Soto-De Leon1, M. Camargo1, R. Sanchez1,2, S. Leon1, M. Urquiza1, J. Acosta1, D. Monsalve1, L. E. Rodriguez1, M. E. Patarroyo1,2 and M. A. Patarroyo1 1) Fundacio´n Instituto de Inmunologia de Colombia (FIDIC) and 2) Universidad Nacional de Colombia, Bogota, Colombia
Abstract The prevalence of human papillomavirus (HPV) infections in 2109 females inhabiting five cities of Colombia was determined. Of the 49.2% with an HPV infection, 59.8% were infected with more than one viral type. Species 7 (of the the genus Alphapapillomavirus) was associated with multiple infections. Analysis of the socio-demographic data revealed a statistically significant protective effect associated with the status of civil union (civil recognition of cohabitation without marriage), and indigenous ethnicity proved to be a risk factor for HPV infection. This is the first study comparing HPV infection among women from geographical regions of Colombia with different socio-cultural structures.
Keywords: Colombia, genotypes, high-risk HPV, human papillomavirus, multiple infections Original Submission: 13 February 2008; Submission: 22 July 2008;
Revised
Accepted: 23 July 2008
Editor: M. Clementi Clin Microbiol Infect 2009; 15: 100–102 10.1111/j.1469-0691.2008.02120.x
Corresponding author and reprint requests: M. A. Patarroyo, Cra 50 # 26-20, Bogota, Colombia E-mail: [email protected]
CMI
Cervical cancer (CC) has been primarily associated with infection with high-risk human papillomavirus (HPV) (HR-HPV) types, mainly with Alphapapillomavirus species 7 (HPV-18, HPV39, HPV-45, HPV-59 and HPV-68) and species 9 (HPV-16, HPV-31, HPV-33, HPV-35, HPV-52, HPV-58 and HPV-67) [1]. Although prior HPV infection is a necessary step for the development of CC, there are several other factors that can increase the risk of developing CC, including age, number of sexual partners, age at first intercourse, pregnancies, smoking habits, contraceptive methods, and co-infection with other pathogens or different HPV types [2]. Co-infection with other pathogens or different HPV types (which ranges in frequency between 30% and 40% around the world [3,4]) especially contributes to the severity of the lesions. Colombia is one of the countries with the highest incidence of CC in the world, the disease affecting 23–48 individuals per 100 000 females, mainly those aged 30–39 years [5]. The present study involved females from five geographical regions, where CC mortality rates ranged between 3.12 and 5.67 [6]: Leticia (Amazon jungle); Chaparral, mainly inhabited by a mestizo population; Tumaco, where most of the black females enrolled in this study live; Bogota, the country’s capital; and Girardot, a tourist destination. The study involved 2109 women (mean age 38.8 years), attending a local CC prevention programme during 2007, from the different regions as follows: 173 women from the League Against Cancer (Leticia, south-east Colombia); 174 women from the Hospital San Juan Bautista (Chaparral region); 921 women from the Hospital de Engativa (Bogota); 334 women from the Nuevo Hospital San Rafael de Girardot-Cundinamarca (central Colombia); and 508 women from the Hospital San Andres de Tumaco-Narin˜o (south-west Colombia). Each patient filled out a questionnaire regarding risk factors and gave her signed consent. The study was supervised and approved by each institution’s ethics committee. All cervical epithelium samples were preserved in 95% ethanol [7]. Samples were washed with phosphate-buffered saline and digested in lysis buffer [8]. To assess cervical sample integrity, the human b-globin gene was amplified using the GH20/PC04 primers, as previously described [9]. Samples yielding DNA of adequate quality were amplified following two PCR protocols using consensus primers GP5+/ GP6+ and MY09/MY11 [10]. HPV DNA-positive samples, according to one or both generic amplification reactions, were assessed by DNA amplification with HR-HPV-16, HR-HPV-31, HR-HPV-33, HR-HPV-45, HR-HPV-58 and HR-HPV-18 typespecific primers [11–13]. The viral types HPV-16, HPV-31, HPV-33 and HPV-58 were grouped into species 9, and viral types HPV-18 and HPV-45 were grouped into species 7 [14].
ª2009 The Authors Journal Compilation ª2009 European Society of Clinical Microbiology and Infectious Diseases, CMI, 15, 93–102