BACKGROUND
Injection of prescription opioids has reached epidemic levels in North America, resulting in an almost fourfold rise in prescription opioid-related substance use disorder treatment admissions in the United States between 2004 and 2014.1,2 3–5 Fig. 1 ).6 7 8
FIGURE 1.: Count and incidence of HIV infection in Middlesex-London and Ontario, 2006–2017. The Ontario and Middlesex-London HIV rates (per 100,000) are shown in the red and blue lines, respectively. Middlesex-London incident HIV cases per year are shown in the blue bars. *Denotes significantly higher (P < 0.05) incidence of HIV in Middlesex-London relative to Ontario.
A study of injection drug users in London identified hydromorphone controlled-release (HMC) capsules [marketed in over 15 countries on 5 continents with several brand names, including Hydromorph Contin or Palladone SR (Purdue Pharma, Stamford, CT) and Jurnista (Janssen-Cilag Pty Ltd, Macquarie Park, New South Wales, Australia)] as the preferred opioid for illicit use.9 9 8
HMC capsules have a low solubility in water, and thus, preparation of the drug for injection requires numerous steps (Fig. 2A ).10 10 10 10,11
FIGURE 2.: Method for preparing hydromorphone controlled-release for injection. A, 1. Open hydromorphone controlled-release capsule and empty time release beads into a pill crusher to crush the beads. Empty the crushed beads into a cooker. 2. Add approximately 1.5 mL of water to the cooker and mix using needle to stir. Allow the cooker to sit for 5 minutes at room temperature to further dissolve the opioid. 3. Place a cotton filter into the solution. 4. Place needle in the filter and draw up the solution into a syringe. Individuals would then inject this solution (injectate). 5. Individuals often perform multiple preparations (washes) to extract residual drug from the filter and cooker. B, HIV may be transmitted when an HIV+ person (person 1) performs a second wash, whereby further water is added to the residual drug in the cooker/filter and the HIV-contaminated needle is used to stir the solution and reintroduced into the same filter; thus both the filter and residual drug in the cooker are contaminated. Person 1 will then draw and reinject the additional injectate. The IDPE may then be shared with an HIV- person (person 2), who would then be exposed after inserting their needle into the contaminated filter, aspirating a contaminated injectate, and injecting it.
Reuse of IDPE is achieved by adding water to the previously used cooker containing residual drug, placing a needle into the previously used filter, and aspirating the residual drug (the “wash”). Multiple PWID may participate in “serial washes,” whereby the same filter/cooker/drug complex and needle/syringe are reused each time, with some PWID performing and sharing as many as 7 washes from a single preparation of HMC.10,11 Fig. 2 ).
Sharing of needles/syringes among PWID has been well established as a means of HIV transmission ,12 13–17 13,18–20 HIV transmission has not been widely studied. The incentives for sharing IDPE despite the existence of free IDPE distribution programs have also not been extensively studied. We recently demonstrated in a case–control study that sharing IDPE was independently associated with HIV infection and that negative beliefs about heating the IDPE were also associated with HIV infection.21
We propose that in a PWID population where IDPE is frequently shared, IDPE may become contaminated with HIV when an HIV-positive PWID reuses their own needle to perform another wash (including using the used needle to stir the solution and placing the needle into the filter to aspirate the fluid) (Fig. 2B ).22,23
METHODS
Experimental Design
The objectives of this study were to clarify the following: (1) the amount of residual hydromorphone in IDPE after the first injection (and thus the incentive for IDPE reuse) as well as the effects of heating the IDPE on the amount of hydromorphone available for injection; and (2) the persistence of HIV in IDPE in the presence of HMC or a major drug excipient and the efficacy of heating to inactivate HIV to prevent transmission associated with IDPE sharing. All experiments simulated the techniques for drug preparation used by local PWID (Fig. 2A ) and were performed in triplicate with standard error bars displayed.
Objective 1: Residual Hydromorphone in IDPE and Effects of Heating
All experiments were performed with unused needles/syringes and IDPE equipment [ie, sterile water, cookers, and filters; (Sterifilt; Apothicom, Paris, France)] that were distributed through the local needle exchange program as part of the provincial harm reduction strategy and thus commonly used by local PWID. Fluid aspirated into the syringe is referred to as the “injectate” because it contains the fluid that would be injected by a PWID.
To quantify the amount of residual drug present in the cooker and filter after an initial injection, we replicated the drug preparation technique described by PWID (Fig. 2A ): crushing the drug, placing it in a cooker, dissolving it in water, and aspirating the solution through a filter. We performed this experiment with HMC capsules (24 mg; Purdue Pharma Canada, Pickering, ON) and hydromorphone immediate-release (HMI) tablets (8 mg; Pharmascience, Inc., Montreal, QC) because these are the doses and preparations identified by local addiction counselors and patient direct reports as those most commonly used locally by PWID. The contents of each HMC capsule or HMI tablet were ground in a pill crusher (Life Brand; Shoppers Drug Mart, London, ON). The ground particles were transferred into a metal cooker, and 2 mL of sterile water was added. Injectate samples were obtained by drawing the resulting mixture through a cotton filter (6 × 6-mm, tightly woven, high-density cotton; Sterifilt, Slovak Republic) with a 25-G needle (BD Canada, Mississauga, ON) into a 3-mL syringe barrel (BD Canada). This equipment is a single unit designed to reduce residual headspace. The effect of heating on HMC and HMI recovery was examined by heating the preparation with a cigarette lighter held under the cooker until the solution is boiled (ie, bubbles formed around the edge of the cooker; <10 seconds).
The drug residue remaining in the used filter and metal cooker was each assayed separately and was each combined with 3-mL methanol. Dimethylsulfoxide (SigmaAldrich, St. Louis, MO) was added at equal volume (3 mL) to methanol, and the resulting mixture was stored for 7 days at room temperature to promote additional dissolution of particles. After additional sonication (Virsonic 100; Virtis, Gardiner, NY), the mixtures were centrifuged at 14,000g at room temperature for 10 minutes, and the supernatants were used for drug analysis.
Hydromorphone concentrations were measured using liquid chromatography–tandem mass spectrometry. Standard curve samples were prepared by diluting hydromorphone (0–100 µg/mL final concentration; Sigma-Aldrich) in water or methanol/dimethylsulfoxide (50%/50%) for assays involving injectate or filter/residue samples, respectively. Injectate samples were diluted 500-fold with water, and an aliquot (50 μL) was combined with internal standard, alprazolam (10 μL, 10 µg/mL in methanol; ThermoFisher Diagnostix, Mississauga, ON), and 50 µL of water. After centrifugation of the sample at 14,000g at 22°C for 10 minutes, the resulting supernatant was diluted 20-fold with 0.1% formic acid in water. Filter/residue samples were diluted 20-fold with 0.1% formic acid in water, and then, 50 μL was combined with alprazolam (10 μL, 10 µg/mL in methanol) and 450 µL water. After centrifugation, the supernatants were diluted 20-fold with 0.1% formic acid in water.
The prepared samples were injected into the liquid chromatograph (Agilent 1200; Agilent Technologies, San Jose, CA), and analytes were separated using reverse phase chromatography on a Thermo Hypersil Gold C18 column (50 × 5 mm; 5 μm) by gradient elution. The mobile phase was delivered at a flow rate of 0.5 mL/min with gradient flow with mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile). For the first 60 seconds, the mobile phase consisted of 2% mobile phase B and then increased linearly to 95% mobile phase B over 3 minutes. Thereafter, a linear decrease to 2% mobile phase B occurred over 1 minute, which was held for an additional 1 minute. The retention times for hydromorphone and alprazolam were 2.9 and 4.1 minutes, respectively.
Analytes were detected by tandem mass spectrometry on a Thermo TSQ Vantage triple quadrupole instrument (ThermoFisher, San Jose, CA) equipped with a heated electrospray ionization probe (HESI-II) set with probe voltage (3000 V), vaporizer temperature (400°C), ion transfer tube temperature (350°C), sheath gas (50 arbitrary units), and auxiliary gas (10 arbitrary units). The following multiple reaction monitoring transitions were used with detection in positive mode and collision gas (1.5 mTorr): hydromorphone (286.3 → 185.2 m/z ) and alprazolam (309.2 → 205.2 m/z ) with collision energy at 34 and 44 V, respectively.
Objective 2: HIV Persistence in IDPE and Effects of Heating
HMC capsules and HMI tablets were prepared as mentioned previously (Fig. 2A ). Two controls were used for each experiment: (1) 250 mg of the excipient microcrystalline cellulose (Sigma-Aldrich) was added to the cookers; or (2) the cookers remained empty. HIV-1 (CXCR4 tropic genotype #1389) at a stock titer of 1 × 108 viral particles per mL was diluted 1:5 in sterile water (Sigma-Aldrich), and 1.5 mL was added to each cooker. This corresponds to approximately 1–5 × 103 infectious units.27
After 5 minutes of room temperature incubation, the solution was aspirated through the filter with a syringe (sans needle per containment laboratory requirements). To replicate reuse at different time points, this injectate preparation protocol was repeated with serial washes at 1, 4, 24, 48, and 72 hours for a total of 6 preparations for each filter. Filters were collected after the final aspiration. The insoluble residual material from the HMC and microcrystalline cellulose cookers was collected after the 24-, 48-, and 72-hour time points (50 mg per time point). To measure HIV viral persistence, injectates were split into aliquots for HIV reverse transcriptase and infectivity assays using Tzm-bl cells. Tzm-bl cells are highly susceptible to HIV infection and were used to quantify viral infectivity in the injectate samples under conditions used by PWID. Protocols for these assays have been previously published.24
All experiments with replication competent HIV-1 were performed in a CSL2+ facility with appropriate safety and waste protocols. HIV reverse transcriptase assays, measuring the activity of reverse transcriptase to synthesize a radiolabeled template, were measured on a STORM 860 Phosphorimager (GE Healthcare, Chicago, IL) after 24 hours of exposure on a phosphor screen. Tzm-bl cell assays, measuring the luciferase activity produced from an HIV responsive promoter, were measured using a Britelite Plus luciferase kit (PerkinElmer, Waltham, MA) and read on a Cytation 5 Plate Reader (BioTek Instruments, Winooski, VT). All experiments were repeated with an alternate strains of HIV-1 (R5 tropic strain #147 at stock concentrations diluted 1:5, 1:10, and 1:20) and lower titers of X4 #1389 virus (stocks diluted 1:10 and 1:20).
RESULTS
Objective 1: Residual Hydromorphone in IDPE and Effects of Heating
Forty-five percent of the total hydromorphone in the HMC capsule remained in the IDPE after the first aspiration and was available for use in a subsequent wash, whereas only 16% remained in the IDPE when using HMI tablets (Fig. 3 ). Heating during the injection preparation did not significantly change the amount of hydromorphone in the solution within the syringe after aspiration or the amount recovered from the IDPE (Fig. 3 ). The preparation reached its boiling point in less than 10 seconds when using a cigarette lighter.
FIGURE 3.: Relative amount of hydromorphone recovered in the syringe (injectate sample) and injection drug preparation equipment [filter and residue (left in cooker)] with and without heating. A, Hydromorphone controlled-release 24 mg and (B) hydromorphone immediate-release 8 mg; doses used were those most commonly injected locally. After preparation of the drug mixtures using the technique commonly used by persons who inject drugs, hydromorphone concentrations were measured using liquid chromatography–tandem mass spectrometry. Error bars represent the standard error.
Objective 2: HIV Persistence in IDPE and Effects of Heating
Simulating drug preparation habits (Fig. 2A ) with a consistent inoculum (∼2–4 × 107 viral particles or ∼1–5 × 103 IU27 Fig. 4 ). When preparing the HMC for injection, infectious HIV was recovered from the 5-minute, 1-, and 4- hour time points, which represented 3 separate washes of HIV-inoculated IDPE (Fig. 4A ).
FIGURE 4.: HIV infectivity and persistence after addition to injection drug preparation equipment. A, Infectivity of HIV-1 in injectates was determined by Tzm-bl cell luciferase production. HIV-1 was inoculated into cookers containing hydromorphone controlled-release or immediate-release, microcrystalline cellulose, or empty cookers to measure viral persistence. Serial washes with sterile water were collected 5 minutes, 1, and 4 hours after addition of virus. Maximal preservation of infectivity over time was seen with hydromorphone controlled-release across all measured time points. B, HIV-1 was added into cookers with varying opioid or drug excipient preparations to measure viral HIV-1 in the injectate solution. HIV-1 reverse transcriptase activity was assessed after 72 hours in the injectate solution (after 6 serial washes of the IDPE). Hydromorphone controlled-release and microcrystalline cellulose showed the greatest reverse transcriptase activity at 72 hours. C, The same method as (B) was used to measure viral HIV-1 in the filter after 72 hours. In the presence of hydromorphone controlled-release, HIV-1 reverse transcriptase activity is significantly greater than hydromorphone immediate-release. It is below the level of detection in the presence of microcrystalline cellulose and water. Dashed lines represent assay detection limits. Error bars represent the standard error. *Significant at P < 0.05, **Significant at P < 0.01 by 2-way ANOVA with the Tukey correction for multiple comparisons. ANOVA, analysis of variance.
Significantly less infectious HIV was recovered from HIV-inoculated IDPE when preparing HMI compared with HMC (Fig. 4A ). Microcrystalline cellulose is a common filler/stabilizer in controlled-release drug formulations including HMC and led to more HIV persistence than sterile water control or HMI, but less than HMC (Figs. 4A, B ).
In the presence of HMC, and to a lesser extent microcrystalline cellulose, HIV reverse transcriptase activity persisted in the IDPE and was present in the fluid aspirated into the syringe (in the injectates) despite a delay of 72 hours after inoculation and 6 serial washes. HIV reverse transcriptase activity in the injectates was significantly reduced in the same conditions when originally mixed with HMI or water (Fig. 4B ). These experiments were repeated at lower infectious titers (500–2500 and 250–1250 IU) and with other strains of HIV-1 (R5 strains #147). The R5 tropic strain of HIV and lower titers of all HIV-1 strains only showed detectable virus by using reverse transcriptase at the 5-minute and 1-hour time points (see Figure 1, Supplemental Digital Content, https://links.lww.com/QAI/B316
There were some visible differences between pure microcrystalline cellulose and the excipients inside the HMC capsule. HMC, when emptied, resembles small beads that have a foam-like resistance compared with the powdered pure cellulose. These differences, and potentially the other ingredients in HMC, seem to allow for the filter to retain HIV when impregnated with HMC. Microcrystalline cellulose retains HIV in the drug residue itself but does not impregnate the filter and thus, on its own, may not result in persistence of infectious HIV. To explore these differences further, we attempted, but failed, to obtain the complete formulation of the HMC capsule without the active opioid from Purdue Pharmaceuticals.
To identify whether HIV was retained in the filters (where the needle tip of a second user would be placed), we measured reverse transcriptase activity in the filters remaining in the cooker after 72 hours. Higher levels of reverse transcriptase activity (ie, HIV) were observed in the filters in the presence of HMC compared with the other compounds (Fig. 4C ). Heating the cookers with the HMC solution to a boil reduced the residual infectious HIV-1 titers in the injectate by 2 logs but did not eliminate infectious HIV. A second round of heating to a boil reduced the HIV-1 titers to the limits of detection (dotted line represents the virus-free control, Fig. 5 ). Further rounds of heating were performed but demonstrated no further reduction in the infectious HIV titer.
FIGURE 5.: Reduction in infectious HIV-1 (measured by Tzm-bl cell luciferase production) after <10 seconds heat treatment. HIV-1 was added to cookers containing 24 mg of hydromorphone controlled-release and a cotton filter and washed with sterile water. Serial washes 1 and 2 were collected 5 minutes and 1 hour after HIV-1 addition, respectively. Cookers were then heated above a flame for <10 seconds (until bubbling) or left at room temperature during each wash. Persisting infectious HIV-1 in the injectate (fluid aspirated into a syringe for injection) was quantified by luciferase production from Tzm-bl cells. Dashed line represents the assay's detection limit. Error bars represent the standard error. **Significant at P < 0.001 by using 2-tailed t test.
DISCUSSION
These results provide the first evidence that 45% of the contents from an HMC capsule remain in the IDPE after an initial use, thereby incentivizing the behavior of frequent IDPE reuse to access retained HMC. The current local street value of a capsule of HMC is approximately $60 CAD ($45 USD), and therefore, disposal of the remaining drug in the IDPE (a $27 CAD value) is very uncommon. This leads to IDPE being commonly shared and sold among PWIDs, despite nearly absent sharing of needles and syringes.10 10,21
We demonstrated prolonged HIV survival in IDPE used in the preparation of controlled-release opioid (up to 3 days) compared with that used for an immediate-release opioid preparation. Although it has been demonstrated that HIV RNA can be found in high titer in IDPE used by PWID, the specific drugs used and their relative abilities to preserve HIV were not described in these studies.22,23 25
Because PWID commonly reinsert their own used needle into the IDPE from multiple washes using a single needle/syringe,10 HIV transmission through this mechanism.11 Figs. 2B and 4 ).
HIV persistence may be related to excipients in the controlled-release preparation because persistence was not seen with HMI tablets. Specifically, excipients in HMC that are not in HMI include ethyl cellulose, hydroxypropyl methylcellulose, and microcrystalline cellulose.26 26
The limiting sensitivity of the Tzm-bl assay prevented us from measuring infectivity beyond 4 hours (ie, third wash). However, previous research has indicated that reverse transcriptase activity in HIV-1 is a strong predictor of virus infectivity and has greater sensitivity/specificity than other surrogate assays such as p24 ELISAs or viral RNA load.27
We demonstrated that heating IDPE until boiling (“cooking the wash”) reduced the amount of virus in the injectate to the limit of detection after 2 rounds of heating (Fig. 5 ). This process was found to be very easily performed, with boiling of the HMC solution occurring in less than 10 seconds when using a cigarette lighter; this demonstrated feasibility for widespread use by PWID and is locally referred to as “cooking your wash”. The campaign emphasized the importance of cooking each wash to get the viral and bacterial28
Using a sterile (ie, previously unused) needle for every aspirate of a drug is another useful strategy in preventing viral contamination of IDPE, but this alone will not reduce the risk of bacterial contamination of the injectate from repeated handling; boiling is necessary to reduce this potential bacterial load.28 28 29
Based on the results of the current study, a “cook your wash” public education campaign was initiated in June 2017. The campaign recommended PWID to heat their IDPE with a cigarette lighter until bubbling for each wash before aspiration. Preliminary data showed a decrease in new HIV diagnoses in the second half of 2017, such that the HIV incidence for 2017 was no longer significantly above the provincial rate (Fig. 1 ). Anecdotal evidence suggests widespread uptake of the recommendation by PWID, but further studies are ongoing to document the changes in behavior associated with the campaign.
A limitation of this study is that we were unable to get access to used IDPE that would be suitable for testing for replication competent virus. This was because there was a strong effort to place all HIV patients on antiretrovirals immediately after diagnosis as per recent recommendations. Therefore, we did not have access to IDPE, which had been used by patients with high plasma viral loads. This meant that it was very unlikely that we would detect replication competent virus in the IDPE as this would have been difficult to do even with plasma.
The recent outbreaks of HIV in PWID emphasize the need for understanding all mechanisms of transmission. London had an outbreak of HIV despite having excellent pre-existing traditional HIV prevention programs for PWID. The outbreak was facilitated by the dual effect of a controlled-release opioid, which not only increases HIV survival in IDPE but also provides an incentive for sharing IDPE because of drug retention. These 2 effects seem to be synergistic in facilitating HIV transmission . Preventing IDPE sharing will be difficult due to the retention of valued drug in the IDPE after use, but promoting the heating of IDPE before each use may be an effective approach to reduce HIV infection. As the popularity of injecting prescription opioids continues to increase,2,10 HIV transmission will become increasingly important.
ACKNOWLEDGMENTS
We would like to thank the staff of the Regional HIV/AIDS Connection including Brian Lester, Sonja Burke, Karen Burton, and Blair Henry for their assistance with patient recruitment and the “cook your wash” campaign, the Middlesex-London Health Unit, and the many patients who volunteered for the study.
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