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 This epidemic has also been associated with several large outbreaks of HIV infection among persons who inject prescription opioids (PWID),3–5 including one in London, Ontario, Canada, that resulted in the declaration of a public health emergency on June 14, 2016 (Fig. 1).6 The control of these outbreaks generally depends on a multipronged approach of distribution of sterile injection equipment, opioid substitution therapy, and provision of antiretroviral therapy.7 The outbreak in London was unusual because it occurred despite the city already having at baseline the highest distribution of sterile needles and syringes per capita in Canada and an opioid substitution therapy prescription rate in the top quartile for Ontario.8 And, London has a government-funded, multidisciplinary HIV care program with access to funded HIV antiretroviral therapy. The reason for this outbreak despite the excellent pre-existing control programs was the subject of the current investigation.
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 Most PWID (79%) reported HMC use in the past 6 months.9 Moreover, prescription rates of HMC capsules were unusually high in London compared with other regions in Ontario, which may indicate widespread availability of the drug for diversion and injection.8
HMC capsules have a low solubility in water, and thus, preparation of the drug for injection requires numerous steps (Fig. 2A).10 The timed-released beads must be removed from the capsule and crushed, mixed with water in a cooker to create a slurry, and then drawn into a syringe through a cotton filter to remove visible particulate matter.10 The filters, cookers, and water are referred to as the injection drug preparation equipment (IDPE). The high cost and large dose of hydromorphone in the controlled-release formulation, and the need to dissolve the capsule in a large volume of water, increase the likelihood that the IDPE will be shared between people.10 The large volume of the solution can require multiple injections, and therefore, PWID are tempted to divide the injections among several people, while sharing costs. In addition, PWID report large amounts of nonsolubilized drugs are retained in the filter and cooker after the first use, thereby encouraging the reuse and sharing of used IDPE.10,11
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 While PWID reuse and share the same IDPE, they often reuse their own, but do not share needles/syringes (Fig. 2).
Sharing of needles/syringes among PWID has been well established as a means of HIV transmission,12 and so distribution of sterile needles/syringes has been a major focus of HIV prevention programs. However, sharing of IDPE is more common than sharing of needles/syringes.13–17 In some centers, sharing of IDPE has been identified as the primary risk factor for hepatitis C virus transmission,13,18–20 but its association with 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 HIV could be transmitted when the contaminated IDPE is shared with a second, HIV-negative PWID placing their own needle into the used filter/cooker to aspirate another wash. We further proposed that drug excipients within HMC could prolong HIV survival within the IDPE and thus facilitating transmission; and heating the contaminated IDPE before each use could inactivate HIV and help prevent transmission.
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 Microcrystalline cellulose was chosen due to its prevalence as an excipient, including a major component of the HMC formulation.
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 To assess the effects of heating on HIV persistence, cookers were heated with an ethanol flame until it reached its boiling point (ie, bubbling around the edge of the cooker, same as previous; <10 seconds), and Tzm-bl assays were conducted. Multiple rounds of heating were performed to investigate the reduction in infectious HIV particles.
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).
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.
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) of HIV-1 added to the IDPE allowed us to quantify relative viral persistence in different IDPE environments (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).
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, http://links.lww.com/QAI/B316). All future time points in these experiments were negative. Lower concentrations of X4-tropic HIV #1389 were also negative after the 5-minute time point.
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.
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 By contrast, the low quantity of residual HMI on the IDPE, because of the lower dose and higher solubility of immediate-release opioids, provides less incentive to reuse the IDPE. These findings likely explain the common behavior of multiple repeated uses of IDPE and the sharing of washes with HMC but not HMI.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 It has been shown that HIV survival is reduced in the presence of heroin, but it is unclear whether this was related to the heating process required for home production or the many caustic chemicals added to produce the solution.25 To the best of our knowledge, this study is the first to assess HIV persistence in the presence of prescribed opioids that do not require heating or the addition of extra chemicals for solubility.
Because PWID commonly reinsert their own used needle into the IDPE from multiple washes using a single needle/syringe,10 this can lead to the IDPE becoming contaminated with HIV. When a second PWID inserts their needle into the same filter, they can become infected through injecting. Although most PWID are careful not to share needles/syringes, they commonly reuse their own needles/syringes because they are unaware that it may contaminate the IDPE and thus increase the risk of HIV transmission through this mechanism.11 Our demonstration that HIV can be detected not only in the injectate aspirated into the syringe from the used IDPE but also in the filter into which the second needle would be inserted suggests that contamination of the second PWID's injection would be very feasible (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 Addition of microcrystalline cellulose alone to the IDPE retained more viable HIV and for longer than IDPE when used with water or with HMI. Cellulose-containing drugs may maintain hydration, resulting in virus preservation. In fact, methylcellulose derivatives are a major constituent in media to maintain hydration and limit diffusion for viral plaque assays used for clinical diagnostics.26 For pharmaceuticals with this agent that may be diverted for injection (eg, opioids), industry should consider seeking alternative excipients given the potential increased risk of bloodborne pathogen transmission.
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 reduction benefits of serial boiling. Cooking the wash did not significantly increase the amount of hydromorphone in the injectate, and thus, should not lead to accidental overdose. And, it did not significantly reduce the amount of drug obtained, and so PWID should find it to be an acceptable harm reduction technique.
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 Boiling until there are bubbles present (for <10 seconds) as performed in this study did markedly reduce the burden of bacterial contamination,28 in another recent study which we reported, and so this degree of boiling was used in this study and in the public health campaign to have a comprehensive intervention that could reduce both bacterial and viral risks. In vitro studies using alternative boiling durations and other pathogens may be helpful to optimize this intervention. Cleaning the IDPE with bleach before reuse has been occasionally recommended as another strategy, although it would not be acceptable in the context of retained drug and injectate being exposed to the caustic effects of bleach. Given that the formulation of HMC is a specific risk factor for reuse, the provision of injectable hydromorphone should also be considered, as per the SALOME trials.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 the implementation of harm reduction strategies that target IDPE sharing as a mechanism of HIV transmission will become increasingly important.
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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