Survival of Hepatitis C Virus in Syringes Is Dependent on the Design of the Syringe-Needle and Dead Space Volume

Background Many people who inject drugs (PWID) use syringes with detachable needles, which have high dead space (HDS). Contaminated HDS blood may substantially contribute to the transmission of HIV, hepatitis C (HCV), and other blood-borne viruses within this population. Newly designed low dead space (LDS) syringe-needle combinations seek to reduce blood-borne virus transmission among PWID. We evaluated the infectivity of HCV-contaminated residual volumes recovered from two LDS syringe-needle combinations. Methods We tested two different design approaches to reducing the dead space. One added a piston to the plunger; the other reduced the dead space within the needle. The two approaches cannot be combined. Recovery of genotype-2a reporter HCV from LDS syringe-needle combinations was compared to recovery from insulin syringes with fixed needles and standard HDS syringe-needle combinations. Recovery of HCV from syringes was determined immediately following their contamination with HCV-spiked plasma, after storage at 22°C for up to 1 week, or after rinsing with water. Results Insulin syringes with fixed needles had the lowest proportion of HCV-positive syringes before and after storage. HCV recovery after immediate use ranged from 47%±4% HCV-positive 1 mL insulin syringes with 27-gauge ½ inch needles to 98%±1% HCV-positive HDS 2 mL syringes with 23-gauge 1¼ inch detachable needles. LDS combinations yielded recoveries ranging from 65%±5% to 93%±3%. Recovery was lower in combinations containing LDS needles than LDS syringes. After 3 days of storage, as much as 6-fold differences in virus recovery was observed, with HCV recovery being lower in combinations containing LDS needles. Most combinations with detachable needles required multiple rinses to reduce HCV infectivity to undetectable levels whereas a single rinse of insulin syringes was sufficient. Conclusions Our study, the first to assess the infectivity of HCV in residual volumes of LDS syringes and needles available to PWID, demonstrates that LDS syringe-needle combination still has the greater potential for HCV transmission than insulin syringes with fixed needles. Improved LDS designs may be able to further reduce HCV recovery, but based on the designed tested, LDS needles and syringes remain intermediate between fixed-needle syringes and HDS combinations in reducing exposure to HCV.

The volume of the residual fluid within the syringes is dependent on several factors, including needle size and length, the amount of space remaining in the hub of the syringe once the needle is attached, and whether or not the needles are detachable from the syringe barrel [10,11,14]. Syringes with fixed needles generally have a 1 mL fluid capacity that retain 5 μl of liquid after the plunger is fully depressed; high dead space (HDS) syringes with detachable needles, on the other hand, come in volumes of 1 mL or larger and retain more liquid after use [10,11,14]. HDS syringes provide a number of advantages for drug preparation and injection. Larger syringe volumes are necessary for the injection of certain drugs, including homemade drug mixtures, viscous liquids such as steroids, and dissolved pharmaceuticals [11,12]. Larger needle sizes and lengths available for HDS syringes allows for access to deeper veins or intramuscular injection [11]. Finally, detachable needles allow for the filtration of drug mixtures and the possibility of needle replacement should clogging or dulling occur [11]. Given the many functional advantages that HDS syringes have over LDS syringes, it is not surprising that PWID in some circumstances have a preference for HDS syringes and are fairly resistant to making the switch to syringes with fixed needles [11][12][13].
There is a recent push by harm reduction agencies, syringe manufacturers and distributors to develop alternatives to HDS syringes that fulfill the various needs of PWID [11]. Designs that seek to create low deep space (LDS) syringe and detachable needle combinations have been manufactured [11]. For instance, the Total Dose™ LDS needles, which attach to 2 mL Nevershare 1 standard syringes, are now available through Exchange Supplies in the United Kingdom [15]. The 2 mL Noloss LDS syringes, in which a piston is attached to the plunger of the syringe that extends through to the tip of the syringe barrel, are available at Apothicom, a French harm reduction supply company [16]. These syringe/needle combinations are shown (Fig 1).
Encouraging PWID to use these LDS syringes and needles requires the demonstration that their use could result in relatively less exposure to HCV in comparison with standard HDS syringes and needles. In this study, we use our previously established microculture assay to determine HCV stability in Total Dose™ LDS needles, used in conjunction with 2 mL Nevershare 1 standard syringes, and in 2 mL Noloss LDS syringes attached to standard Precision-Glide™ needles. Comparison was made to standard HDS combinations and to fixed needle syringes intended for insulin injection.
We tested PrecisionGlide™ standard needles (Becton, Dickson and Company, Franklin Lakes, NJ), which attached to standard tuberculin and Noloss LDS syringes, and Total Dose™ LDS needles (Exchange Supplies, United Kingdom), which attach only to the Nevershare 1 syringes. The PrecisionGlide™ standard needle sizes tested were 27-gauge ½ inch (27G½"), 25-gauge ⅝ inch (25G⅝") and 23-gauge 1¼ inch (23G1¼") while the Total Dose™ LDS needle sizes were 25G⅝" and 23G1¼". The higher the gauge, the smaller the inner diameter of the needle bore [23], therefore, the order of bore diameters was, from smallest to largest: 27G, 25G, and 23G.

Residual liquid in syringes-needle combinations
We estimated the residual liquid in the hub of the different syringe-needle combinations using a 0.003 g/mL solution of brilliant yellow dye (Sigma-Aldrich, St. Louis, MO). Briefly, the dye solution was introduced into different syringe-needle pairs, the plunger was fully depressed, and the syringes were rinsed with 1 mL of distilled water, which was transferred into quartz cuvettes (World Precision Instruments, Sarasota, FL). Absorbance measurements were made at 260nm using a spectrophotometer (GeneQuant™ Pro, Amersham Biosciences, GE Healthcare, Piscataway, NJ). The relative volumes of residual liquid in the syringe-needle combinations were calculated from the respective concentrations of brilliant yellow in the 1 ml rinse water by comparison to a standard curve based on 1:2 serial dilutions of the dye solution. Ten syringes were tested for each syringe-needle pair and data is shown as residual volume ±SD.

Viability of HCV in syringes
The flow diagram (Fig 2) outlines how experiments were conducted. We tested for HCV infectivity in the syringe-needle combinations immediately after contaminating them with HCV and after storage at room temperature for up to 1 week as described in previous studies [20,21,24]. Syringes were loaded with plasma spiked with HCV and stored for up to 7 days at room temperature. The syringe-needle combinations were then flushed with 200 μL of cell culture media, which was then used to infect Huh-7.5 cells seeded at 1.5x10 4 cells per well in 96-well plates on the previous day. After 5 hours of incubation at 37°C, the cells were washed once with 100 μL sterile phosphate-buffered saline (PBS, Invitrogen, Life Technologies, Grand Island, NY) and 100 μL of fresh cell culture media was added. Viral supernatant was harvested after incubation for 3 days at 37°C and residual infectivity was measured as a function of the amount of luciferase produced in a luciferase-based reporter assay (Promega, Madison, WI). Luminescence measurements were made with a luminometer (Synergy HT, BioTek, Winooski, VT). Outputs from the luminometer are reported as relative luciferase units (RLUs), which quantify the amount of light generated by the virally-encoded luciferase. Previous work has established that the relationship between RLU and HCV infectivity are linear over a range of at least four orders of magnitude of HCV concentration [20]. Half-life data from the storage experiments were obtained by fitting one-phase exponential decay curves to the percent-positive syringe data using GraphPad Prism 6.0 (GraphPad Software, San Diego, CA).
In addition, we tested for HCV infectivity after rinsing contaminated syringes with water. The syringe-needle combinations were loaded with plasma spiked with HCV and then rinsed once or twice with sterile distilled water. The syringes were rinsed with water loaded up to approximately half the volume marked on the syringe barrel, i.e., 2 mL syringes were rinsed with 1 mL of water and 1 mL syringes were rinsed with 0.5 mL of water. The syringe-needle combinations were then flushed with 200 μL of cell culture medium that was added to Huh 7.5 cells. Cultures were maintained and viral infectivity was determined after 3 days as described above.
These experiments were repeated at least three times with a set of 10 syringes tested per condition for each experiment. Residual infectivity was determined as the infectivity above the pre-determined cutoff of 1000 RLU; twice the average background RLU measurements [21,24]. The results are reported first as the number and percentage of HCV-positive syringes. For those syringes with residual infectivity above the cut-off, the mean residual infectivity (RLU) was calculated across experiments and the data presented with 95% confidence intervals.

Statistical Analyses
Comparisons of residual volumes between different syringe combinations were made using ttests. Pairwise comparisons of the proportion of syringes yielding viable HCV in LDS and HDS syringe-needle combinations were made using chi-square tests. Comparisons of residual infectivity in LDS syringe-needle combinations to their HDS counterpart were made using t-tests. Statistical calculations were done with GraphPad Prism 6.0.
Decay rates were calculated for the loss of infectivity with storage for all the different syringe-needle combinations ( Table 2). Because of the limited number of replications, the 95% confidence intervals all overlap, so the decay rates cannot be construed to differ across conditions. However, it is worth noting that, as expected, the point estimate for the insulin syringes is the lowest and that LDS needles shortened half-lives compared to standard needles on the Nevershare syringes.

Effect of rinsing HCV-contaminated syringes with water
We determined HCV recovery after rinsing the syringe-needle combinations once or twice with water (Fig 6). In agreement with our previous report [24], one rinse with water reduced HCV infectivity to levels below the limit of detection in 1 mL insulin syringes with fixed 27G½" needles ( Fig 6). The remaining syringe-needle combinations required multiple rinses to reduce residual infectivity to low levels (Fig 6). The 1 mL tuberculin syringes attached to Preci-sionGlide™ 27G½" standard needles and the 2 mL Nevershare 1 syringes with Total Dose™ LDS 23G1¼" needles were the only two syringe-needle combinations with detachable needles that yielded no HCV-positive syringes after two rinses with water (Fig 6).

Discussion
Syringes with fixed needles, which retain the smallest volume after use, may not always be useful to or acceptable among PWID. Attempts to deal with this reality have led to the manufacture of detachable syringe-needle combinations that seek to reduce the dead space found in traditional detachable needle-syringe combinations. Our study confirms that the 1 mL insulin syringes with fixed 27G½" needles were the most effective of all injection equipment we tested at reducing HCV retention in syringes, outperforming all detachable syringe and needle combinations tested. This held true whether HCV recovery was assessed immediately after use, after storage at room temperature, or after rinsing with water. Our findings suggest that despite the changes in syringe and needle design to offer low dead space options, more improvements are needed if HCV transmission is to be reduced. We have demonstrated that the Noloss LDS syringes with standard needles and the standard Nevershare 1 syringes with Total Dose™ LDS needles retained levels of viable HCV that were comparable to their HDS counterparts immediately after use. Upon storage at room temperature, however, distinctions between the detachable LDS syringe-needle pairs were observed. The Nevershare 1 standard syringes attached to the Total Dose™ LDS needles showed a faster decline over time in HCV recovery at room temperature than the Noloss LDS syringes, consistently yielding the lowest proportion of HCV-positive among the syringes with detachable needles during the week of storage at room temperature. These results suggest possible differences in the impact of the LDS syringes and needles on reducing infectious HCV in syringes, but only upon substantial lag times between uses.
Overall, however, the lack of a consistent pattern across all three sets of experimentsimmediate testing, storage, and rinsing with water-and the variance between experiments preclude the recommendation of any one LDS syringe-needle combination over the others or in place od HDS syringes. Given that the differences in cost between standard and the lower dead space syringe/needle combinations are minimal and that the lower dead space syringes cost more without providing much benefit suggest little or no economic benefit in providing the lower dead space syringes as an HCV prevention measure. Instead, HCV prevention efforts should emphasize the importance of traditional harm reduction approaches that include preparing drugs and injecting them only with new, sterile equipment, minimizing contamination of injection locales, and hypochlorite bleach disinfection [21,[24][25][26].
Our study has several limitations. First, our experiments were done solely at room temperature, which potentially restricts the relevance of our findings to PWID who are able to store their used syringes at approximately 22°C. Based on our previously published work, we predict that storage at lower temperatures would slow the rate of loss of HCV viability and higher temperatures would increase the rate [20,21,24,27]. We hope to address the effect of temperature in future studies. Second, our use of plasma instead of blood could affect our findings as the thicker consistency of blood could influence virus retention or survival in the dead space of the syringes. Third, the use of a genotype 2a laboratory clone of HCV may have survival characteristics different from other genotypes. One this last point, a previous study with HCV clones of different genotypes has shown HCV thermostability to be relatively similar across genotypes [28], suggesting that our findings could be generalized. Fourth, we tested limited quantities and specific brands of LDS injection equipment, so we are unable to determine if other designs outperform those we tested.
The duration of HCV recovery from tuberculin syringes appeared shorter than in studies we previously published [20]. We have no firm explanation for the more rapid decline. Possible explanations include batch-to-batch variations in HCV titer, differences in permissibility of the Huh-7.5 subclones grown out from storage, or variations in the human plasma used to dilute HCV stocks and load syringes and growth factors contained in the fetal bovine serum used to supplement culture medium. It should be noted that the earlier set of experiments, which involved far more syringes per condition, revealed a biphasic decay pattern in which the second phase was far less steep, accounting for the prolonged infectivity in a small percentage of syringes. The decay rates we report herein for tuberculin syringes are consistent with the decay rates for the first phase we reported in 2010.
There remains much that is unknown about the overall impact of syringe type on HCV transmission among PWID. Our findings suggest that the low volume fixed needle 1 mL insulin syringe continues to be the best option for limiting HCV transmission. PWID who require the of use syringes with larger volumes and detachable needles need to be made aware that they are at increased risk for HCV transmission compared to fixed needle syringes if injection equipment is reused, even when using two-part LDS syringes and even after several rinses with water. Additional efforts are needed to improve the design of LDS needles and syringes to reduce the risk of HCV transmission and further laboratory research is necessary to determine whether LDS syringe use could be beneficial in reducing the transmission of other blood borne pathogens, especially HIV, among PWID.