- Email: [email protected]
Hepatitis C virus transmission during colonoscopy evidenced by phylogenetic analysis
Journal of Clinical Virology 57 (2013) 263–266
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Hepatitis C virus transmission during colonoscopy evidenced by phylogenetic analysis Verónica Saludes a,b,1 , María Esteve c , Irma Casas c , Vicente Ausina a,d , Elisa Martró a,b,∗ a Microbiology Service, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain b CIBER en Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain c Department of Preventive Medicine, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain d CIBER Enfermedades Respiratorias (CIBERES), Bunyola, Spain
a r t i c l e
i n f o
Article history: Received 3 December 2012 Received in revised form 1 February 2013 Accepted 10 March 2013 Keywords: HCV Colonoscopy E1–E2 region Phylogenetic analysis Nosocomial person-to-person Transmission Antiviral treatment
a b s t r a c t Background: Nosocomial transmission events still play an important role in hepatitis C virus (HCV) spreading. Among most reported medical procedures involved in nosocomial transmission, endoscopy procedures remain controversial and might be underestimated. Objective: The aim of the study was to investigate a case of nosocomial person-to-person transmission of HCV in an endoscopy unit. Study design: An acute HCV infection was detected in a person that had undergone a colonoscopy after an HCV-infected patient. Serum samples from both persons were subjected to a molecular epidemiology study. The HCV NS5B genetic region was amplified and directly sequenced and the E1–E2 region was amplified, cloned and sequenced (20 clones per specimen). All sequences were subjected to phylogenetic analyses. A conventional epidemiological investigation was performed to determine the most likely cause of HCV transmission. Results: NS5B sequence analysis revealed that both persons were infected with closely related HCV-1b strains. Furthermore, phylogenetic analysis of E1–E2 sequences evidenced a direct transmission between patients. The epidemiological investigation pointed out to anesthetic procedures as the most likely source of HCV transmission. The index case, not having spontaneously cleared the infection 10 months after infection, required antiviral treatment, which resulted in a sustained virological response. Conclusions: The molecular epidemiology study performed provided evidence of a person-to-person transmission of HCV during a colonoscopy procedure, and the anesthetic procedure was the most likely source of HCV transmission. This study highlights the importance of strictly following standard precautions by healthcare workers in order to prevent nosocomial HCV transmission. © 2013 Elsevier B.V. All rights reserved.
1. Background Although rarely reported, nosocomial transmission of hepatitis C virus (HCV) continues occurring in developed countries.1,2 Several medical procedures have been associated with HCV transmission, being haemodialysis the most frequently reported one.3–5 Transmission during colonoscopy procedures has also been reported.6–10 However, the direct transmission by the use of a
contaminated colonoscope remains controversial and might be underestimated. 2. Objectives The aim of the study was to investigate a case of nosocomial person-to-person transmission of HCV in an endoscopy unit by conventional and molecular epidemiology analyses. 3. Study design
Abbreviations: HCV, hepatitis C virus; RNA, ribonucleic acid. ∗ Corresponding author at: Microbiology Service, Hospital Universitari Germans Trias i Pujol, Ctra. del Canyet s/n, 08916 Badalona, Spain. Tel.: +34 934 978 894; fax: +34 934 978 895. E-mail address: [email protected] (E. Martró). 1 Present address: Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain. 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.03.007
An acute HCV infection was diagnosed in February 2010 in a person that had undergone a colonoscopy in a Spanish hospital. The Hospital Director and the Preventive Medicine Group initiated an investigation. The suspicion of nosocomial transmission was reported to the Public Health Authority.
264
V. Saludes et al. / Journal of Clinical Virology 57 (2013) 263–266
Medical records of the patient were reviewed and data on risk factors, serological status, diagnosis, diagnostic procedures, surgical interventions, medications and other treatments were collected. A preliminary epidemiological investigation identified a colonoscopy in November 2009 as the only risk factor for HCV infection. The review of the list of patients that had undergone a colonoscopy on the same date at that endoscopy unit revealed that an HCV-infected patient had been submitted to this procedure right before the index case. Thus, this patient was considered to be the possible source of the transmission. No other patients had undergone a colonoscopy after the index case on the same day. In order to confirm the possible epidemiological link between the putative source patient and the index case, a molecular epidemiology study of the virus infecting both patients was carried out. Serum samples from the source patient (November 2009 and February 2010) and the index case (December 2009) were analyzed. Extracted viral RNA was reverse transcribed using random hexamers and the M-MLV reverse transcriptase (Invitrogen). The HCV genotype and subtype were determined by sequencing and phylogenetic analyses of a 222-bp fragment belonging to the NS5B region as previously described.11 Obtained sequences were aligned together with reference sequences from the LANL HCV Database (http://hcv.lanl.gov/) and from 37 unrelated patients with chronic hepatitis C followed at the same hospital using ClustalW. A maximum-likelihood phylogenetic tree was obtained with PHYML.12 Bootstrap support was determined by 1000 resamplings of the sequences in PHYML. Additionally, a phylogenetic analysis of the more variable HCV genetic region E1–E2 was carried out to further characterize the relationship between both viral strains. To this end, hemi-nested PCR amplification of the E1–E2 region, cloning and sequencing were performed as previously described.13 Nineteen to 20 clones per serum sample belonging to a region of 532-bp were subjected to phylogenetic analyses as described above, together with sequences from 39 unrelated patients with chronic hepatitis C followed at the same hospital. All sequences generated were deposited in the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) with accession numbers HF558299–HF558360.
4. Results HCV NS5B sequence analysis revealed that both persons were infected with subtype 1b, and that both viral strains were closely related (data not shown). Additionally, the phylogenetic analysis of the more variable HCV genetic region E1–E2 evidenced that the viral sequences obtained from the source and the index patients were more closely related to each other than to the rest of sequences obtained from other patients (Fig. 1). Since the sequences from both patients formed a well-supported monophyletic group (84% bootstrap support), the direct transmission between them was confirmed. Among the E1–E2 sequences from the index case only two different viral variants or haplotypes were identified, one of them being the major variant with a frequency of 18/19. The sequences from the two serum samples of the source case collected three months apart were genetically similar (distinct groups could not be identified in the phylogenetic tree), and both showed a higher degree of genetic heterogeneity (19 and 20 haplotypes were found among the 20 cloned sequences for each serum sample). Once the nosocomial patient-to-patient HCV transmission was phylogenetically confirmed, an epidemiological investigation was performed to find out the most likely unsafe medical practice related to that colonoscopy. All procedures of the endoscopy unit were reviewed and staff members were asked to go over the disinfection methods used for colonoscopies, the procedures that
involved administration of parenteral anesthesia and adherence to standard universal precautions. The investigation pointed out to the anesthetic procedure through the use of monodose propofol vials as the most likely source of HCV transmission, either in the course of intravenous drug administration or catheter handling due to a sporadic unintended human error. Although HCV transmission during the colonoscopy procedure itself could not entirely be ruled out, it would be less likely because a biopsy was not performed in this patient. Recommendations given to the health-care personnel in order to prevent further episodes included the strict adherence to standard precautions, the adequate manipulation of the administration equipment and the adequate use of monodose anesthetic vials to avoid parenteral transmission. The source case had a viral load of 7,141,501 IU/mL (Abbott RealTime HCV, Abbott Molecular) in February 2010 and was not receiving antiviral treatment. The index case was initially tested for HCV-specific antibodies (November 2009) resulting in a gray-zone result (VITROS anti-HCV assay, Ortho Clinical Diagnostics); a few days later an HCV-RNA determination revealed a viral load of 313,277 IU/mL. HCV seroconversion was confirmed in February 2012, when viral load had declined to 42 IU/mL. Not having spontaneously cleared the virus ten months after infection (HCV-RNA levels fluctuated at low levels), the index patient finally initiated antiviral therapy (pegylated interferon-␣ plus ribavirin) in September 2010, with a pretreatment viral load of 152 IU/mL. Given that the patient showed a rapid virological response at week 4 but HCV RNA was again detectable at week 12 of therapy, treatment was extended to 72 weeks. The patient had a 100% treatment adherence and finally responded successfully showing a sustained virological response in August 2012.
5. Discussion The molecular epidemiology study performed provided evidence of a person-to-person transmission of HCV during a colonoscopy procedure. The sequences from both patients formed a well-supported monophyletic group in the phylogenetic tree of the HCV E1–E2 genomic region. Sequences from the source case showed a high degree of genetic heterogeneity, which is a typical pattern observed in chronic infections,6 while sequences from the index case showed a lower genetic variability; this observed pattern is compatible with a recent infection, especially when the inoculum is small. Furthermore, the virus was almost cleared by the host’s immune system during the acute infection; the viral load had decreased to 42 IU/mL (3.9 log decline) three months after the transmission event. Thus, the viral population transmitted from the source patient to the index case was subjected to a strong selective pressure resulting in a bottleneck effect that narrowed the original spectrum of variants down to a few that were finally able to persist in the new host (only two variants were identified and one of them was clearly dominant). The E1–E2 region studied includes the hypervariable region 1, which is the most variable region in the whole HCV genome as it is targeted by host neutralizing antibodies and plays a role in immune escape. This could explain a rapid divergence of the E1–E2 sequences found in the index case from those seen in the source patient. The epidemiological investigation pointed out to anesthetic procedures as the most likely source of HCV transmission, either in the course of intravenous drug administration or catheter handling, due to a sporadic unintended human error. Although no biopsies had been taken, transmission during the colonoscopy procedure itself could not be completely ruled out. This is in line with previous reports on patient-to-patient HCV transmission events associated with a colonoscopy procedure.6,9 In contrast, several studies have
V. Saludes et al. / Journal of Clinical Virology 57 (2013) 263–266
265
2639 FN676756
Outgroup sequences
3313 FN676904 1746 FN676459 2449 FN676716 0953 FN676185 1957 FN676560 1119 FN676272 3580 FN675985 2121 FN676577 0955 1667 FN676414 3906 FN676051 0587 FN676116 1313 FN676294 3612 FN676010 2395 FN676686 3249 FN676872 3403 FN676917 3227 FN676828 1616 FN676372 2196 FN676622 1852 FN676491 3946 0747 FN676128 4051 FN676076 0801 FN676149 1955 FN676534 0183 FN676078 2349 FN676639 2570 FN676755 1459 FN676336 3247 FN676853 1711 FN676452 2193 FN676599 1066 FN676220 2357 FN676670 1434 FN676312 1634 FN676394 1548 FN676351
100
84
71
0.05
Fig. 1. Maximum likelihood phylogenetic tree of the E1–E2 region. For each serum sample, 19–20 sequences were obtained by cloning and sequencing. Hasegawa, Kishino and Yano substitution model (proportion of invariable sites: 0.400, Gamma shape parameter: 0.952). Nodes supported with a bootstrap value > 75% (1000 replicates) are indicated. Sequences from other patients are identified with isolate and accession numbers. The bar represents substitutions per nucleotide position. Triangle, index case; circle, first serum sample of the source patient; square, second serum sample of the source patient.
266
V. Saludes et al. / Journal of Clinical Virology 57 (2013) 263–266
strongly associated unsafe injection practices during the anesthetic procedure with HCV transmission in endoscopy units.7,8,10 The case described here highlights the importance of strictly following standard precautions by healthcare workers in order to prevent HCV transmission events through unsafe medical practices. In addition, this study shows the usefulness of phylogenetic analyses of viral genomic regions with a high level of variability for resolving viral transmission events. Authorship/contributors VS, ME, IC, VA and EM contributed to the study conception and design, data acquisition, analysis and interpretation. VS, ME and EM drafted the article and IC and VA critically revised it. All authors approved the final version to be published. Funding This study was funded by Hospital Universitari Germans Trias i Pujol, and was carried out with the support of grant CP09/00044 from “Ministerio de Economía y Competitividad”, within the “Plan Nacional de Investigación científica, Desarrollo e Innovación Tecnológica (I+D+I)” (EM). Competing interests None declared. Ethical approval Not required.
References 1. Cornberg M, Razavi HA, Alberti A, Bernasconi E, Buti M, Cooper C, et al. A systematic review of hepatitis C virus epidemiology in Europe, Canada and Israel. Liver Int 2011;31:30–60. 2. Perz JF, Grytdal S, Beck S, Fireteanu AM, Poissant T, Rizzo E, et al. Case–control study of hepatitis B and hepatitis C in older adults: do healthcare exposures contribute to burden of new infections? Hepatology 2012 [Epub ahead of print]. 3. Lanini S, Abbate I, Puro V, Soscia F, Albertoni F, Battisti W, et al. Molecular epidemiology of a hepatitis C virus epidemic in a haemodialysis unit: outbreak investigation and infection outcome. BMC Infect Dis 2010;10:257. 4. Thompson ND, Novak RT, Datta D, Cotter S, Arduino MJ, Patel PR, et al. Hepatitis C virus transmission in hemodialysis units: importance of infection control practices and aseptic technique. Infect Control Hosp Epidemiol 2009;30:900–3. 5. Centers for Disease Control and Prevention (CDC). Hepatitis C virus transmission at an outpatient hemodialysis unit – New York, 2001–2008. MMWR Morb Mortal Wkly Rep 2009;58:189–94. 6. González-Candelas F, Guiral S, Carbó R, Valero A, Vanaclocha H, González F, et al. Patient-to-patient transmission of hepatitis C virus (HCV) during colonoscopy diagnosis. Virol J 2010;7:217. 7. Fischer GE, Schaefer MK, Labus BJ, Sands L, Rowley P, Azzam IA, et al. Hepatitis C virus infections from unsafe injection practices at an endoscopy clinic in Las Vegas, Nevada, 2007–2008. Clin Infect Dis 2010;51:267–73. 8. Gutelius B, Perz JF, Parker MM, Hallack R, Stricof R, Clement EJ, et al. Multiple clusters of hepatitis virus infections associated with anesthesia for outpatient endoscopy procedures. Gastroenterology 2010;139:163–70. 9. Bronowicki JP, Venard V, Botté C, Monhoven N, Gastin I, Choné L, et al. Patientto-patient transmission of hepatitis C virus during colonoscopy. N Engl J Med 1997;337:237–40. 10. Tallis GF, Ryan GM, Lambert SB, Bowden DS, McCaw R, Birch CJ, et al. Evidence of patient-to-patient transmission of hepatitis C virus through contaminated intravenous anaesthetic ampoules. J Viral Hepat 2003;10:234–9. 11. Martró E, González V, Buckton AJ, Saludes V, Fernández G, Matas L, et al. Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5 noncoding and nonstructural 5b genomic regions. J Clin Microbiol 2008;46:192–7. 12. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003;52:696–704. 13. Saludes V, Bracho MA, Valero O, Ardèvol M, Planas R, González-Candelas F, et al. Baseline prediction of combination therapy outcome in hepatitis C virus 1b infected patients by discriminant analysis using viral and host factors. PLoS ONE 2010;5:e14132.
Contents lists available at SciVerse ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Hepatitis C virus transmission during colonoscopy evidenced by phylogenetic analysis Verónica Saludes a,b,1 , María Esteve c , Irma Casas c , Vicente Ausina a,d , Elisa Martró a,b,∗ a Microbiology Service, Fundació Institut d’Investigació en Ciències de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain b CIBER en Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain c Department of Preventive Medicine, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Badalona, Spain d CIBER Enfermedades Respiratorias (CIBERES), Bunyola, Spain
a r t i c l e
i n f o
Article history: Received 3 December 2012 Received in revised form 1 February 2013 Accepted 10 March 2013 Keywords: HCV Colonoscopy E1–E2 region Phylogenetic analysis Nosocomial person-to-person Transmission Antiviral treatment
a b s t r a c t Background: Nosocomial transmission events still play an important role in hepatitis C virus (HCV) spreading. Among most reported medical procedures involved in nosocomial transmission, endoscopy procedures remain controversial and might be underestimated. Objective: The aim of the study was to investigate a case of nosocomial person-to-person transmission of HCV in an endoscopy unit. Study design: An acute HCV infection was detected in a person that had undergone a colonoscopy after an HCV-infected patient. Serum samples from both persons were subjected to a molecular epidemiology study. The HCV NS5B genetic region was amplified and directly sequenced and the E1–E2 region was amplified, cloned and sequenced (20 clones per specimen). All sequences were subjected to phylogenetic analyses. A conventional epidemiological investigation was performed to determine the most likely cause of HCV transmission. Results: NS5B sequence analysis revealed that both persons were infected with closely related HCV-1b strains. Furthermore, phylogenetic analysis of E1–E2 sequences evidenced a direct transmission between patients. The epidemiological investigation pointed out to anesthetic procedures as the most likely source of HCV transmission. The index case, not having spontaneously cleared the infection 10 months after infection, required antiviral treatment, which resulted in a sustained virological response. Conclusions: The molecular epidemiology study performed provided evidence of a person-to-person transmission of HCV during a colonoscopy procedure, and the anesthetic procedure was the most likely source of HCV transmission. This study highlights the importance of strictly following standard precautions by healthcare workers in order to prevent nosocomial HCV transmission. © 2013 Elsevier B.V. All rights reserved.
1. Background Although rarely reported, nosocomial transmission of hepatitis C virus (HCV) continues occurring in developed countries.1,2 Several medical procedures have been associated with HCV transmission, being haemodialysis the most frequently reported one.3–5 Transmission during colonoscopy procedures has also been reported.6–10 However, the direct transmission by the use of a
contaminated colonoscope remains controversial and might be underestimated. 2. Objectives The aim of the study was to investigate a case of nosocomial person-to-person transmission of HCV in an endoscopy unit by conventional and molecular epidemiology analyses. 3. Study design
Abbreviations: HCV, hepatitis C virus; RNA, ribonucleic acid. ∗ Corresponding author at: Microbiology Service, Hospital Universitari Germans Trias i Pujol, Ctra. del Canyet s/n, 08916 Badalona, Spain. Tel.: +34 934 978 894; fax: +34 934 978 895. E-mail address: [email protected] (E. Martró). 1 Present address: Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain. 1386-6532/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jcv.2013.03.007
An acute HCV infection was diagnosed in February 2010 in a person that had undergone a colonoscopy in a Spanish hospital. The Hospital Director and the Preventive Medicine Group initiated an investigation. The suspicion of nosocomial transmission was reported to the Public Health Authority.
264
V. Saludes et al. / Journal of Clinical Virology 57 (2013) 263–266
Medical records of the patient were reviewed and data on risk factors, serological status, diagnosis, diagnostic procedures, surgical interventions, medications and other treatments were collected. A preliminary epidemiological investigation identified a colonoscopy in November 2009 as the only risk factor for HCV infection. The review of the list of patients that had undergone a colonoscopy on the same date at that endoscopy unit revealed that an HCV-infected patient had been submitted to this procedure right before the index case. Thus, this patient was considered to be the possible source of the transmission. No other patients had undergone a colonoscopy after the index case on the same day. In order to confirm the possible epidemiological link between the putative source patient and the index case, a molecular epidemiology study of the virus infecting both patients was carried out. Serum samples from the source patient (November 2009 and February 2010) and the index case (December 2009) were analyzed. Extracted viral RNA was reverse transcribed using random hexamers and the M-MLV reverse transcriptase (Invitrogen). The HCV genotype and subtype were determined by sequencing and phylogenetic analyses of a 222-bp fragment belonging to the NS5B region as previously described.11 Obtained sequences were aligned together with reference sequences from the LANL HCV Database (http://hcv.lanl.gov/) and from 37 unrelated patients with chronic hepatitis C followed at the same hospital using ClustalW. A maximum-likelihood phylogenetic tree was obtained with PHYML.12 Bootstrap support was determined by 1000 resamplings of the sequences in PHYML. Additionally, a phylogenetic analysis of the more variable HCV genetic region E1–E2 was carried out to further characterize the relationship between both viral strains. To this end, hemi-nested PCR amplification of the E1–E2 region, cloning and sequencing were performed as previously described.13 Nineteen to 20 clones per serum sample belonging to a region of 532-bp were subjected to phylogenetic analyses as described above, together with sequences from 39 unrelated patients with chronic hepatitis C followed at the same hospital. All sequences generated were deposited in the EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl/) with accession numbers HF558299–HF558360.
4. Results HCV NS5B sequence analysis revealed that both persons were infected with subtype 1b, and that both viral strains were closely related (data not shown). Additionally, the phylogenetic analysis of the more variable HCV genetic region E1–E2 evidenced that the viral sequences obtained from the source and the index patients were more closely related to each other than to the rest of sequences obtained from other patients (Fig. 1). Since the sequences from both patients formed a well-supported monophyletic group (84% bootstrap support), the direct transmission between them was confirmed. Among the E1–E2 sequences from the index case only two different viral variants or haplotypes were identified, one of them being the major variant with a frequency of 18/19. The sequences from the two serum samples of the source case collected three months apart were genetically similar (distinct groups could not be identified in the phylogenetic tree), and both showed a higher degree of genetic heterogeneity (19 and 20 haplotypes were found among the 20 cloned sequences for each serum sample). Once the nosocomial patient-to-patient HCV transmission was phylogenetically confirmed, an epidemiological investigation was performed to find out the most likely unsafe medical practice related to that colonoscopy. All procedures of the endoscopy unit were reviewed and staff members were asked to go over the disinfection methods used for colonoscopies, the procedures that
involved administration of parenteral anesthesia and adherence to standard universal precautions. The investigation pointed out to the anesthetic procedure through the use of monodose propofol vials as the most likely source of HCV transmission, either in the course of intravenous drug administration or catheter handling due to a sporadic unintended human error. Although HCV transmission during the colonoscopy procedure itself could not entirely be ruled out, it would be less likely because a biopsy was not performed in this patient. Recommendations given to the health-care personnel in order to prevent further episodes included the strict adherence to standard precautions, the adequate manipulation of the administration equipment and the adequate use of monodose anesthetic vials to avoid parenteral transmission. The source case had a viral load of 7,141,501 IU/mL (Abbott RealTime HCV, Abbott Molecular) in February 2010 and was not receiving antiviral treatment. The index case was initially tested for HCV-specific antibodies (November 2009) resulting in a gray-zone result (VITROS anti-HCV assay, Ortho Clinical Diagnostics); a few days later an HCV-RNA determination revealed a viral load of 313,277 IU/mL. HCV seroconversion was confirmed in February 2012, when viral load had declined to 42 IU/mL. Not having spontaneously cleared the virus ten months after infection (HCV-RNA levels fluctuated at low levels), the index patient finally initiated antiviral therapy (pegylated interferon-␣ plus ribavirin) in September 2010, with a pretreatment viral load of 152 IU/mL. Given that the patient showed a rapid virological response at week 4 but HCV RNA was again detectable at week 12 of therapy, treatment was extended to 72 weeks. The patient had a 100% treatment adherence and finally responded successfully showing a sustained virological response in August 2012.
5. Discussion The molecular epidemiology study performed provided evidence of a person-to-person transmission of HCV during a colonoscopy procedure. The sequences from both patients formed a well-supported monophyletic group in the phylogenetic tree of the HCV E1–E2 genomic region. Sequences from the source case showed a high degree of genetic heterogeneity, which is a typical pattern observed in chronic infections,6 while sequences from the index case showed a lower genetic variability; this observed pattern is compatible with a recent infection, especially when the inoculum is small. Furthermore, the virus was almost cleared by the host’s immune system during the acute infection; the viral load had decreased to 42 IU/mL (3.9 log decline) three months after the transmission event. Thus, the viral population transmitted from the source patient to the index case was subjected to a strong selective pressure resulting in a bottleneck effect that narrowed the original spectrum of variants down to a few that were finally able to persist in the new host (only two variants were identified and one of them was clearly dominant). The E1–E2 region studied includes the hypervariable region 1, which is the most variable region in the whole HCV genome as it is targeted by host neutralizing antibodies and plays a role in immune escape. This could explain a rapid divergence of the E1–E2 sequences found in the index case from those seen in the source patient. The epidemiological investigation pointed out to anesthetic procedures as the most likely source of HCV transmission, either in the course of intravenous drug administration or catheter handling, due to a sporadic unintended human error. Although no biopsies had been taken, transmission during the colonoscopy procedure itself could not be completely ruled out. This is in line with previous reports on patient-to-patient HCV transmission events associated with a colonoscopy procedure.6,9 In contrast, several studies have
V. Saludes et al. / Journal of Clinical Virology 57 (2013) 263–266
265
2639 FN676756
Outgroup sequences
3313 FN676904 1746 FN676459 2449 FN676716 0953 FN676185 1957 FN676560 1119 FN676272 3580 FN675985 2121 FN676577 0955 1667 FN676414 3906 FN676051 0587 FN676116 1313 FN676294 3612 FN676010 2395 FN676686 3249 FN676872 3403 FN676917 3227 FN676828 1616 FN676372 2196 FN676622 1852 FN676491 3946 0747 FN676128 4051 FN676076 0801 FN676149 1955 FN676534 0183 FN676078 2349 FN676639 2570 FN676755 1459 FN676336 3247 FN676853 1711 FN676452 2193 FN676599 1066 FN676220 2357 FN676670 1434 FN676312 1634 FN676394 1548 FN676351
100
84
71
0.05
Fig. 1. Maximum likelihood phylogenetic tree of the E1–E2 region. For each serum sample, 19–20 sequences were obtained by cloning and sequencing. Hasegawa, Kishino and Yano substitution model (proportion of invariable sites: 0.400, Gamma shape parameter: 0.952). Nodes supported with a bootstrap value > 75% (1000 replicates) are indicated. Sequences from other patients are identified with isolate and accession numbers. The bar represents substitutions per nucleotide position. Triangle, index case; circle, first serum sample of the source patient; square, second serum sample of the source patient.
266
V. Saludes et al. / Journal of Clinical Virology 57 (2013) 263–266
strongly associated unsafe injection practices during the anesthetic procedure with HCV transmission in endoscopy units.7,8,10 The case described here highlights the importance of strictly following standard precautions by healthcare workers in order to prevent HCV transmission events through unsafe medical practices. In addition, this study shows the usefulness of phylogenetic analyses of viral genomic regions with a high level of variability for resolving viral transmission events. Authorship/contributors VS, ME, IC, VA and EM contributed to the study conception and design, data acquisition, analysis and interpretation. VS, ME and EM drafted the article and IC and VA critically revised it. All authors approved the final version to be published. Funding This study was funded by Hospital Universitari Germans Trias i Pujol, and was carried out with the support of grant CP09/00044 from “Ministerio de Economía y Competitividad”, within the “Plan Nacional de Investigación científica, Desarrollo e Innovación Tecnológica (I+D+I)” (EM). Competing interests None declared. Ethical approval Not required.
References 1. Cornberg M, Razavi HA, Alberti A, Bernasconi E, Buti M, Cooper C, et al. A systematic review of hepatitis C virus epidemiology in Europe, Canada and Israel. Liver Int 2011;31:30–60. 2. Perz JF, Grytdal S, Beck S, Fireteanu AM, Poissant T, Rizzo E, et al. Case–control study of hepatitis B and hepatitis C in older adults: do healthcare exposures contribute to burden of new infections? Hepatology 2012 [Epub ahead of print]. 3. Lanini S, Abbate I, Puro V, Soscia F, Albertoni F, Battisti W, et al. Molecular epidemiology of a hepatitis C virus epidemic in a haemodialysis unit: outbreak investigation and infection outcome. BMC Infect Dis 2010;10:257. 4. Thompson ND, Novak RT, Datta D, Cotter S, Arduino MJ, Patel PR, et al. Hepatitis C virus transmission in hemodialysis units: importance of infection control practices and aseptic technique. Infect Control Hosp Epidemiol 2009;30:900–3. 5. Centers for Disease Control and Prevention (CDC). Hepatitis C virus transmission at an outpatient hemodialysis unit – New York, 2001–2008. MMWR Morb Mortal Wkly Rep 2009;58:189–94. 6. González-Candelas F, Guiral S, Carbó R, Valero A, Vanaclocha H, González F, et al. Patient-to-patient transmission of hepatitis C virus (HCV) during colonoscopy diagnosis. Virol J 2010;7:217. 7. Fischer GE, Schaefer MK, Labus BJ, Sands L, Rowley P, Azzam IA, et al. Hepatitis C virus infections from unsafe injection practices at an endoscopy clinic in Las Vegas, Nevada, 2007–2008. Clin Infect Dis 2010;51:267–73. 8. Gutelius B, Perz JF, Parker MM, Hallack R, Stricof R, Clement EJ, et al. Multiple clusters of hepatitis virus infections associated with anesthesia for outpatient endoscopy procedures. Gastroenterology 2010;139:163–70. 9. Bronowicki JP, Venard V, Botté C, Monhoven N, Gastin I, Choné L, et al. Patientto-patient transmission of hepatitis C virus during colonoscopy. N Engl J Med 1997;337:237–40. 10. Tallis GF, Ryan GM, Lambert SB, Bowden DS, McCaw R, Birch CJ, et al. Evidence of patient-to-patient transmission of hepatitis C virus through contaminated intravenous anaesthetic ampoules. J Viral Hepat 2003;10:234–9. 11. Martró E, González V, Buckton AJ, Saludes V, Fernández G, Matas L, et al. Evaluation of a new assay in comparison with reverse hybridization and sequencing methods for hepatitis C virus genotyping targeting both 5 noncoding and nonstructural 5b genomic regions. J Clin Microbiol 2008;46:192–7. 12. Guindon S, Gascuel O. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 2003;52:696–704. 13. Saludes V, Bracho MA, Valero O, Ardèvol M, Planas R, González-Candelas F, et al. Baseline prediction of combination therapy outcome in hepatitis C virus 1b infected patients by discriminant analysis using viral and host factors. PLoS ONE 2010;5:e14132.