May 4, 2012

Standardization of Terminology of Virological Response in the Treatment of Chronic Hepatitis C

From Journal of Viral Hepatitis

Panel Recommendations

M. Jacobson; F. Poordad; R. S. Brown Jr; P. Y. Kwo; K. R. Reddy; E. Schiff

Posted: 05/03/2012; J Viral Hepat. 2012;19(4):236-243. © 2012 Blackwell Publishing

Abstract and Introduction
Abstract

Summary. The treatment paradigm for hepatitis C virus (HCV) infection is at a critical point in its evolution. The addition of a protease inhibitor to peginterferon plus ribavirin has become the new standard-of-care treatment for most patients. Data from clinical trials of new antivirals have been difficult to interpret and compare, partly because of heterogeneity in trial design, and partly because of inconsistencies in terminology used to define viral responses and the populations evaluated. Present definitions of viral responses for treatment with peginterferon and ribavirin are insufficient for novel treatment paradigms. Further, categorization of prior patient treatment experience in clinical trials, particularly of nonresponders to prior therapy, is inconsistent. Existing terms and definitions must be updated, standardized and/or redefined for easier interpretation of data and effective communication among clinicians. A panel of experts in HCV infection treatment met on 3 December 2009. Goals of the panel were to evaluate terms and definitions used traditionally in treatment with peginterferon and ribavirin, to refine and clarify definitions of existing terms that have varying meanings and to propose new terms and definitions appropriate for novel treatment paradigms emerging with development of new agents. A number of recommendations were accepted unanimously by the panel. Adoption of these terms would improve communication among investigators, enhance comparability among clinical trials, facilitate development of therapeutic guidelines and provide a standardized terminology for use in clinical practice.

Introduction

Over the past decade, the standard of care for the treatment of infection with HCV – 48 weeks of peginterferon alfa-2a or 2b plus ribavirin – yielded overall sustained virological response (SVR) rates of 42–46%.[1,2] The recent approval of direct-acting antiviral agents (DAA), such as the protease inhibitors telaprevir and boceprevir, represent a new standard of care for treatment-naïve and experienced patients. Many other DAAs are in development as well.

Not unexpectedly, these new agents have brought with them novel study designs, patient categorizations and treatment paradigms, which has led to some confusion over the terminology used at scientific meetings and in published articles, especially for terms relating to on-treatment virological response. The desirability of a standardized set of terms to allow comparisons between clinical trials led to the roundtable discussion among the investigators described herein. The purpose of the discussion was to evaluate, refine and standardize the definitions of existing terminology and to propose, where necessary, new terms and definitions appropriate for the novel treatment paradigms resulting from the next generation of antiviral agents.

Diagnostic Precision of Hepatitis C Virus RNA Assays

When seeking to apply standardized terminology that focuses on the presence, absence or degree of virological response experienced in patients with HCV, it is important to consider the diagnostic tests used to detect and quantify viral load. Previous qualitative HCV assays (e.g. Roche HCV Amplicor 2.0, Pleasanton, CA, USA) that measured viral load using endpoint polymerase chain reaction (PCR) have been replaced by quantitative assays that utilize real-time PCR (e.g. Roche COBAS TaqMan, Pleasanton, CA, USA; Abbott Realtime HCV RNA, Des Plaines, IA, USA) and transcription-mediated amplification technology (Quest Diagnostics Heptimax, Madison, NJ, USA). These newer commercial assays allow for the more accurate representation of HCV RNA levels given their high sensitivity, broad dynamic range and improved lower limits of detection (Table 1).[3–7] Given these differences, it is important to consider which HCV RNA assay is being used when utilizing the nomenclature or making cross-study comparisons of efficacy.

Table 1. Commercially available diagnostic assays for HCV3–7

 

Diagnostic assay* Lower limit of detection (IU/mL) Dynamic range of quantitation (IU/mL)
Roche HCV Amplicor 2.0 50 600–500 000
Roche COBAS TaqMan 2.0 HCV 10 25–390 000 000
Abbott Realtime HCV RNA 12 12–100 000 000 begin_of_the_skype_highlighting 12–100 000 000 end_of_the_skype_highlighting
Quest Diagnostics Heptimax 5 5–69 000 000

HCV, hepatitis C virus. *The COBAS Ampliprep is often used for sample preparation (i.e. automated vs manual RNA extraction) in conjunction with the listed diagnostic assays.

Current Terminology in the Context of Novel Therapies

In the two decades since the advent of interferon-based therapy for hepatitis C, investigators have developed terminology to characterize patient response to treatment. The term 'SVR' has been defined as being HCV RNA negative 6 months following treatment cessation. This became the standard endpoint for clinical trials and, given exceedingly low rates of relapse after that time point, has been interpreted as a 'cure'.[8] Other terms are associated with specific milestones in viral response that have been shown to be predictive of eventual SVR and are listed in Table 2.[8]

Table 2. Current definitions for virological response8

 

Virological response Definition Clinical utility
Rapid virological response (RVR) HCV RNA negative at treatment week 4 by a sensitive PCR-based quantitative assay May allow shortening of course for genotype 2 and 3 and possibly genotype 1 with low viral load
Early virological response (EVR) ≥2 log reduction in HCV RNA level compared with baseline HCV RNA level (partial EVR) or HCV RNA negative at treatment week 12 (complete EVR) Predicts lack of SVR
End-of-treatment response (ETR) HCV RNA negative by a sensitive test at the end of 24 or 48 weeks of treatment  
Sustained virological response (SVR) HCV RNA negative 24 weeks after cessation of treatment Sustained clearance or cure
Breakthrough Reappearance of HCV RNA in serum while still on therapy  
Relapse Reappearance of HCV RNA in serum after therapy is discontinued  
Nonresponder Failure to clear HCV RNA from serum after 24 weeks of therapy  
Null responder Failure to decrease HCV RNA by >1 log10 at 4 weeks or >2 log10 at 12 weeks of therapy  
Partial responder 2-log10 decrease in HCV RNA, but still HCV RNA positive at week 24  

HCV, hepatitis C virus; PCR, polymerase chain reaction.

The existing terminology has, for the most part, proved adequate for use in the development of study designs, communication of results and patient management in the context of peginterferon and ribavirin treatment. However, with new treatment paradigms and the introduction of new terms in recent studies of novel drugs, limitations of the current terminology have become apparent.

One example of this new terminology involves phase 3 studies of the protease inhibitor telaprevir, in which treatment-naïve patients evaluated response-guided therapy, with HCV RNA levels measured at weeks 4 and 12 (Fig. 1a,b). Patients with undetectable levels at each time point were said to have achieved 'extended rapid viral response' (eRVR),[9,10] which was required to stop therapy after a 24-week course instead of the 48-week course non-eRVR patients received.

762505-fig1

Figure 1. (a) ADVANCE and (b) ILLUMINATE study design. eRVR, extended rapid viral response (undetectable hepatitis C virus RNA at week 4 and week 12); PEG, peginterferon alfa-2a; RBV, ribavirin; TVR, telaprevir.

Lead-in dosing with peginterferon and ribavirin, used in the phase 3 development programme for boceprevir and incorporated into its approved treatment regimen, also poses particular terminological challenges because of potential ambiguity in designating response at various time points (Fig. 2a,b).[11,12] In the phase 3 SPRINT-2 trial, rapid virological response (RVR) criteria differed from the standard used in peginterferon/ribavirin trials: HCV RNA negativity by PCR after 4 weeks of peginterferon plus ribavirin treatment.[12] Instead, the SPRINT-2 trial criterion for the equivalent of 'RVR' was HCV RNA negativity at week 4 of boceprevir treatment, which was overall treatment week 8. For patients who achieved this milestone and remained negative at treatment week 24, all therapy was stopped. Meanwhile, patients with detectable HCV RNA at week 4 of boceprevir treatment to week 20, yet had undetectable HCV RNA at week 24, continued to receive peginterferon plus ribavirin alone for a total treatment duration of 48 weeks (Fig. 2b).[12] Thus, referring to response at week 4 of boceprevir as RVR may be confusing because that term refers to response at week 4 of treatment overall.

762505-fig2

Figure 2. (a) SPRINT-1 and (b) SPRINT-2 study design.11,12 Patients in all arms were followed for 24 weeks after the end of treatment. Lead-in = peginterferon alfa-2b (1.5 μg/kg/week) + ribavirin (800–1400 mg/day) for 4 weeks. BOC, boceprevir; PEG, peginterferon alfa-2b; RBV, ribavirin; TID, 3 times daily; 4/24/48, 4-week/24-week/48-week treatment duration; TW, treatment week.

With such challenges in mind, the panel proposed recommendations for terminology to be used in the reporting of clinical data pertaining to the treatment of chronic HCV infection, particularly data generated from studies of the new antiviral agents

Recommendations
Terminology for New Treatment Paradigms in Development

Two general terms have been widely used to describe new antiviral agents for the treatment of HCV infection. One is 'specifically targeted antiviral therapy for hepatitis C' (STAT-C), and the other is 'DAAs'. While STAT-C has appeal from the standpoint of pronunciation and specificity for the context of HCV therapy, the term 'DAA' has been adopted by the European Medicines Agency (EMEA) as its term of choice.[13] The US Food and Drug Administration has also used 'DAA' in the HIV arena and, more recently, in the HCV arena.[14] In recognition of the need to align with the terminology that appears likely to be adopted by regulatory agencies and others, the panel supports the future use of 'DAA'.

Terminology Relating to Treatment Experience

While the panel agreed that the term 'treatment naïve' is clear, 'treatment failure' was the subject of some debate. First, it does not adequately describe patients who stop therapy for reasons other than lack of response, such as discontinuation of treatment because of adverse events. Second, patient advocates argue that the word 'failure' should be avoided because of its pejorative connotations. While an alternative term 'treatment experienced' was discussed by the panel, it was decided to be too vague with regard to prior treatment success/failure. Therefore, the panel recommended that the term 'treatment failure' be retained but that physicians remain aware that this term may be perceived to have a negative connotation by patients or their families.

Recommendation 1: The terms 'treatment naïve' and 'treatment failure' should be retained in their current usages. The term 'treatment failure' may be refined with specific information about the nature of the failure (e.g. the regimen on which the patient failed and the nature of the failure – relapse, nonresponse and premature discontinuation for adverse events).

Definitions of Response

Sustained Virological Response The most common primary endpoint for clinical trials is SVR, which is defined as undetectable HCV RNA at 24 weeks after the end of treatment. Panel members felt that adding a number to the end of the acronym to represent the time of last-confirmed viral negativity, as has already been adopted in some presentations, would provide added clarity (e.g. SVR12 would stand for viral negativity at week 12 post-treatment). This may be important as the duration of therapy continues to shorten and late relapse may be seen beyond 24 weeks. The panel believes that a minimum follow-up period of 12 weeks is required before any terminology related to 'SVR' is used.

Recommendation 2: SVR is defined as undetectable HCV RNA levels at 24 weeks post-treatment. The term may be modified by adding a number to the end to indicate the time of the last documented negative HCV RNA result (e.g. SVR12 would mean negative HCV RNA levels at 12 weeks post-treatment).

End-of-treatment Response End-of-treatment response has been defined previously as HCV RNA negativity at the completion of treatment. The panel supported the continued use of this definition and agreed that the abbreviation 'end-of-treatment response' (ETR) is most appropriate.

Recommendation 3: An ETR is defined as undetectable HCV RNA levels at the end of treatment regardless of treatment duration.

Rapid Virological Response and Complete Early Virological Response Rapid virological response is generally defined as undetectable HCV RNA using a sensitive PCR assay at week 4 of therapy, while a complete early virological response (EVR) is defined as undetectable HCV RNA at week 12 of therapy. To simplify the nomenclature, the panel suggested that a new term be established: complete virological response (CVR), defined as an undetectable level of HCV RNA while the patient is still on treatment. To clarify the time point at which a patient achieves CVR, a number can be added to indicate the week of treatment (e.g. CVR4 and CVR12). Under this scheme, the term RVR will be replaced by CVR4, and complete EVR will be replaced by CVR12. These new terms will be very relevant to clinical studies of the DAAs, because in many trials complete viral suppression within a certain time frame will be required to allow continuation of therapy. Given the central role accorded 'extended' RVR (eRVR, attainment of RVR with maintenance of HCV RNA undetectability at subsequent time points) in studying response-guided therapy in major DAA trials to date, the term CVR allows for greater precision because it may be followed by a designation of the weeks at which HCV undetectability is required to have been demonstrated. Thus, HCV RNA undetectability at weeks 4 and 12 would be designated 'CVR4,12', while its undetectability at multiple time points, for example, weeks 4, 12, 16, 20, could be designated 'CVR4–20'.

Recommendation 4: A complete virological response (CVR#) is defined as an undetectable HCV RNA level during treatment, where # is the total treatment week at which time a negative HCV RNA level is first documented. CVR4 should thus replace the term 'RVR', and CVR12 should replace the term 'complete EVR'. The term eRVR should be replaced by CVR at the intended time points, starting with the first time point at which HCV RNA became undetectable.

Partial Early Viral Response and Partial Responder The panel agreed that that the term partial EVR has been a useful clinical tool. Clinicians have typically stopped peginterferon/ribavirin treatment for patients who fail to achieve at least a partial EVR because their likelihood of achieving an SVR is extremely low, but patients who do achieve a partial EVR still have a chance for SVR and may benefit from an extended treatment period. A partial responder has usually been defined as a patient who achieves at least a 2-log10 decline in HCV RNA level at treatment week 12 but who does not achieve an undetectable viral level by the end of treatment. However, this does not specify the duration of therapy to which the term applies. The panel proposed that the terms 'partial EVR' and 'partial responder' be supplanted by the more precise term 'partial virological response' (PVR), defined as a 2-log10 decline in HCV RNA level with detectable viraemia at a given treatment week. A number can be added to indicate the first or any subsequent treatment week at which the latest HCV RNA level was documented (e.g. PVR12). Using this nomenclature, the term 'partial EVR' is replaced by 'PVR12'. A partial response that persists to a subsequent time point can be designated by the addition of a second number, for example, a partial response that occurred at week 12 and persisted to week 24 could be designated as PVR12, 24. Note that the designation of PVR# can be adapted to novel treatment regimens using other criteria for degree of viral decline that may be more suitable in the context of such therapies.

Recommendation 5: A partial response (PVR#) is defined as a ≥ 2-log10 decrease in HCV RNA level but with detectable viraemia at treatment week #. A 'partial EVR' on peginterferon and ribavirin should be referred to as a PVR12.

Slow responder The term 'slow responder' has been used to describe a patient with detectable viraemia at treatment week 12 (generally with at least a 2-log10 decline) and whose HCV RNA level is undetectable at treatment week 24. Studies have suggested that extending therapy to 72 weeks can increase the chance of SVR in such patients.[15–17] However, some studies evaluating prolongation of extended therapy have used different criteria, such as failure to attain HCV RNA undetectability by week 4[18] or initial undetectability at week 12 after HCV RNA was positive at week 8.[19] The panellists decided there was no need for a term that is open to various interpretations when the time point can be specified in the term itself. The proposed term CVR24 clearly indicates that the first documentation of an undetectable HCV RNA level occurred at treatment week 24. In this context, a 'slow responder' would be referred to as 'PVR12, CVR24'. This terminology can be readily adapted to the study of other time points for initial HCV RNA undetectability as a determinant of treatment duration.

Recommendation 6: Use the term 'CVR24' to indicate the initial attainment of a complete response by treatment week 24 instead of 'slow responder'.

Nonresponder The panel agreed that the term should be defined as a patient who never achieved an undetectable (i.e. CVR) level of HCV RNA during or at the end of treatment.

Recommendation 7: A nonresponder is defined as any patient who never achieved undetectable serum HCV RNA level on treatment or at the end of treatment.

Null Responder The historical definition of a null responder has been either a patient who achieves less than a 1-log10 decline in HCV RNA level at treatment week 4 or one who achieves less than a 2-log10 decline in HCV RNA at treatment week 12. The panel proposed using the term 'null response' (NuR) followed by a number indicating the last time point of evaluation, with the following definitions:

Recommendation 8: NuR is defined as:

  • NuR4 = <1-log10 decline in HCV RNA level at treatment week 4.
  • NuR12 = <2-log10 decline in HCV RNA level at treatment week 12.

The patient should be categorized by last time point of evaluation. When used in clinical trials on retreatment, study investigators should define patients' degree of exposure to prior treatment. The use of these terms allows for additional refinement of patient groups. Thus, the term NuR4PVR12 denotes a patient with <1-log decline in HCV RNA at treatment week 4 but a ≥ 2-log decline by week 12.

Breakthrough and Viral Rebound The panel noted that there has been much confusion about the precise definition of the terms 'breakthrough' and 'viral rebound'. In the past, both 'breakthrough' and 'viral rebound' have been defined as greater than a 1-log10 increase in HCV RNA from nadir and a minimum level of 1000 IU/mL. Others have used the term 'breakthrough' to indicate at least a 2-log10 increase in HCV RNA level from nadir and a minimum level of 50 000 IU/mL. Still another definition of 'breakthrough' has been a greater than 1-log10 increase in HCV RNA from nadir or an increase to >100 IU/mL, provided that the HCV RNA level had been undetectable at some point during treatment. The panel suggested that the key difference between the terms 'breakthrough' and 'rebound' is whether the patient has achieved HCV RNA negativity (a CVR) at any point on treatment. A patient who has had a CVR on treatment but then becomes viraemic would fall into the category of breakthrough, but a patient who has had a decline in HCV RNA levels that stops short of a CVR and then experiences a rise in HCV RNA level would fall into the category of viral rebound.

Recommendation 9: Breakthrough is defined as the on-treatment presence of detectable HCV RNA on 2 consecutive serum tests conducted after a previous on-treatment serum test showed an undetectable level of HCV RNA with a real-time quantitative PCR or similarly sensitive test. The HCV RNA level must be at least 100 IU/mL on the second positive serum test.

Recommendation 10: Viral rebound is defined as an on-treatment 1-log10 increase in HCV RNA level from nadir and an absolute level of at least 1000 IU/mL in a patient who has not achieved an undetectable HCV RNA level during the current treatment regimen.

Terminology for Agents Utilizing the Lead-in Strategy

There was much discussion among the panel members about clarifying the terminology for the lead-in strategy. There was a consensus that the language must facilitate comparisons of clinical trial results among various agents, both those that are dosed with and without a lead-in strategy of peginterferon and ribavirin alone. Further, it was unanimously agreed that the new terminology should not create the misperception that treatment begins with the initiation of the targeted antiviral; the panel was definitive that treatment begins at the start of the lead-in period. It was agreed that the abbreviation Li4 (lead-in 4) before an abbreviation for response (e.g. Li4-CVR8) would be a clear way to indicate the exact time point during treatment at which the HCV RNA test was conducted. Another example of defining patterns of response using the terminologies proposed in this manuscript would be the term Li4-NuR4 (see 'recommendation 8'). This would indicate that at the end of a 4-week lead-in phase, the patient has had <1 log decline in HCV RNA. To stratify treatment outcomes between patients with intrinsically poor vs better interferon responsiveness, one could apply the terms Li4-NuR4 or Li4-R4, where the latter denotes a ≥ 1 log decline in HCV RNA after 4 weeks of lead-in therapy.

Recommendation 11: The abbreviation Li4 should be added as a prefix to on-treatment response terminology when the clinical study utilizes the lead-in strategy in which patients receive 4 weeks of treatment with peginterferon and ribavirin before the addition of the DAA to the regimen. Under this system, Li4-CVR8 would indicate an undetectable level of HCV RNA at triple therapy week 4 and total treatment week 8. As another example, Li4-CVR8, 24 would indicate an absence of detectable HCV RNA at total treatment weeks 8 and 24 after 4 weeks of lead-in therapy followed by the addition of a protease inhibitor, as is currently required to stop all therapy at total treatment week 28 in a response-guided therapy regime containing boceprevir. Finally, Li4-NuR4 would indicate a failure of HCV RNA to decline by at least 1 log after 4 weeks of lead-in therapy.

A summary of recommended terminology, with definitions, is presented in Table 3.

 

Table 3. Summary of recommendations for updated terminology

Term Definition
Treatment failure Patient who failed to achieve sustained virological response
Sustained virological response (SVR) Undetectable HCV RNA level at 24 weeks post-treatment
End-of-treatment response (ETR) Undetectable HCV RNA level at end of treatment regardless of treatment duration
Complete virological response (CVR); number at end represents week at which HCV RNA negativity is noted Undetectable HCV RNA level during treatment CVR4 should replace the old term RVR CVR12 should replace the old term cEVR CVR24 should replace the old term slow responder CVRx,y or x-y should replace the term eRVR
Partial virological response (PVR) ≥2-log10 decrease in HCV RNA level but with detectable viraemia at treatment week no. PR12 should replace the old term pEVR
Nonresponder Any patient who never achieved undetectable serum HCV RNA level on treatment or at the end of treatment
Null response (NuR) NuR4 = <1-log10 decline in HCV RNA level at treatment week 4 NuR12 = <2-log10 decline in HCV RNA level at treatment week 12
Breakthrough On-treatment presence of detectable HCV RNA on two consecutive serum tests conducted after a previous on-treatment serum test showed an undetectable level of HCV RNA with a real-time quantitative PCR or similarly sensitive test. The HCV RNA level must be at least 100 IU/mL on the second positive serum test
Viral rebound On-treatment 1-log10 increase in HCV RNA level from nadir and an absolute level of at least 1000 IU/mL in a patient who has not achieved an undetectable HCV RNA level during the current treatment regimen
Lead-in 4 (Li4) The abbreviation Li4 should be added as a prefix to on-treatment response terminology when the clinical study utilizes the lead-in strategy in which patients receive 4 weeks of treatment with peginterferon and ribavirin before the addition of the DAA to the regimen. Li4-CVR8 indicates an undetectable level of HCV RNA at triple therapy week 4 and total treatment week 8

DAA, direct-acting antiviral agent; HCV, hepatitis C virus; PCR, polymerase chain reaction; eRVR, extended rapid viral response.

Conclusions

The changing treatment landscape of HCV infection has highlighted a number of difficulties with the current definitions and terminology used in the standard-of-care HCV treatment paradigm. The authors of this report have presented recommendations that are intended both to clarify historical terminology and introduced new terms. The definitions contained in this report were designed to reflect current and future clinical practice and to standardize clinical trial design. It is of note that unanimous agreement was obtained on all issues in the present report. Although this proposal is not intended to represent guidelines for diagnosis or treatment, it is the hope of the panel that these recommendations will prove valuable for the development of a language common to clinical trials, the dissemination and comparison of clinical trial data, the development of new clinical guidelines, as well as for everyday use in clinical practice. It is very possible that future treatment paradigms will require a further modification of the nomenclature used to describe various scenarios in the treatment of hepatitis C.

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Source

Inactivation and Survival of Hepatitis C Virus on Inanimate Surfaces

J Infect Dis. (2011) 204 (12): 1830-1838. doi: 10.1093/infdis/jir535 First published online: October 19, 2011

Juliane Doerrbecker1, Martina Friesland1, Sandra Ciesek1,2, Thomas J. Erichsen2, Pedro Mateu-Gelabert5, Jörg Steinmann3, Jochen Steinmann4, Thomas Pietschmann1 and Eike Steinmann1

Author Affiliations

1Division of Experimental Virology, Twincore, Centre for Experimental and Clinical, Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI)

2Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School

3Institute of Medical Microbiology, University Hospital Essen

4MikroLab GmbH, Bremen, Germany

5National Development Research Institutes, New York

Correspondence: Eike Steinmann, PhD, Division of Experimental Virology, Twincore Center for Experimental and Clinical Infection Research, Feodor-Lynen-Straße 7-9, 30625 Hannover, Germany (eike.steinmann@twincore.de).

Abstract

(See the editorial commentary by Hagan, on pages 1819–21 and the brief report by Thibault et al, on pages 1839–42.)

Background. Hepatitis C virus (HCV) cross-contamination from inanimate surfaces or objects has been implicated in transmission of HCV in health-care settings and among injection drug users. We established HCV-based carrier and drug transmission assays that simulate practical conditions to study inactivation and survival of HCV on inanimate surfaces.

Methods. Studies were performed with authentic cell culture derived viruses. HCV was dried on steel discs and biocides were tested for their virucidal efficacy against HCV. Infectivity was determined by a limiting dilution assay. HCV stability was analyzed in a carrier assay for several days or in a drug transmission assay using a spoon as cooker.

Results. HCV can be dried and recovered efficiently in the carrier assay. The most effective alcohol to inactivate the virus was 1-propanol, and commercially available disinfectants reduced infectivity of HCV to undetectable levels. Viral infectivity on inanimate surfaces was detectable in the presence of serum for up to 5 days, and temperatures of about 65–70°C were required to eliminate infectivity in the drug transmission assay.

Conclusions. These findings are important for assessment of HCV transmission risks and should facilitate the definition of stringent public health interventions to prevent HCV infections.

Hepatitis C virus (HCV) is an enveloped virus that, at present, chronically infects ∼130 million people worldwide [1]. One hallmark of HCV is its high degree of sequence variability, which likely contributes to its ability to establish chronic infections. Different patient isolates are grouped into 7 genotypes and more than 100 subtypes within the genus Hepacivirinae of the family Flaviviridae [2]. Persistent infection is associated with a variable degree of liver damage often progressing in severity over the course of decades. Accordingly, a large number of patients are at risk of severe sequelae including life-threatening conditions like cirrhosis and hepatocellular carcinoma [3]. The best available treatment, a combination of polyethylene glycol (PEG)-conjugated interferon alpha (IFN-α) and ribavirin, is not effective in every patient and can be associated with severe adverse effects [4]. A prophylactic or therapeutic vaccine is so far not available.

Hepatitis C is a blood-borne viral infection transmitted mainly through intravenous drug use, blood transfusions, accidental needle sticks, and other parental exposures, including nosocomial transmissions [59]. With the implementation of routine testing of blood products for HCV, transfusion-transmitted infections became rare [10]. However, outbreaks in health-care settings have been consistently reported primarily attributed to contaminated medications or equipment and breaches in aseptic techniques in the United States, Europe, and Japan [1115]. Furthermore, cross-contamination continues to occur among injection drug users (IDUs) by the sharing of drug preparation equipment [1618]. The seroprevalence of HCV among IDUs in the United States is high, ranging between 30% and 85%, with current estimates suggesting more than over 60% of newly acquired infections occur in individuals who have injected drugs [19, 20]. The adequate assessment of transmission risks and the evaluation of the mechanisms of transmission have been difficult due to the lack of cell culture systems and animal models permissive to HCV infection. This obstacle has been overcome with the development of an HCV cell culture system based on the Japanese fulminant hepatitis (JFH1) HCV isolate, which reproduces the complete viral replication cycle in vitro [2123]. This infection system was recently applied to evaluate the environmental stability of HCV and its susceptibility to chemical biocides in liquid suspensions [24]. Furthermore, Paintsil et al [25] analyzed in 2010 the survival of HCV in contaminated syringes and the duration of potential infectiousness; however, both studies did not analyze viability and infectivity of dried HCV.

Therefore, simulating realistic practical conditions, we established an HCV-based carrier and drug transmission assay to test inactivation and stability of HCV on inanimate surfaces. These results allow the further exploration of viral transmission from contaminated surfaces, objects, or devices and the potential for recommendations for effective measures interrupting this transmission.

MATERIALS AND METHODS

Plasmids and Viruses

The plasmid pFK-Jc1 has been described recently [26]. Construct Luc-Jc1 encodes a chimeric HCV polyprotein that consists of codons 1-846 derived from J6/CF [27] combined with codons 847-3033 of JFH1. In this genome the HCV polyprotein-coding region is located in the second cistron and is expressed via an internal ribosomal entry side element derived from the encephalomyocarditis virus. The first cistron contains the firefly luciferase reporter gene fused to the JFH1-derived 5'NTR and coding region of the N-terminal 16 amino acids of JFH1 core [28].

Chemical Biocides

The alcohol substances 1-propanol, 2-propanol and ethanol were purchased from Carl Roth, Karlsruhe, Germany. Six commercially available biocides for surface disinfection were chosen to study the efficacy against dried HCV: Product A (based on ethanol, 2-propanol), product B (based on ethanol, 1-propanol), product C (based on glutaraldehyde), product D and E (based on quaternary ammonium compounds), and product F (based on peroxide compounds).

Cell Culture

Huh7.5 cells were cultured in Dulbecco modified Eagle medium (DMEM, Invitrogen) with 10% fetal bovine serum, 1× nonessential amino acids (Invitrogen), 100 μg/mL streptomycin (Invitrogen) and 100 IU/mL penicillin (Invitrogen).

In Vitro Transcription, Electroporation, and Production of Cell Culture-Derived HCV

Infectious HCV particles were produced as described elsewhere [28]. Briefly, Jc1 or Luc-Jc1 plasmid DNA was linearized and transcribed into RNA, which was then electroporated into Huh7.5 cells. Virus-containing culture fluids were harvested after 48 or 72 hours filtered through a 0.45 μm pore size filter. For determination of viral infectivity cell-free supernatants were used to infect naive Huh7.5 target cells.

Determination of HCV Infectivity

Titers of infectious virus were determined by using a limiting dilution assay on Huh7.5 cells with a few minor modifications and tissue culture infectious dose 50 (TCID50) was determined as described elsewhere [23]. For determination of Luc-Jc1 reporter activity, infected cells were washed with phosphate-buffered saline (PBS) and lysed in luciferase lysis buffer (1 % Triton X-100, 25 mmol/L glycylglycine, 15 mmol/L MgSO4, 4 mmol/L EGTA, and 1 mmol/L DTT, pH 7.8). Firefly luciferase activity was measured as described previously [28].

Preparation of the Carrier

Stainless steel discs with grade 2B finish on both sides (20 mm diameter, GK Formblech GmbH) were incubated in a 5% (vol/vol) Decon 90-solution (Decon Laboratories Ltd) for 1 hour. Afterward the discs were rinsed off twice with freshly distilled water for 10 seconds, ensuring that the carriers did not dry to any extent, and were then placed in 70% ethanol (vol/vol) for 15 minutes. Finally, the carriers were dried by evaporation in sterile petri dishes under a biological safety cabinet.

Experimental Procedure of HCV Carrier Assay

In total, 50 μL of the virus inoculum were pipetted in the center of each pretreated carrier and dried in a desiccator or under a laminar flow for about 1–3 hours at room temperature. After drying, the virus contaminated discs were transferred with forceps into 25 mL plastic vial holders (Sarstedt AG & Co KG), which were previously filled with 0.5 g of sterile glass beads (0.25–0.50 mm diameter, Carl Roth GmbH) to increase virus recovery by mechanical abrasion. Then, 100 μL of the test substance were pipetted on the dried virus inoculum and incubated for 1 or 5 minutes. Control carriers received 100 μL of water instead of the chemical biocide. In order to neutralize the test substance, 900 μL of culture medium were immediately added at the end of the chosen exposure time. The vials were directly vortexed for 1 minute to recover the residual virus, before the eluate was diluted to measure viral infectivity. To determine cytotoxicity of the biocides, 1 part of PBS was mixed with 9 parts of the biocide and used to inoculate, permissive Huh7.5 cells. Cytotoxicity was determined by examining permissive cells by microscopy for any significant changes in the cell monolayer and calculated analogously to virus titer (TCID50/mL).

For testing HCV stability and inactivation in the presence of serum, whole blood samples of healthy donors were centrifuged for 5 minutes at 5000 rpm to obtain serum. The effect of serum on HCV stability was tested by mixing serum and virus suspension in a ratio 1:1 in a total volume of 0.1 mL before the drying procedure.

Experimental Procedure for HCV Drug Transmission Assay

To test the effect of different temperatures on HCV infectivity in a drug preparation simulation, viral suspensions of 800 μL were used as inoculum of a standard household spoon (stainless steel). A heating procedure was started with a tea candle with a distance of ∼4 cm between the spoon and the top of the flame. Temperatures of the suspensions were measured at specific time intervals using a thermometer for small liquids (YEW pocket thermometer 2542). At given temperatures, 70 μL of the viral suspension was sampled. To judge the influence of human serum on virus stability in the drug transmission assay, virus suspension was diluted in a ratio of 1:8 with serum or water. Viral infectivity was determined by a luciferase reporter assay as described elsewhere [28].

RESULTS

Development of a HCV-Based Carrier Test

In general, the carrier test method is designed to evaluate the ability of chemical biocides to inactivate vegetative bacteria, viruses, fungi, mycobacteria and bacterial spores on inanimate surfaces [29]. Here, the experimental procedure of the carrier assay was used for the first time to test the virucidal activity of biocides against dried HCV. First, stainless steel discs were inoculated with a virus preparation of the HCV genotype 2a chimera Jc1 [26] and dried under a laminar flow (Figure 1A). After drying, the virus-contaminated discs were transferred into plastic vial holders, which were previously filled with glass beads to increase virus recovery by mechanical abrasion. Next, the tested biocides were distributed onto the dried virus and incubated for 1 or 5 minutes. In order to neutralize the test substance, culture medium was immediately added at the end of the exposure time. The vials were directly vortexed to recover residual infectivity, before the eluate was diluted to determine viral infectivity using a limiting dilution assay.

It has been described that depending on which virus type is dried on the carrier the amount of infectivity recovered might vary [29]. Therefore, to determine the recovery efficiency for HCV, we titrated Jc1 incubated 1 hour in suspension and a virus inoculum that was dried for the same time on a carrier disc. As depicted in Figure 1B, the infectivity of HCV recovered from the carrier surface by our procedure was about 10-fold lower compared with the HCV stored in a liquid environment. Thus, ∼10% of the viral infectivity was recovered in the carrier assay.

Virucidal Efficacy of 1-Propanol, 2-Propanol, and Ethanol Against Dried HCV

Surface disinfectants used in health care and other medical settings often contain 1-propanol, 2-propanol or ethanol as active ingredients for decontamination of surfaces. To assess the virucidal efficacy of these alcohols at concentrations ranging from 10% to 60% on contaminated surfaces, we incubated each alcohol for 1 minute (Figure 2A) and 5 minutes (Figure 2B) on dried HCV. The most effective alcohol to inactivate HCV was 1-propanol, reducing viral titers to background levels at a concentration of 30% with both incubation times (Figure 2). For 2-propanol, a concentration of 30% decreased infectivity about 10-fold, and complete inactivation was observed at an alcohol content of 50% with a 1-minute exposure time and 40% with 5 minutes incubation, respectively. Ethanol showed the lowest virucidal efficacy with a required concentration of 50% to reduce viral titers to undetectable levels in the 5-minute exposure (Figure 2B).

Effect of Commercially Available Surface Disinfectants Against Dried HCV

To directly determine the efficacy of commercially available surface disinfectants, we chose 6 different chemical biocides with different virucidal substances as ingredients. Products A and B were both based on ethanol and 2-propanol or 1-propanol, respectively. Product C contained glutaraldehyde as active ingredient. Product D and E were on the basis of quaternary ammonium compounds, whereas for product F peroxide compounds were used as virucidal substance. The alcohol-based biocides were tested as recommended with an incubation time of 5 minutes in the concentrations of 10%, 50%, and 100%. As depicted in Figure 3A, a concentration of 50% for product A reduced viral titers about 50-fold. In an undiluted preparation no infectivity could be detected; however, at a 100% concentration also cytotoxicity was visible. Product B containing ethanol and 1-propanol demonstrated a higher virucidal efficacy than product A reducing viral titers to background levels already at a concentration of 50%, thus confirming the previous results that 1-propanol is superior over 2-propanol as biocide for HCV. The other commercially available disinfectants were tested at concentrations of 0.025%, 0.25%, and 0.5% in the carrier test (Figure 3B). A complete inactivation could be achieved by all products at the highest concentration with only slight cytotoxicity for products C, D, and E. These results show that ingredients like glutaraldehyde, quaternary ammonium, and peroxide compounds have a high virucidal efficacy against HCV.

Survival of Dried HCV on Inanimate Surfaces

Recently, it could be shown that HCV can be stable for several weeks in a liquid environment or in syringes [24, 25]. To evaluate the stability of nonliquid HCV, Jc1 virus was dried on carrier discs and incubated for several days at room temperature. As HCV infection is typically transmitted via blood, the effect of healthy serum on the stability of dried HCV was analyzed in parallel. Infectivity of dried virus in the presence of serum was reduced 10-fold after 2 days and reached undetectable levels after 6 days. Furthermore, the addition of serum resulted in reduced viral titers compared with the virus without serum (Figure 4A). In the latter case, we still could measure infectious HCV with a titer of about 30 TCID50/mL after 7 days of incubation demonstrating a stability of dried HCV for more than a week on the carrier surface. In the next set of experiments, we analyzed if the addition of serum before the drying procedure influences the ability of the different biocides to inactivate HCV as reported for other viruses. The different alcohols or commercial disinfectants that were used in a concentration completely inactivated HCV as shown before (compare Figures 2 and 3). All tested biocides were able to inactivate HCV infectivity to undetectable levels in the presence or absence of serum (Figure 4B), indicating that serum cannot confer viral resistance to the tested biocides.

Heat Stability of HCV in a Drug Transmission Assay

Epidemiologic studies indicate that the sharing of the drug preparation equipment among IDUs is an important risk factor for HCV transmission [18, 30]. Spoons and/or cookers are used to heat diluted heroin into solution. Cookers are mostly used in the United States, whereas spoons are mostly used in Europe. During the drug preparation, spoons are often reused and shared between users. The drug dilution from the spoon is drawn into a syringe, and blood contaminated with HCV can be exposed to the drug dilution by insertion of an HCV-contaminated syringe into spoons that are shared. Therefore, blood on spoons/cookers could be source for contamination with infectious HCV, and the ability of the virus to survive on such surfaces can have a strong impact on cross-transmissions. To evaluate the transmission risk via this route, we contaminated a spoon with Jc1 reporter virus (Figure 5A). With the use of a tea candle, increasing temperatures were simulated with the cooker device. At indicated time intervals, aliquots were taken and used to determine infectivity by luciferase reporter assay. Viral infectivity started to decrease at a temperatures of ∼50°C and was below the detection limit at about 65–70°C in 9 independent measurement series (Figure 5B). The time required to reach certain temperatures depends highly on the experimental setup, but in our case ∼80–95 seconds were necessary when small bubbles start to appear on the spoon. The half-life of HCV at different temperatures did not differ significantly between reporter virus and authentic wild-type HCV Jc1 (data not shown). Next, we tested the impact of water and serum in this drug transmission assay. As depicted in Figure 5C, the addition of water or serum to the virus solution did not influence HCV stability. Again, ∼65°C was the temperature required to inactivate viral infectivity to background levels.

DISCUSSION

For better understanding and prevention of HCV transmission in medical settings and in the environment, experimental system simulating practical conditions are highly relevant. In this study, we addressed HCV inactivation and stability profiles on inanimate surfaces to mimic viral cross-transmissions among IDUs and in health-care settings where HCV infections continue to occur. We demonstrated that HCV could be dried and recovered efficiently in a carrier assay that can therefore be used to validate chemical biocides in their virucidal efficacy against HCV. Importantly, it also confirms that reusing HCV contaminated cookers could lead to infection even if using sterile syringes. Furthermore, by simulating the procedure for heating drugs into solution, we showed that HCV could be eliminated at temperatures of 65–70°C. These data can be used for the design of public health recommendations and prevention of viral spread among IDUs. Until recently, experimental data about the environmental stability of HCV were not reported or performed with surrogate markers (antigens, RNA, enzyme activity) for the presence or absence of infectious particles. The HCV infection system used here is based on human hepatoma cells and viruses generated in vitro [2123], and substantial progress has been made in HCV basic and translational research with this model [31]. However, limitations are that in vivo hepatocytes and patient-derived particles might be slightly different or that not all genotypes can be grown in cell culture.

In the environment, viruses are normally found on surfaces and/or embedded in body fluids like excrements, serum, blood, or other excretions, and the risk of viral transmission depends on the contact number, time, body parts, and how readily the virus is released from such surfaces. The carrier test method for HCV developed here allows predicting the activity of chemical biocides simulating practical conditions. Dried HCV was exposed to a test product for a defined contact time. At the end of the contact time, the virus-biocide mixture was recovered from the surface of the carrier and titrated to determine the degree of loss in virus infectivity. We could previously show in a quantitative suspension assay that 1-propanol is the most effective alcohol in activating HCV [24]. However, whereas in a suspension test a concentration of 20% 1-propanol was sufficient to eliminate Jc1 with a viral titer of 106 TCID50/mL, higher concentration of the alcohol are needed to inactivate dried HCV due to a stronger challenge for the disinfectant [29]. Importantly, we could demonstrate that commercially available surface disinfectants have a high virucidal efficacy at concentrations recommended by the manufacturers as previously shown for hand antiseptics [24]. While dried virus in the presence of serum could survive for up to 5 days at room temperature, we could show that HCV in suspension could survive for even 3 weeks [24], and in syringes infectivity was detected for up to 63 days [25]. Kamili and colleagues [32] demonstrated in a chimpanzee animal model that dried HCV derived from patient sera could survive for at least 16 hours but was not detectable after storage of 4 or 7 days. Differences in the viral dose, storage conditions, or determination of infectivity in vitro or in vivo [33, 34] might account for the different survival times between these studies. We used here a highly sensitive detection assay and were able to determine precise survival times of the virus on dried surfaces in the presence or absence of serum. The transmission patterns for hepatitis B virus (HBV) are very similar to HCV, and high stability in the environment has been reported for this hepatotropic virus as well [35]. In line with our results, infectivity after drying of HBV-positive human plasma could be detected for at least 1 week while no longer incubation times were analyzed [35]. In summary, these reports showed that HCV could remain viable for a prolonged time in the environment indicating that blood-contaminated surfaces can serve as HCV reservoirs. Consequently, effective disinfection of surfaces is crucial in the prevention of HCV transmission.

Transmission of HCV remains high among IDUs in recent years, with incidence rates ranging from 16% to 42% per year [36]. Furthermore, the risk of HCV transmission estimated per exposure to a contaminated syringe is 5-fold to 20-fold higher than that of HIV [3739]. Recently, Paintsil et al [25] contributed to the understanding of biological mechanisms of HCV transmission by studying contaminated syringes with HCV cell culture derived virus [40]. They found that HCV survival was dependent on syringes type, time, and temperature. Infectivity could be detected for up to 63 days in high void volume tuberculin syringes. These results suggest that this long survival contributes to the high prevalence of HCV in comparison to HIV among IDUs in spite of successful syringe exchange programs. Besides syringes, the sharing of drug cookers and cotton for filtration was also significantly associated with HCV infection independent of sharing needles and syringes [18, 30]. We show here that HCV on a spoon as cooker can survive temperatures up to 65°C, which corresponds to a heating time of 80–95 seconds in this assay setup, indicating that virus survival on cookers could also be a potential source of infectious HCV aside from syringes.

In summary, we show that infectious HCV can persist as a dried sample for up to 1 week. The most effective alcohol to inactivate the virus was 1-propanol, and commercially available disinfectants reduced HCV infectivity to undetectable levels, emphasizing strict hygiene measurements. These experimental developments should facilitate testing the virucidal activity against HCV of chemical biocides used for surface disinfection. In addition, these results will further improve the understanding of HCV cross-contaminations and its prevention in health-care settings and among injection drug users.

Notes

Acknowledgments.

We are grateful to Takaji Wakita and Jens Bukh for JFH1 and J6CF isolates, respectively and to Charles Rice for Huh7.5 cells and the E9E10 monoclonal antibody. Moreover, we thank Heiner Wedemeyer, Britta Becker, and Thomas Magulski for support and would also like to thank all members of the Department of Experimental Virology, Twincore, for helpful suggestions and discussions.

Financial support.

E. S. was supported by the DFG (STE 1954/1-1) and intramural young investigator award of the Helmhotlz Centre for Infection Research. P. M.-G. has been funded by the National Institute on Drug Abuse, National Institutes of Health (1R21DA026328-01 and R01 DA19383). T. P. was supported by a grant from the Helmholtz Association (SO-024).

Potential conflicts of interest.

All authors: No reported conflicts.

All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.

Received February 3, 2011. Accepted June 13, 2011.

References

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HCV Outside the Body: How Well Does It Survive on Surfaces, in Syringes, and in the Lab?

— Liz Highleyman
HCV Advocate

The hepatitis C virus (HCV) is relatively hardy compared with some other viruses, which has implications for transmission and prevention. HCV may survive outside the body for days in dried blood on surfaces, or for months in a liquid medium under favorable conditions. Paradoxically, however, HCV has proven difficult to maintain in laboratory cell cultures, which has hampered research on potential treatments.

Survival in the Lab

Due to the difficulty of maintaining viable HCV in the laboratory, researchers have relied on “replicon” models of a specific strain of HCV (JFH-1 genotype 2a) in a liver cancer cell line. But this year researchers from Massachusetts Institute of Technology and Rockefeller University finally found a way to sustain viral replication in healthy liver cells for up to three weeks.

As described in the February 16, 2010 Proceedings of the National Academy of Sciences, Sangeeta Bhatia and colleagues developed a way to maintain healthy hepatocytes, or liver cells, for four to six weeks by precisely arranging them on a specially patterned plate and mixing them with fibroblast cells that support their growth. The liver cells could then be infected with HCV for two to three weeks, giving time to study response to candidate drugs. The HCV strain used for this research came from a Japanese patient with fulminant hepatitis; the researchers hope to modify their system to maintain a genotype 1 HCV strain, the most common type in the U.S. and the hardest to treat.

Survival on Surfaces

According to the U.S. Centers for Disease Control and Prevention (CDC), HCV can survive on environmental surfaces at room temperature for at least 16 hours but no longer than four days. The more fragile HIV virus, in contrast, only lives on surfaces for a few hours, while influenza viruses may survive for several hours up to about a day.

The CDC estimate is based on a study by Kris Krawczynski and colleagues, presented at the 2003 American Society for the Study of Liver Diseases (AASLD) meeting and published in the May 2007 issue of Infection Control and Hospital Epidemiology. The researchers examined the stability of genotype 1a HCV in dried blood plasma from an infected chimpanzee.

Plasma samples were dried in test tubes overnight (for about 16 hours) then either rehydrated immediately using sterile water and stored at -70ºC (about -160ºF, the temperature of biomedical research freezers), or put in a controlled environment chamber with 42% humidity at 25ºC (77ºF, room temperature) for four or seven days before rehydration. The rehydrated virus was then injected into a different chimp to see whether it remained infectious.

HCV RNA (genetic material) was detectable in plasma dried overnight and stored for seven days, though viral load decreased by 1 log, or ten-fold, compared with the original plasma sample. The test chimpanzee injected with virus dried overnight developed detectable HCV RNA and elevated alanine aminotransferase (ALT), became HCV antibody positive, and had detectable HCV antigen in liver cells. Injection of rehydrated virus stored for four or seven days, however, did not lead to infection. The researchers therefore concluded that HCV could remain infectious on surfaces outside the body somewhere between 16 hours and four days.

Viability outside the body, however, can vary widely depending on conditions. Viruses survive longer on hard surfaces such as stainless steel and less time on soft surfaces like fabric. HCV can live longer at cooler temperatures and prefers humidity to dry conditions.

Survival in Liquid

HCV survives longer in liquids than it does when dried on surfaces. In one recent study, described in the June 15, 2010 Journal of Infectious Diseases, Sandra Ciesek from Hannover Medical School in Germany and colleagues looked at the environmental stability and infectivity of HCV grown in a laboratory cell culture, as well as its susceptibility to chemical disinfectants. The researchers measured changes in viral load and introduced recovered HCV RNA into cultured Huh7.5 liver cancer cells to test for infectiousness.

In a liquid environment, HCV was detectable for up to five months at lower temperatures. However, the researchers noted that the risk of HCV infection may not accurately be reflected by measuring HCV RNA levels, because viral infectivity and viral load were not directly correlated. Further, they found that various alcohols and commercially available antiseptics reduced HCV to undetectable levels, though diluting hand disinfectants reduced their virucidal activity.

In another study published in the February 2010 Virology Journal, Hongshuo Song from Peking University and colleges found that JFH-1 cell culture-derived HCV could survive in liquid culture medium for two days at 37ºC (98ºF, body temperature) and 16 days at 25ºC, but was relatively stable at 4ºC (about 40º, average refrigerator temperature) without major loss of infectivity for at least six weeks.

This cell culture-derived HCV was vulnerable to heat; infectious virus could be inactivated in four minutes at 65ºC (about 150ºF) or eight minutes at 60ºC (140ºF), but this took 40 minutes at 56ºC (about 130ºF). Ultraviolet light efficiently inactivated HCV within two minutes. Exposures to formaldehyde and various detergents destroyed infectious HCV effectively in both culture medium and human serum.

HCV’s ability to live for a prolonged period in liquid blood underlies its transmission via nasal drug use (HCV RNA was detected on 5% of straws from hepatitis C patients who “snorted air”—simulating drug use—in one recent study), tattooing, sharing personal care equipment such as razors, childbirth, certain sexual activities, and re-use of medical equipment in healthcare settings.

Epidemiologic studies show that hepatitis C prevalence is higher among people who have undergone various medical procedures including kidney dialysis, indicating that HCV can spread from one patient to another via contaminated equipment if proper infection control practices are not followed. In 2008, for example, several patients at a Las Vegas endoscopy clinic contracted hepatitis C when clinicians gave multiple people injections from the same vials of anesthesia medication.

Survival in Syringes

HCV survival in blood in syringes is a key concern, given that sharing needles for drug injection is the most common route of hepatitis C transmission. In a presentation at the 17th Conference on Retroviruses and Opportunistic Infections in February, Elijah Paintsil from Yale University School of Medicine reported findings from a laboratory study looking at how long HCV can live in syringes.

The researchers first filled syringes with HCV-infected blood and depressed the plunger, simulating what happens when a user “boots,” or draws blood up into a syringe to mix with drugs and then reinjects it. Either immediately or after storing for up to two months at various temperatures, the team flushed out the syringes and attempted to grow recovered virus in genotype 2 HCV in cell cultures. They analyzed both low-volume (2 microliter) insulin syringes with permanently attached needles and high-volume (32 microliter) tuberculin syringes with detachable needles.

In the low-volume syringes, the likelihood of finding infectious HCV declined rapidly, with no viable virus recovered after one day of storage at 37ºC or three days at 22ºC (72ºF). At 4ºC, viable virus could be detected in two-thirds of syringes after one day of storage, about 25% after three days, and about 5% after seven days.

But in high-volume syringes, infectious HCV could still be recovered from nearly all syringes stored at 4ºC for seven days, from about half of those stored for 35 days, and from about 10% even after 63 days. At higher temperatures of 22ºC or 37ºC, viable HCV could still be recovered from a small percentage of syringes after two months.

The longer survival of HCV in syringes helps explain why HCV transmission occurs ten times more often than HIV transmission from accidental needle sticks, and why harm reduction measures such as needle exchange have reduced HIV incidence more than new HCV incidence.

At an accompanying press conference Paintsil said that while it might be advisable for needle exchange programs to offer smaller insulin syringes, some individuals (for example, transgender people who inject hormones) want larger syringes, and the most important thing is to provide enough so that people never have to share.

Understanding how long HCV can survive outside the body can inform practices to reduce the risk of viral transmission. According to Krawczynski and colleagues, “The potential for HCV to survive in the environment re-emphasizes the importance of cleaning and disinfection procedures, safe therapeutic injection practices, and harm reduction counseling and services for injection drug users.”

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