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We have determined that the duration of survival of HIV-1 in syringes typically used by injectors of illicit drugs can exceed 6 weeks. The percentage of syringes with viable virus varied with the volume of blood remaining in the syringes and the temperature at which syringes were stored. These experiments underscore the need for needle exchange programs and other HIV prevention interventions that promote the availability of clean syringes and the removal of potentially infectious ones from circulation.
The HIV-1 epidemic in the United States is, at present, fueled by injection drug use. While injection drug use is the sole factor in only about one quarter of all AIDS cases, it may account for more than half of all new infections. The implementation of effective prevention programs aimed at reducing syringe-borne HIV-1 has been hampered primarily by legal prohibitions, which limit the availability of clean syringes. But it has also been hampered by the lack of comprehensive data on the risks of HIV-1 transmission in the drug injection process. Despite the epidemiologic data linking sharing of syringes to HIV-1 infection, little is known, virologically, about the ability of syringes to serve as effective vectors for transmission. This has led to the dissemination of confusing and unfocused prevention messages.
Nevertheless, one prevention message is obvious: a new, sterile syringe should be used for each injection by those who continue to inject illicit drugs. Reliable sources for sterile syringes include pharmacies in the states that permit sales without a prescription and syringe exchange programs (SEPs) in those jurisdictions in which they are permitted to operate. The unfortunate reality is that the public health dictum of "1 shot, 1 syringe" is hard for illicit drug injectors to live by. Syringes remain scarce in many jurisdictions because of prohibitions on syringe availability. Syringes supplied by SEPs account for fewer than 2% of those needed to meet the standard of 1 syringe per injection. Furthermore, in places where syringes are legally available, injectors reduce their access, rapidly discarding their syringes because those they carry can be used by the police to arrest or intimidate injectors. As a result, syringes continue to be shared by many injectors.
The failure to fully implement SEPs in the United States has had dire consequences. It has been estimated that this failure has resulted in 10,000 to 20,000 HIV-1 infections that could have been prevented. The effectiveness of SEPs in limiting the spread of HIV-1 among drug injectors can be seen in the myriad reports that SEP use is associated with decreases in risky injection practices (for review, see Normand et al and Vlahov and Junge) and by works providing ecologic and individual-level evidence of reduced transmission. SEPs result in a quicker removal of used syringes from circulation, and with this removal is a decreased probability that a contaminated syringe will be shared and that HIV-1 will be transmitted. Implicit is the notion that the virus remains alive within syringes long enough for the decrease in circulation time to matter. We wanted to study the durability of HIV-1 in syringes to make explicit the relationship between circulation and decreased transmission.
In order to accurately determine the survival of HIV-1 in syringes, it was necessary to unite 2 disparate fields of research: virology and anthropology. Virology was necessary to develop a reliable assay for the propagation of HIV-1 from the small volumes of blood that remain associated with used syringes and other injection paraphernalia. Anthropology was necessary to obtain accurate information on how drugs are prepared and injected.
The virologic assay capable of propagating HIV-1 from syringes was developed over the course of several years in our laboratory. A suitable assay had to, first and foremost, be sensitive enough to accept as inputs the small volumes of blood remaining within syringes, which range from less than 1 µL to as much as 35 µL. Standard quantitative HIV-1 culture assays, such as those used in AIDS Clinical Trials Group virologic studies, begin with 10 peripheral blood mononuclear cells (PBMCs) from the infected patient. This number of PBMCs is 2 to 3 orders of magnitude greater than that found in a used syringe. Using 96-well microtiter plates, we miniaturized the culture assay to reliably propagate HIV-1 from as little as 0.4 µL of infected blood.
Before we could use the microculture assay to determine how long the virus remained alive within syringes, it was important to learn how injectors handled their syringes. Qualitative ethnographic fieldwork to document real-world drug preparation and injection practices was begun in the 1970s and 1980s by anthropologists such as Michael Agar. Before this, studies of injectors attributed syringe sharing to ritual, implying that sharing was a conscious part of a collective injection drug user (IDU) belief system. The newer research discovered that injectors balanced risks and opportunities. The researchers combined investigations of the risk at the level of the individual with a knowledge of political economy to demonstrate how the structural conditions, including laws limiting syringe sales and possession, influence people's actions. Their conclusions were that the scarcity of syringes results in frequent syringe reuse.
In our studies from several cities in the United States, we have reported that in the absence of an operational SEP as a regular and reliable source of clean syringes, injectors, on average, reuse their syringes a half dozen times or more. Each time they inject intravenously, they introduce blood into the barrel of the syringe. After the syringe is loaded with the drug solution and the needle is inserted, the plunger is drawn back. The appearance of blood in the barrel of the syringe indicates that the syringe is "registered" in a vein. Only then are the drugs injected. Once this is done, many injectors pull up on the plunger again to mix the last bit of drug with blood and reinject, a process called "booting." Registering introduces some blood into the syringe, and booting ensures that what remains in the syringe is almost entirely blood. These practices, and the risks they entail, were brought to the attention of the pub-lic health community by ethnographers, anthropologists trained in the study of the behaviors of individuals and communities.
For the purposes of understanding HIV survival in syringes, there was another important set of ethnographic observations centered on the syringe. Field observations noted that drug injectors will use syringes of different sizes and needles of different gauges and lengths for injection. Because the majority of American injectors favor disposable syringes with fixed needles, syringes and needles are often thought of as synonymous. Nevertheless, the difference in syringe and needle sizes and the use of syringes with attached or detachable needles could have important consequences for HIV transmission. Our research confirmed earlier findings that the commonly used 1-mL insulin syringes with attached 27-gauge cannulae contain, on average, a residuum of approximately 2 µL, whereas syringes paired with detachable needles contain an average of approximately 20 µL.
We have integrated the ethnographic findings on injection practice and syringe choice into our laboratory simulations in determining the duration of HIV-1 survival in syringes. First, to mirror the practice of booting, which leaves virtually undiluted blood within syringes, we partially filled insulin syringes with HIV-1-infected blood and expelled it. Second, we tested syringes containing a variety of residual volumes. Complete depression of the plunger resulted in the retention of approximately 2 µL of blood. Use of syringes with detachable needles was simulated by retaining approximately 20 µL of blood within used syringes. We also strove to achieve conditions most closely simulating the parenteral transmission of HIV-1 via the sharing of syringes. The blood used in these studies contained HIV-1 with between 100 and 5000 infectious units per milliliter, characteristic of what might be found in the blood of untreated, HIV-1-infected drug injectors. The HIV-1 isolates were obtained from patients in and out of antiretroviral treatment. These isolates were subjected to minimal manipulation; they were neither filtered nor centrifuged after expansion in culture. All viral cultures were performed using stimulated PBMCs obtained from seronegative donors.
Syringes were loaded with the HIV-1-infected blood and stored for periods ranging from 1 to 48 days. In these studies, the effects of 3 variables -- HIV-1 titer, volume of blood, and storage temperature -- on the length of viral survival were assessed. Each of these variables had a significant effect on the survival of HIV-1 in syringes.
HIV-1 Titer. The titer of HIV-1 in the blood was measured independently by end-point serial dilution. Within each experiment, higher titer was significantly associated with more syringes yielding viable HIV-1 at a particular period of storage. However, the variability of the microculture assay resulted in the loss of a significant association when the data from multiple experiments were analyzed together.
Volume of Blood. The volume of HIV-1-infected blood was either approximately 2 µL or approximately 20 µL. Almost 800 syringes stored at room temperature were tested. A strong association was noted between the volume of blood and the probability of recovering viable HIV-1 from syringes following any duration of storage (Figure 1). For syringes containing approximately 2 µL of blood, viable HIV-1 was recovered from 52% of syringes after 1 day of storage, from 26% of syringes after 2 to 10 days of storage, from 10% of syringes after 11 to 21 days of storage, and from 3% of syringes after 22 to 35 days of storage. No viable HIV-1 was recovered from 68 syringes stored for longer than 35 days. For syringes containing approximately 20 µL of blood, viable HIV-1 was recovered from 86% of syringes after 1 day of storage, from 77% of syringes after 2 to 10 days of storage, from 53% of syringes after 11 to 21 days of storage, from 30% of syringes after 22 to 35 days of storage, and from 8% of syringes stored for 36 to 48 days. At all intervals of storage, more blood resulted in a higher percentage of syringes with viable HIV-1.
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Survival of HIV-1 in syringes stored at room temperature. Syringes were loaded with 2 µL (gray circles) or 20 µL (solid boxes) of HIV-1-infected blood on day 1 and stored for up to 48 days at room temperature. Syringe contents were then introduced into an HIV-1 microculture assay. The percentage of syringes from which HIV-1 p24 antigen increased at least 4-fold during the course of culture was calculated for each interval of storage. The number of syringes tested for each interval is given above the data point.
Storage Temperature. The temperature at which the syringes were stored is an important variable because many injectors do not have the luxury of keeping their syringes in a temperature-controlled environment. Injectors are likely to keep their syringes at ambient temperatures ranging from the inside of unheated apartments in the cold of northern winters to the glove compartments of cars parked in the heat of desert summers. In simulating this variation, we stored syringes at constant temperatures ranging from 4[ring]C (39.2[ring]F) to 37[ring]C (98.6[ring]F) for 1, 7, 14, 21, 28, 35, or 42 days. We did not attempt to simulate diurnal variations in temperature. Generally speaking, there was an association between lower storage temperature and longer HIV-1 survival in syringes (Figure 2). While the residual volume of blood in the syringe had an effect on the percentage of syringes from which viable HIV-1 was recovered, some syringes with either 2 or 20 µL of blood stored at 4[ring]C yielded viable HIV-1 even at 42 days of storage, the longest duration tested. At the other extreme, at 37[ring]C, syringes with either 2 or 20 µL of blood failed to yield viable HIV-1 if stored for 7 days or longer. At intermediate temperatures, the amount of blood in the syringe affected the duration of viability. At 22[ring]C (71.6[ring]F), viable HIV-1 was recovered from some syringes with approximately 2 and 20 µL of blood for up to 21 and 42 days, respectively. At both 27[ring]C (80.6[ring]F) and 32[ring]C (89.6[ring]F), viable HIV-1 was recovered from some syringes with approximately 2 and 20 µL of blood for 1 and 7 days, respectively.
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(click image to zoom) Effect of storage temperature on survival of HIV-1 in syringes. Syringes were loaded with 2 µL (gray) or 20 µL (black) of HIV-1-infected blood on day 1 and stored for up to 48 days at different temperatures. Syringe contents were then introduced into an HIV-1 microculture assay. The final day on which any of at least 8 syringes contained viable HIV-1 was determined.
In summary, the results from testing more than 1500 syringes revealed that HIV-1 viability in blood slowly decays during storage in syringes. The reduction in viability is more rapid when there is less blood and a lower titer of virus in the blood and when the temperature is higher. Because viable HIV-1 can be recovered from syringes even after periods of storage in excess of 1 month, it is best to assume for the purposes of designing prevention messages that once a syringe becomes contaminated, it remains so. This demonstration of the longevity of HIV-1 in syringes reinforces the importance of SEPs that act to remove contaminated syringes from circulation as well as to serve as a source for clean syringes in obviating the scarcity that leads to syringe sharing.
In addition to direct sharing of syringes, there is a set of drug preparation practices that puts injectors at risk. These have been labeled syringe-mediated drug sharing by their discoverer, Jean-Paul C. Grund. These practices involve the use of syringes to divide drug solutions. When injectors pool their scant resources to buy drugs such as heroin or cocaine or both for making "speedballs," they need to divide the drug or drugs. The easiest way to accomplish this is to dissolve the drug in a "cooker" with a known volume of water, measured out using the markings on the barrel of the syringe. The dissolved drug is then drawn through a "cotton" into the barrel of a syringe, and shares are apportioned. If the syringe used to measure the water or mete out the drug solution is contaminated, HIV-1 may be spread among people who may each have had his or her own syringe and who would report not having shared syringes.
Of 3 studies analyzing the associations among HIV-1 prevalence, syringe-mediated drug sharing, and other risky behaviors, 2 found significant associations between HIV-1 prevalence and syringe-mediated drug sharing, even when adjusting for other behaviors. Thus, it is likely that the practices of syringe-mediated drug sharing place injectors at high risk for HIV-1 infection. This conclusion is supported by our virologic analysis, which demonstrates long-term viability of HIV-1 in used syringes that might be employed in syringe-mediated sharing.
HIV-1 is only 1 of the infections transmitted among drug injectors. In most locales, HIV-1 prevalence is lower -- in many cases far lower -- than the prevalence of hepatitis B and C viruses. We cannot, at present, perform equivalent virologic studies of hepatitis B and C viruses to assay their survival in syringes. However, the hepatitis viruses appear to be more easily transmitted then HIV-1 following parenteral exposure. It may be because their survival in syringes is the same as, or more prolonged than, HIV-1 or because the titers of the hepatitis viruses exceed that of HIV-1. In either event, recommendations based on our studies might also serve to diminish transmission of the hepatitis viruses as well.
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