GHOST-assisted molecular testing revealed a large network of HCV transmission that facilitated the HIV outbreak in Indiana. This analysis shows transmissions of HCV among PWID predated the HIV outbreak, with multiple introductions of the virus into the community over several years. Widespread HCV transmission was revealed when the HIV outbreak was detected, and findings explain a large proportion of HIV-positive cases being co-infected with HCV.

Evidence of rising number of HCV cases in the region, which likely reflected a rise in the number of people abusing injection drugs, was an early signal that the community was at risk of additional public health issues, including an HIV outbreak. Similar at-risk communities across the nation might forestall massive HIV outbreaks, by keeping close tabs on acute HCV infections.

This was the first real time field application of GHOST, the cyber-based HCV molecular surveillance technology, to a disease outbreak of such magnitude. GHOST integrates bioinformatics and web-based technologies with Next Generation Sequencing, and enables users, regardless of their computational expertise, to conduct independent, cost-effective, and accurate hepatitis molecular surveillance. Genetic testing coupled with new computational technologies enables rapid identification of complex transmission networks operating among PWID. Timely detection of these networks is important for disease surveillance and development of targeted public health interventions to contain infection transmission.

Hepatitis C virus (HCV) infection is a global public health problem, with 62.5–79.4 million persons chronically infected worldwide [1]. HCV is the most common blood-borne infection in the United States, resulting in an estimated 3.5 million individuals infected with the virus [2,3]. From 2003 to 2013, national HCV-related mortality surpassed that from human immunodeficiency virus (HIV) infection [4], combined with deaths associated with the 59 other nationally notifiable infectious conditionS [4]. HCV is efficiently transmitted via percutaneous exposure to blood and blood products. A large proportion of new HCV infections are associated with injection drug use (IDU) [[5], [6], [7], [8]]. Elevated risk of HCV infection among young persons who inject drugs (PWID) has been reported from many countries, including the United States, for more than a decade [[9], [10], [11]].

Over the last two decades, the opioid epidemic has led to an increase in IDU and associated morbidity and mortality in the United States [12,13]. Increase in acute HCV infections was recently reported, with the largest increase in Central Appalachia [14]. Surveillance data from Kentucky, Tennessee, Virginia, and West Virginia showed a 364% increase in the number of acute HCV infections from 2006 to 2012 among PWID aged ≤30 years [15]. Increase in new HCV infections was highly correlated with the epidemic of prescription opioid abuse in this region [16]. During 2010–2015, HCV incidence rates increased by 167% with the highest rates among young PWID [14]. Prevalence of HIV infection is historically low in the Mid-West [17]. However, in January 2015, the Indiana State Department of Health recorded a cluster of 11 newly diagnosed HIV infections among residents of a small rural community in Scott County, which led to detection of 181 cases of HIV infection diagnosed from November 2014 to November 2015 linked to injection use of oxymorphone [18]. This study showed that the rapid transmission of HIV was initiated by introduction of a single HIV-1 strain. Additionally, serological and molecular analyses showed that 92.3% of the HIV patients were co-infected with HCV [18], suggesting a difference in the epidemic history of HCV infection in this community.

Here, we report results of complex genetic analyses that reveal long-standing and continued HCV transmission within the affected community in Indiana. A dense and dynamic network of HCV transmission among PWID presaged the explosive HIV outbreak.

To the best of our knowledge, this is the first comprehensive genetic evaluation of an HCV transmission network among PWID during an HIV outbreak in the United States. In the current HIV-driven field investigation, application of GHOST revealed a long-lasting local HCV epidemic. Analysis conducted using NS5B sequences indicates that each patient was infected with a unique HCV variant that belonged to one of four subtypes from three genotypes, suggesting multiple introductions of HCV into this rural PWID community. Such diversity can be achieved only by exposure of the entire community to HCV from several outside sources for many years. A long history of HCV infection among PWID in Indiana, prior to the recent introduction of a single HIV strain that caused the large HIV outbreak in 2015–2016, is supported by the detection of four HCV-positive patients, specimens from whom were collected in 2010. All four are involved in the major HCV transmission cluster of 130 cases, indicating that some HCV strains identified during the HIV outbreak were already circulating in this community for at least five years. Additionally, a long-term persistence of HCV infection among Indiana residents is strongly suggested by the observation of large endemic clusters of closely related HCV strains identified by NS5B sequence analyses. Extensive sampling from these clusters suggests a certain temporal pattern of HCV introduction when ancestral strains for each cluster have established local infections before all other detected HCV strains, so that the diverse progeny of these ancestors became dominant and more readily sampled from the affected PWID community. The earlier HCV strains, having infected this community before any other strain detected here, belonged to subtypes 1a and 3a. However, more recently introduced HCV strains belonged to subtype 1a predominantly. These findings imply a substantial increase in the introduction of new HCV strains over time, indicating a recent influx of HCV 1a infections and emphasizing possible wide-ranging contacts of the community with outside HCV sources.

The epidemiological investigation of the Indiana outbreak showed rapid spread of HIV by IDU, and occurred among PWID who shared syringes to inject the extended-release formulation of the prescription oral opioid oxymorphone [18]. Thus, our hypothesis of a recent increase in the introduction of HCV strains into this community is supported by recent reports of epidemics of prescription opioid abuse in the US [12,13], and increased numbers of patients with acute HCV infection in Central Appalachia [14]. A significant finding of our study is the detection of 51 cases (18%) with HCV mixed-genotype infections among the 281 persons tested using NGS. Spontaneous clearance of viruses from one subtype and persistence of another subtype after mixed infection was observed here in the persons who had a follow-up visit. Considering that HCV superinfections usually result in a rapid (<30 days) strain switch in affected persons [30], most probably owing to replicative exclusion [31], such a high rate of infections with multiple HCV strains indicates an extremely high rate of transmission of HCV in this rural PWID community. Co-transmission of more than a single HCV strain can also occur by the persistence of many mixed-genotype infections among PWID. However, in this case, the rate of HCV transmission in this community should have been expected to be even greater than suggested by superinfections alone, since a long-term co-existence of multiple HCV strains in a single person has been reported to be rare [31]. Detection of rapid succession of infections with different HCV strains in the patients with longitudinal follow-up specimens strongly supports the extraordinarily high rate of HCV transmission in this community of PWID. The complex and dynamic intra-host viral population is indicative of the evolving HCV epidemic, and underscores the need for a comprehensive and consistent assessment of the community in the following years. Combined, these data highlight significant epidemics of HCV among PWID in Indiana who previously have had limited access to HCV-related public health services, and how these gaps paved way for infiltration of HIV.

This is the first report where GHOST was field tested for an investigation of this scale, and our results offered high-resolution details about the HCV transmission dynamics. GHOST analysis showed that ~70% of the NGS-tested persons were organized into 23 transmission clusters, with the largest cluster including ~46% of all patients. This finding indicates the existence of a vast and highly modular HCV transmission network, reflecting the underlying network of contacts relevant to dissemination of blood-borne infections, that likely facilitated the explosive spread of HIV during the outbreak in the Indiana PWID community. Our analysis shows that these co-circulating HCV lineages preceded HIV in this community, with both viruses exploring the same transmission network among PWID, because 92.3% of all HIV-infected persons were found co-infected with HCV [18], but only 26.8% of HCV-infected cases tested here were co-infected with HIV. Segregation of 81.6% of HCV variants from HIV-co-infected patients into NS5B clusters (Fig. 1) indicates similarity in patterns of transmission between these two viruses. The major transmission cluster identified by GHOST includes 58.1% of HCV/HIV co-infected persons tested by NGS, while the cluster itself constitutes 46.3% of all HCV cases in the network. These results suggest that HIV dissemination was facilitated by the existing transmission network, which perpetuated HCV in the community for many years. The distribution of HIV co-infections predominantly among HCV patients organized into tight clusters suggests that both viral infections have similar routes of dissemination and explore the same contact network, with HCV having established infections earlier than HIV. Thus, the pattern of HCV dissemination reflects the pattern of HIV introduction to PWID in Indiana.

With the growing opioid crisis, the dual epidemics of HCV and HIV demand an integrated response. Our finding of similar transmission patterns across the PWID network suggests that targeted surveillance in PWID networks may permit a more efficient detection of new HIV infections in PWID communities infected with HCV. The high burden of HCV and HIV/HCV co-infection, coupled with lack of awareness and low-access to HCV services, highlights the need for sustained molecular surveillance of high-risk groups to understand better the landscape of HCV and HIV infections in the United States. HIV testing of persons, specifically from HCV clusters in HIV-naïve PWID populations, may facilitate early detection of HIV introduction into the community and allow early intervention to prevent future outbreaks of this magnitude. Early detection and prevention of HIV transmission would produce enormous public health and economic savings. Outcomes from the current investigation suggest that GHOST could facilitate prioritization of integrated intervention services for people at risk for both HIV and HCV infection. Implementing GHOST testing in rural high-risk communities, with PWID affected by the opioid epidemic where large HCV transmission networks with multiple genotypes are likely to exist, could help reduce existing and new HCV infections, and potentially prevent HIV introduction and transmission, if accompanied by network-targeted public health interventions such as Syringe Services Program (SSP), Medication Assisted Treatment (MAT), and HCV Direct Acting Antiviral therapy.

The limitation of this study is that GHOST testing was not conducted locally, which reduced identification of cases linked to the transmission network. Indeed, the identification of 29.5% of all cases and 21.6% of HIV-co-infected cases outside of the detected transmission clusters despite the expected high rate of HCV transmission suggests incomplete sampling from the local transmission network. At the population level, the infection network can be wider than the cases studied here, with unknown and unknowable transmissions occurring. GHOST integrates bioinformatics and web-based technologies with NGS, and enables users, regardless of their computational expertise, to conduct independent, cost-effective, and accurate hepatitis molecular surveillance. Participation of local public health institutions in GHOST-based surveillance is crucial for the most efficient detection of HCV outbreaks and transmission networks and for guiding targeted public health interventions.

In conclusion, while a single HIV strain caused the recent large HIV outbreak, numerous HCV strains have been introduced into the Indiana PWID community over many years prior to the HIV outbreak, with the rate of introductions likely increasing over time. Our investigation highlights the intersection of three major epidemics - opioid abuse, HCV, and HIV. GHOST offered detailed analysis of the genetic data and identified a vast HCV transmission network, frequent mixed-genotype infections and superinfections, indicating a complex, high-rate of HCV transmission in this community. The existing dense contact network that perpetuated HCV infections for years, likely enabled an increase in HIV infections in this population. As the heroin and opioid epidemic continues to worsen in the United States, it is imperative that susceptible populations are quickly identified to prevent new HIV outbreaks. Because the prevalence of HIV infection is generally low in the Mid-West, initiatives to identify high-risk HCV transmission networks may guide early detection of HIV outbreaks and interventions for interrupting HCV and HIV transmission among PWID in this, and other, geographic regions.

SR and YK lead the study. SR, HT, FJ, YL, LP, LGR, GV and HR performed specimen management and laboratory testing. SR, GX, ZD, EC, WS, PS and AS analyzed data. DC, IR, SS provided computational support. SR and YK coordinated the investigation in partnership with PP, RG, PPo, JG, SB, JL, WS, ET and JW. SR and YK wrote the manuscript, and all authors contributed to the critical revision of the manuscript and approved the final version.