Survival of hepatitis C virus in syringes: implication for transmission among injection drug users

Abstract

Background: We hypothesized that the high prevalence of hepatitis C virus (HCV) among injection drug users might be due to prolonged virus survival in contaminated syringes.

Methods: We developed a microculture assay to examine the viability of HCV. Syringes were loaded with blood spiked with HCV reporter virus (Jc1/GLuc2A) to simulate 2 scenarios of residual volumes: low void volume (2 microL) for 1-mL insulin syringes and high void volume (32 microL) for 1-mL tuberculin syringes. Syringes were stored at 4 degrees C, 22 degrees C, and 37 degrees C for up to 63 days before testing for HCV infectivity by using luciferase activity.

Results: The virus decay rate was biphasic (t1/2alpha= 0.4 h and t1/2beta = 28 hh). Insulin syringes failed to yield viable HCV beyond day 1 at all storage temperatures except 4 degrees , in which 5% of syringes yielded viable virus on day 7. Tuberculin syringes yielded viable virus from 96%, 71%, and 52% of syringes after storage at 4 degrees, 22 degrees, and 37 degrees for 7 days, respectively, and yielded viable virus up to day 63.

Conclusions: The high prevalence of HCV among injection drug users may be partly due to the resilience of the virus and the syringe type. Our findings may be used to guide prevention strategies.

Figure 1

Linear dynamic range of microculture assay. 100-μl aliquots of serial 1:2 dilutions of stock virus were used to infect Huh 7.5 cell. After 3 days of incubation, the culture supernatant was harvested and the concentration of virus determined as a function of relative luciferase activity as described in the “Materials and Methods” section. The relative luciferase units (RLU) were plotted as a function of the concentration of input virus (TCID₅₀/ml) on a logarithmic scale. The experiment was performed on two separate occasions in triplicate and the data were combined for analysis.

Figure 2

Hepatitis C virus decay rate at room temperature. Aliquots (100- μl) of the virus were stored in room temperature from 0 to 96 h. Aliquots were removed from room temperature every 6 h or less and stored at −80°C. The stored aliquots were thawed and used to infect Huh-7.5 cells. The relative infectivity was determined by measuring the relative luciferase units (RLU) after 3 days of infection as described in the “Materials and Methods” section. Each value is the mean of 2 independent experiments.

Figure 3

Survival of HCV in low void insulin syringes. Syringes were loaded with HCV-spiked blood to simulate “booting”. Syringes were stored at 4°C, 22°C, and 37°C for up to 14 days before contents were flushed to infect Huh-7.5 cells. HCV survival, a function of infectivity, was determined by the relative luciferase units (RLU) after 3 days of culture. There were 15 syringes at each time point. (A) The percentage of HCV positive syringes. (B) HCV infectivity per positive low void syringe. Each value is mean ± SEM from at least 3 independent experiments.

Figure 3

Survival of HCV in low void insulin syringes. Syringes were loaded with HCV-spiked blood to simulate “booting”. Syringes were stored at 4°C, 22°C, and 37°C for up to 14 days before contents were flushed to infect Huh-7.5 cells. HCV survival, a function of infectivity, was determined by the relative luciferase units (RLU) after 3 days of culture. There were 15 syringes at each time point. (A) The percentage of HCV positive syringes. (B) HCV infectivity per positive low void syringe. Each value is mean ± SEM from at least 3 independent experiments.

Figure 4

Survival of HCV in high void tuberculin syringes. Syringes were loaded with HCV-spiked blood to simulate “booting”. Syringes were stored at 4°C, 22°C, and 37°C for up to 63 days before contents were flushed to infect Huh-7.5 cells. HCV survival, a function of infectivity, was determined by the relative luciferase units (RLU) after 3 days of culture. There were 15 syringes at each time point. (A) The percentage of HCV positive syringes at each time point. (B) HCV infectivity per positive high void syringe after 1 through 63 days of storage. Each value is mean ± SEM from at least 3 independent experiments.

Figure 4

Survival of HCV in high void tuberculin syringes. Syringes were loaded with HCV-spiked blood to simulate “booting”. Syringes were stored at 4°C, 22°C, and 37°C for up to 63 days before contents were flushed to infect Huh-7.5 cells. HCV survival, a function of infectivity, was determined by the relative luciferase units (RLU) after 3 days of culture. There were 15 syringes at each time point. (A) The percentage of HCV positive syringes at each time point. (B) HCV infectivity per positive high void syringe after 1 through 63 days of storage. Each value is mean ± SEM from at least 3 independent experiments.