| |
Hepatitis C Virus Maintains Infectivity for Weeks after Drying on Inanimate Surfaces at Room Temperature: Implications for Risks of Transmission
|
| |
| |
Download the PDF here
Journal of Infectious Diseases Advance Access published November 23, 2013
"In our simulation of real world risks of HCV transmission in settings conducive to exposure to HCV-contaminated fomites, we observed that HCVcc (cell culture derived HCV) could maintain infectivity for up to 6 weeks at 4° and 22°C......the commercially available antiseptics reduced the infectivity of HCVcc on surfaces only when used at the recommended concentrations 25,27, but not when further diluted......Almost 100% of the contaminated spots stored at 4° and 22°C remained positive for HCVcc through three weeks of storage (Figure 2A). At 37°C, 100% of the spots were positive till 10 days of
storage and then declined to 40% and 0% at days 14 and 21.....After 1 min exposure to bleach (1:10 dilution), cavicide (1:10), and ethanol (70%), the percentage of positive contaminated HCVcc spots were 0%, 3 ± 6%, and 13 ± 6%, respectively (Figure 3B)."
" Of infection control relevance is the fact that all the HCVcc-contaminated spots
dried at room air within 4 hours, becoming inconspicuous and therefore more likely to cause accidently exposures to HCV. HIV was also reported to dry at room temperature within 3 hours and retain infectivity for up to 7 days 33,34. ........This finding supports our hypothesis that the increasing incidence of nosocomial HCV infections may be due to accidental contact with HCV-contaminated fomites and other hospital equipment even after prolonged periods following their deposition. Moreover, we found that HCVcc infectivity was influenced by HCVcc viral titer and the temperature and humidity of the storage environment. Furthermore, the commercially available antiseptics reduced the infectivity of HCVcc on surfaces only when used at the recommended concentrations 25,27, but not when further diluted."
"The fact that under these conditions
we found HCVcc to be infectious for up to 6 weeks, consistent with our previous report that HCVcc survived in tuberculin syringes for up to 63 days 20, is of public health concern. Taken together, these studies show that HCVcc remains potentially infectious for prolonged periods of time, ranging from 16 hours to 6 weeks depending on the assay system."
"Finally, given the infection control implications of our findings, we decided to
investigate if commonly used antiseptics are effective against HCV. We demonstrated that bleach, cavicide, and ethanol are effective at their recommended concentrations 25-27.
It is possible that the efficacy of cavicide at 1:10 is overestimated because the disinfectant itself reduced host cell viability by 70%. Further dilution of each antiseptic proved suboptimal (Figures 3B and C). The finding for ethanol paralleled that of Ciesek et al., who found that HCV titers decreased at concentrations of 30% and 40% but complete inactivation did not occur at an exposure time of 5 min29. However, undiluted concentrations of several hand antiseptics (based on povidone-iodine, chlorhexidine digluconate, and triclosan) reduced HCV infectivity to undetectable levels 29. Thus, there are several commercially available antiseptics that are effective against HCV."
"Our study, which sought to improve upon prior studies, still has some limitations.
First, the assay employs a genetically modified HCV laboratory clone derived from a genotype 2a virus that may not reflect survival characteristics of human isolates. However, the thermostability pattern of our virus is similar to that of other genotypes 32. Second, the spiking of HCVcc-seronegative blood might not sufficiently replicate the biological factors present in the blood of HCV-infected individuals that could moderate HCV transmission and infectivity.
However, the consistency of our results with previous in vitro studies and epidemiologic studies reporting transmission of HCV in healthcare setting and through sharing of injection paraphernalia39-43 support our findings."
In conclusion, we have demonstrated that HCVcc can remain infectious at room
temperature for up to 6 weeks. Our hypothesis of potential transmission from fomites was supported by the experimental results and provides the biological basis for recent observational studies reporting increasing incidence of nosocomial HCV infections and continued high incidence among people who inject drugs."
---------------------
Hepatitis C Virus Maintains Infectivity for Weeks after Drying on Inanimate Surfaces at Room Temperature: Implications for Risks of Transmission
Journal of Infectious Diseases Advance Access published November 23, 2013
Elijah Paintsil1, Mawuena Binka2, Amisha Patel2, Brett D. Lindenbach3, and Robert
Heimer2
1Departments of Pediatrics & Pharmacology, Yale School of Medicine, New Haven, CT
2Department of the Epidemiology of Microbial Diseases, Yale School of Public Health,
New Haven, CT
3Microbial Pathogenesis, Yale School of Medicine, New Haven, CT
Abstract
Background. Healthcare workers may come into contact with fomites containing infectious HCV during preparation of plasma, or following placement or removal of venous lines. Similarly, injection drugs users may come into contact with fomites. Hypothesizing that prolonged viability of HCV in fomites may contribute significantly to incidence; we determined the longevity of virus infectivity and the effectiveness of antiseptics.
Methods. We determined the volume of drops misplaced during transfer of serum or plasma. Aliquots equivalent to the maximum drop volume of plasma spiked with 2a HCV reporter virus were loaded into 24-well plates. Plates were stored uncovered at three temperatures: 4°, 22°, and 37°C for up to 6 weeks before viral infectivity was determined in a microculture assay.
Results. The mean volume of an accidental drop was 29 μl (min - max of 20 - 33 μl). At storage temperatures 4° and 22°C, we recovered viable HCV from the low titer spots for up to 6 weeks of storage. The rank order of HCV virucidal activity of commonly used antiseptics was bleach (1:10)>cavicide (1:10)>ethanol (70%).
Conclusions. The hypothesis of potential transmission from fomites was supported by the experimental results. The anti-HCV activity of commercial antiseptics varied.
INTRODUCTION
The global burden of morbidity and mortality from hepatitis C virus (HCV)
infection is truly pandemic with more than 170 million people currently infected
1_ENREF_1. Since there is currently no vaccine for HCV and available treatment
regimens are limited by efficacy, cost, and side effects, prevention of HCV transmission remains the primary strategy for curbing the HCV epidemic. HCV is transmitted primarily through parenteral exposure to blood or body fluids contaminated with HCV. Injecting drug use (IDU), mother-to-child transmission, multiple heterosexual partners, accidental needle injuries, and transfusion of blood or blood products are among the most relevant risk factors for HCV acquisition2-4.
The epidemiology of HCV has changed in the last decade. Transmission from
blood transfusions and surgical procedures have all but disappeared in developed countries 5. There have been modest but insufficient declines in incidence among IDUs in locations with broad implementation of syringe exchange programs6-12. Nosocomial transmissions of HCV increasingly account for a large proportion of new HCV infections (i.e., 20% to 50%) in developed countries13-17. Thus, the relative impact and burden of nosocomial HCV transmission might be greater now than a decade ago. In an Italian
study of 214 patients with acute HCV infections18, the most relevant associated risk factors were: history of medical procedures (32%) (e.g., hospitalization, surgery, endoscopy, dialysis, blood transfusion, dental treatment, or other invasive procedures) and intravenous drug use (30%). Interestingly, among the patients classified under medical procedures almost half of them did not have surgery or any invasive procedures while on admission. This has been corroborated by a study from Spain where the investigators found that the only documented risk factor among patients with acute HCV
infection was hospital admission19. One can speculate that these patients might have been exposed to HCV-contaminated surfaces during hospitalization. We hypothesized that occupational and iatrogenic HCV infections may be due in part to the ability of the virus to remain viable on fomites and other hospital equipment for prolonged periods.
We recently established a microculture assay for propagation of cell culture
derived HCV (HCVcc) in small volumes by using a genetically engineered reporter virus derived from the HCVcc clone20,21. Using our microculture assay system, we performed a set of experiments to replicate the circumstances in which healthcare workers or patients may come into contact with HCV dried upon surfaces that include preparation of plasma, handling of hemodialysis equipment, and following placement or removal of venous lines. To our knowledge this constitutes the first study to closely simulate conditions leading to nosocomial transmission of HCV.
RESULTS
Volume of accidentally misplaced HCVcc contaminated plasma
The experiment was done on three occasions, and on each occasion 10 drops were weighed. The mean volume of the drops, calculated on the basis that 1 ml weighs 1 gram, was 29 ± 5 μl and the range was 18 to 33 μl. Since the maximum drop volume of 33 μl presents the most risk of transmission, we used 33 μl throughout our study.
Time to drying of HCVcc contaminated drops at different temperatures
Dried droplets of serum contaminated with HCV may be inconspicuous and,
therefore, more likely than a liquid droplet to cause accidental exposures to HCV. We determined how long it took a drop of HCVcc contaminated plasma to dry at 4°, 22° and 37°C. We determined the mean temperature and relative humidity in the refrigerator, the benchtop, and the incubator over a week. The temperature was 4 ± 1°, 22 ± 0°, and 37 ± 0°C in the refrigerator, the benchtop, and the incubator, respectively. The humidity was 53 ± 10%, 44 ± 5%, and 82 ± 1% at 4°, 22° and 37°C, respectively. The order of time to dryness was 4, 24, and 28 hrs at 22° (benchtop), 4° (refrigerator), and 37°C (incubator), respectively. Thus time to dryness correlated positively with the humidity of the storage condition.
Infectivity of dried HCVcc on surfaces at different temperatures
We investigated the infectivity of HCVcc after drying on surfaces at different
temperatures. Aliquots of 33 μl of HCVcc contaminated serum were pipetted into 24-well plates and stored for up to six weeks. Twenty spots of dried HCVcc for each combination of storage time and temperature were reconstituted with culture media after storage and introduced into our assay system 20. The proportion of HCVcc positive dried spots and the infectivity per HCVcc dried spot were determined. The results presented here came from at least three independent experiments.
First, we used a low titer stock of HCVcc, (i.e., equivalent to 104 infectious
units/mL) to determine the infectivity of HCVcc after drying and storage for up to 6 weeks. We observed a negative correlation between storage temperature and HCVcc infectivity (Figure 1A). With an assay detection limit of 1000 RLA (2-3 times over the background luciferase activity), we recovered viable HCVcc from dried spots stored at 37°C until day 7 of storage. In contrast, at storage temperatures 4° and 22C°, we recovered replicating HCVcc from all the spots for up to 6 weeks of storage. The infectivity, measured by RLA of the reconstituted spots, declined rapidly over time inversely to the storage temperature (Figure 1B). At storage temperatures of 4° and 22°C, we observed a sharp decline in infectivity over the first two weeks followed by persistent but lower infectivity through week six (Figure 1B). This is consistent with our previous
report of biphasic decay rate of HCVcc20.
By using a high titer stock of HCVcc (equivalent to106 infectious units/mL), we
observed a prolonged infectivity of HCVcc at all storage temperatures. Almost 100% of the contaminated spots stored at 4° and 22°C remained positive for HCVcc through three weeks of storage (Figure 2A). At 37°C, 100% of the spots were positive till 10 days of storage and then declined to 40% and 0% at days 14 and 21, respectively (Figure 2A). The infectivity of the HCVcc recovered from the high titer HCVcc contaminated spots was in general 2 to 3-fold higher than the RLA of the low titer HCVcc at each time point. Infectivity was inversely proportional to the storage temperature. We observed a 50% reduction in infectivity at day 3, 14 and 21 for storage temperatures 37°, 22°, and 4°, respectively (Figure 2B).
Effect of antiseptics on infectivity of HCVcc on surfaces
To investigate the virucidal effect of bleach, ethanol, and cavicide, we first
determined the effects of these antiseptics on the growth of Huh-7.5 cells by using the alamarBlue® assay. When we tried undiluted bleach and cavicide, which were diluted 1:10 before addition to the tissue culture system, we found they were uniformly ctytotoxic to Huh-7.5 cells whereas 70% ethanol had no significant effect on cell growth (Figure 3A). Cell growth was almost restored to control levels with a 1:10 dilution of bleach and a 1:20 dilution of cavicide following passage of the solution through MicroSpin S-400 HR columns (Figure 3A). Cavicide at a 1:10 dilution reduced growth by 70% relative to the control.
Based on the cytotoxicity results, experiments using bleach diluted 1:10 and 1:100, cavicide diluted 1:10 and 1:20, and ethanol at 70% and 7% were conducted by using MicroSpin S-400 HR columns29 prior to adding eluate to the microculture system. After 1 min exposure to bleach (1:10 dilution), cavicide (1:10), and ethanol (70%), the percentage of positive contaminated HCVcc spots were 0%, 3 ± 6%, and 13 ± 6%,
respectively (Figure 3B). Further dilutions of bleach (1:100), cavicide (1:20), and ethanol (7%) resulted in 17 ± 6%, 43 ± 6%, and 90 ± 17% positive spots, respectively. For certain viruses, passage through a MicroSpin column could reduce viral infectivity30, therefore, we performed a control experiment comprising HCVcc without exposure to any antiseptic and with or without passage through a MicroSpin column prior to infection of Huh 7.5 cells. The infectivity was 80 ± 10% and 100% for HCVcc with and without passage through MicroSpin column, respectively (Figure 3B). We next tested the infectivity of HCVcc without MicroSpin column after exposure to antiseptic at concentrations that are least cytotoxic. After 1 min exposure to bleach (1:100 dilution), cavicide (1:20), 70% ethanol, and 7% ethanol, the percentage of positive contaminated
HCVcc spots were 30 ± 10%, 60 ± 36%, 30 ± 35%, and 93 ± 12%, respectively (Figure 3B). The infectivity of residual HCVcc after passage through MicroSpin column (Figure 3C) was correlated with the likelihood of recovery of viable HCVcc. RLA was highest for 7% ethanol (with 27 of 30 spots yielding viable HCVcc) and lowest for 1:10 cavicide (1 of 30 spots yielding viable HCV).
DISCUSSION
In our simulation of real world risks of HCV transmission in settings conducive to exposure to HCV-contaminated fomites, we observed that HCVcc could maintain infectivity for up to 6 weeks at 4° and 22°C. This finding supports our hypothesis that the increasing incidence of nosocomial HCV infections may be due to accidental contact with HCV-contaminated fomites and other hospital equipment even after prolonged periods following their deposition. Moreover, we found that HCVcc infectivity was influenced by HCVcc viral titer and the temperature and humidity of the storage environment. Furthermore, the commercially available antiseptics reduced the infectivity of HCVcc on surfaces only when used at the recommended concentrations 25,27, but not when further diluted.
Although there have been two previous studies on infectivity and stability of HCV
on surfaces28,31, to our knowledge, this is the first study that closely simulates the natural events likely to cause transmission of HCV. First, Kamili et al. reported that 100 μl aliquots of chimpanzee plasma contaminated with HCV was still infectious when dried and stored at room temperature for up to 16 hours 31. Transmission of infection did not occur after 16 hours to up 7 days of storage. More recently, Doerrbecker et al. demonstrated that 50 μl of cell culture-derived HCV dried on steel discs remains infectious for up to 5 days at room temperature28. The limitations of these previous studies include simulation of HCV transmission under artificial drying conditions.
Furthermore, Doerrbecker et al. found that the infectivity of the virus recovered from the carrier system was 10-fold lower than that stored in liquid media. Therefore, one can speculate that the duration of infectivity observed in their study could be an underestimation. Moreover, differences in the three assay systems (e.g., in vivo versus in vitro assay; artificial versus passive desiccation) might account for the different durations of survival reported. Our study sought to overcome some of these limitations by determining the exact size of accidentally misplaced HCVcc-contaminated plasma and allowing the drops to dry under natural conditions. The fact that under these conditions we found HCVcc to be infectious for up to 6 weeks, consistent with our previous report that HCVcc survived in tuberculin syringes for up to 63 days 20, is of public health concern. Taken together, these studies show that HCVcc remains potentially infectious for prolonged periods of time, ranging from 16 hours to 6 weeks depending on the assay system. We previously reported on the biphasic decay rate of our genotype 2a HCVcc at room temperature; a rapid decline of infectivity within the first 6 h followed by a second phase of a relatively slow exponential decay 20. This is consistent with recent report on thermostability of 7 genotypes including 2a genotype 32. Such prolonged infectivity could contribute to the increasing incidence of nosocomially acquired HCV infections.
Of infection control relevance is the fact that all the HCVcc-contaminated spots
dried at room air within 4 hours, becoming inconspicuous and therefore more likely to cause accidently exposures to HCV. HIV was also reported to dry at room temperature within 3 hours and retain infectivity for up to 7 days 33,34. The infectivity of HCVcc and HIV when stored at room temperature for several days is consistent with that of other envelope viruses 35,36. The prolonged infectivity of these viruses has been attributed, in part, to their lipid envelope, which resists drying and protects the viral capsid from the deleterious effects of dehydration 37. Hepatitis B virus, another lipid-enveloped hepatotropic virus, was reported to survive up to 7 days at room temperature; further time points were not available due to a laboratory mishap 38. The resilience of these viruses at room temperature raises the possibility of their being transmitted through fomites. Our
findings support the surveillance data on increasing incidence of nosocomial
transmissions of HCV in developed countries 13-17. Interestingly, most of the patients who acquired HCV in the hospital had no surgeries or invasive procedures; their only risk was hospital admission18,19. Fomites could, therefore, be an important vehicle for transmission of HCV in the hospital and household settings.
Finally, given the infection control implications of our findings, we decided to
investigate if commonly used antiseptics are effective against HCV. We demonstrated that bleach, cavicide, and ethanol are effective at their recommended concentrations 25-27. It is possible that the efficacy of cavicide at 1:10 is overestimated because the disinfectant itself reduced host cell viability by 70%. Further dilution of each antiseptic proved suboptimal (Figures 3B and C). The finding for ethanol paralleled that of Ciesek et al., who found that HCV titers decreased at concentrations of 30% and 40% but complete inactivation did not occur at an exposure time of 5 min29. However, undiluted concentrations of several hand antiseptics (based on povidone-iodine, chlorhexidine digluconate, and triclosan) reduced HCV infectivity to undetectable levels 29. Thus, there
are several commercially available antiseptics that are effective against HCV.
Our study, which sought to improve upon prior studies, still has some limitations.
First, the assay employs a genetically modified HCV laboratory clone derived from a genotype 2a virus that may not reflect survival characteristics of human isolates. However, the thermostability pattern of our virus is similar to that of other genotypes 32. Second, the spiking of HCVcc-seronegative blood might not sufficiently replicate the biological factors present in the blood of HCV-infected individuals that could moderate HCV transmission and infectivity. However, the consistency of our results with previous in vitro studies and epidemiologic studies reporting transmission of HCV in healthcare setting and through sharing of injection paraphernalia39-43 support our findings.
In conclusion, we have demonstrated that HCVcc can remain infectious at room
temperature for up to 6 weeks. Our hypothesis of potential transmission from fomites was supported by the experimental results and provides the biological basis for recent observational studies reporting increasing incidence of nosocomial HCV infections and continued high incidence among people who inject drugs.
|
|
| |
| |
|
|
|
