A Phylogenetic Analysis of Hepatitis C Virus Transmission, Relapse, and Reinfection Among People Who Inject Drugs Receiving Opioid Agonist Therapy.

BACKGROUND: Understanding hepatitis C virus (HCV) transmission among people who inject drugs (PWID) is essential for HCV elimination. We aimed to differentiate reinfections from treatment failures and to identify transmission linkages and associated factors in a cohort of PWID receiving opioid agonist therapy (OAT). METHODS: We analyzed baseline and follow-up specimens from 150 PWID from 3 OAT clinics in the Bronx, New York. Next-generation sequencing data from the hypervariable region 1 of HCV were analyzed using Global Hepatitis Outbreak and Surveillance Technology. RESULTS: There were 3 transmission linkages between study participants. Sustained virologic response (SVR) was not achieved in 9 participants: 7 had follow-up specimens with similar sequences to baseline, and 2 died. In 4 additional participants, SVR was achieved but the participants were viremic at later follow-up: 2 were reinfected with different strains, 1 had a late treatment failure, and 1 was transiently viremic 17 months after treatment. All transmission linkages were from the same OAT clinic and involved spousal or common-law partnerships. CONCLUSION: This study highlights the use of next-generation sequencing as an important tool for identifying viral transmission and to help distinguish relapse and reinfection among PWID. Results reinforce the need for harm reduction interventions among couples and those who report ongoing risk factors after SVR.

Estimated Community Seroprevalence of SARS-CoV-2 Antibodies - Two Georgia Counties, April 28-May 3, 2020.

Transmission of SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19), is ongoing in many communities throughout the United States. Although case-based and syndromic surveillance are critical for monitoring the pandemic, these systems rely on persons obtaining testing or reporting a COVID-19-like illness. Using serologic tests to detect the presence of SARS-CoV-2 antibodies is an adjunctive strategy that estimates the prevalence of past infection in a population. During April 28-May 3, 2020, coinciding with the end of a statewide shelter-in-place order, CDC and the Georgia Department of Public Health conducted a serologic survey in DeKalb and Fulton counties in metropolitan Atlanta to estimate SARS-CoV-2 seroprevalence in the population. A two-stage cluster sampling design was used to randomly select 30 census blocks in each county, with a target of seven participating households per census block. Weighted estimates were calculated to account for the probability of selection and adjusted for age group, sex, and race/ethnicity. A total of 394 households and 696 persons participated and had a serology result; 19 (2.7%) of 696 persons had SARS-CoV-2 antibodies detected. The estimated weighted seroprevalence across these two metropolitan Atlanta counties was 2.5% (95% confidence interval [CI] = 1.4-4.5). Non-Hispanic black participants more commonly had SARS-CoV-2 antibodies than did participants of other racial/ethnic groups (p<0.01). Among persons with SARS-CoV-2 antibodies, 13 (weighted % = 49.9; 95% CI = 24.4-75.5) reported a COVID-19-compatible illness,* six (weighted % = 28.2; 95% CI = 11.9-53.3) sought medical care for a COVID-19-compatible illness, and five (weighted % = 15.7; 95% CI = 5.1-39.4) had been tested for SARS-CoV-2 infection, demonstrating that many of these infections would not have been identified through case-based or syndromic surveillance. The relatively low seroprevalence estimate in this report indicates that most persons in the catchment area had not been infected with SARS-CoV-2 at the time of the survey. Continued preventive measures, including social distancing, consistent and correct use of face coverings, and hand hygiene, remain critical in controlling community spread of SARS-CoV-2.

Poldip2 knockdown inhibits vascular smooth muscle proliferation and neointima formation by regulating the expression of PCNA and p21.

Polymerase delta-interacting protein 2 (Poldip2) is a multi-functional protein with numerous roles in the vasculature, including the regulation of cell apoptosis and migration, as well as extracellular matrix deposition; however, its role in VSMC proliferation and neointimal formation is unknown. In this study, we investigated the role of Poldip2 in intraluminal wire-injury induced neointima formation and proliferation of vascular smooth muscle cells in vitro and in vivo. Poldip2 expression was observed in the intima and media of human atherosclerotic arteries, where it colocalized with proliferating cell nuclear antigen (PCNA). Wire injury of femoral arteries of Poldip2+/+ mice induced robust neointimal formation after 2 weeks, which was impaired in Poldip2+/‒ mice. PCNA expression was significantly reduced and expression of the cell cycle inhibitor p21 was significantly increased in wire-injured arteries of Poldip2+/‒ animals compared to wild-type controls. No difference was observed in apoptosis. Downregulation of Poldip2 in rat aortic smooth muscle cells significantly reduced serum-induced proliferation and PCNA expression, but upregulated p21 expression. Downregulation of p21 using siRNA reversed the inhibition of proliferation induced by knockdown of Poldip2. These results indicate that Poldip2 plays a critical role in the proliferation of VSMCs.

GHOST: global hepatitis outbreak and surveillance technology.

BACKGROUND: Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections associated with unsafe injection practices, drug diversion, and other exposures to blood are difficult to detect and investigate. Effective HCV outbreak investigation requires comprehensive surveillance and robust case investigation. We previously developed and validated a methodology for the rapid and cost-effective identification of HCV transmission clusters. Global Hepatitis Outbreak and Surveillance Technology (GHOST) is a cloud-based system enabling users, regardless of computational expertise, to analyze and visualize transmission clusters in an independent, accurate and reproducible way. RESULTS: We present and explore performance of several GHOST implemented algorithms using next-generation sequencing data experimentally obtained from hypervariable region 1 of genetically related and unrelated HCV strains. GHOST processes data from an entire MiSeq run in approximately 3 h. A panel of seven specimens was used for preparation of six repeats of MiSeq libraries. Testing sequence data from these libraries by GHOST showed a consistent transmission linkage detection, testifying to high reproducibility of the system. Lack of linkage among genetically unrelated HCV strains and constant detection of genetic linkage between HCV strains from known transmission pairs and from follow-up specimens at different levels of MiSeq-read sampling indicate high specificity and sensitivity of GHOST in accurate detection of HCV transmission. CONCLUSIONS: GHOST enables automatic extraction of timely and relevant public health information suitable for guiding effective intervention measures. It is designed as a virtual diagnostic system intended for use in molecular surveillance and outbreak investigations rather than in research. The system produces accurate and reproducible information on HCV transmission clusters for all users, irrespective of their level of bioinformatics expertise. Improvement in molecular detection capacity will contribute to increasing the rate of transmission detection, thus providing opportunity for rapid, accurate and effective response to outbreaks of hepatitis C. Although GHOST was originally developed for hepatitis C surveillance, its modular structure is readily applicable to other infectious diseases. Worldwide availability of GHOST for the detection of HCV transmissions will foster deeper involvement of public health researchers and practitioners in hepatitis C outbreak investigation.

Accurate Genetic Detection of Hepatitis C Virus Transmissions in Outbreak Settings.

Hepatitis C is a major public health problem in the United States and worldwide. Outbreaks of hepatitis C virus (HCV) infections are associated with unsafe injection practices, drug diversion, and other exposures to blood and are difficult to detect and investigate. Here, we developed and validated a simple approach for molecular detection of HCV transmissions in outbreak settings. We obtained sequences from the HCV hypervariable region 1 (HVR1), using end-point limiting-dilution (EPLD) technique, from 127 cases involved in 32 epidemiologically defined HCV outbreaks and 193 individuals with unrelated HCV strains. We compared several types of genetic distances and calculated a threshold, using minimal Hamming distances, that identifies transmission clusters in all tested outbreaks with 100% accuracy. The approach was also validated on sequences obtained using next-generation sequencing from HCV strains recovered from 239 individuals, and findings showed the same accuracy as that for EPLD. On average, the nucleotide diversity of the intrahost population was 6.2 times greater in the source case than in any incident case, allowing the correct detection of transmission direction in 8 outbreaks for which source cases were known. A simple and accurate distance-based approach developed here for detecting HCV transmissions streamlines molecular investigation of outbreaks, thus improving the public health capacity for rapid and effective control of hepatitis C.

Cryptic Hepatitis B and E in Patients With Acute Hepatitis of Unknown Etiology.

BACKGROUND: Up to 30% of acute viral hepatitis has no known etiology. To determine the disease etiology in patients with acute hepatitis of unknown etiology (HUE), serum specimens were obtained from 38 patients residing in the United Kingdom and Vietnam and from 26 healthy US blood donors. All specimens tested negative for known viral infections causing hepatitis, using commercially available serological and nucleic acid assays. METHODS: Specimens were processed by sequence-independent complementary DNA amplification and next-generation sequencing (NGS). Sufficient material for individual NGS libraries was obtained from 12 HUE cases and 26 blood donors; the remaining HUE cases were sequenced as a pool. Read mapping was done by targeted and de novo assembly. RESULTS: Sequences from hepatitis B virus (HBV) were detected in 7 individuals with HUE (58.3%) and the pooled library, and hepatitis E virus (HEV) was detected in 2 individuals with HUE (16.7%) and the pooled library. Both HEV-positive cases were coinfected with HBV. HBV sequences belonged to genotypes A, D, or G, and HEV sequences belonged to genotype 3. No known hepatotropic viruses were detected in the tested normal human sera. CONCLUSIONS: NGS-based detection of HBV and HEV infections is more sensitive than using commercially available assays. HBV and HEV may be cryptically associated with HUE.

Intra-host diversity and evolution of hepatitis C virus endemic to Côte d'Ivoire.

Hepatitis C virus (HCV) infection presents an important, but underappreciated public health problem in Africa. In Côte d'Ivoire, very little is known about the molecular dynamics of HCV infection. Plasma samples (n = 608) from pregnant women collected in 1995 from Côte d'Ivoire were analyzed in this study. Only 18 specimens (∼3%) were found to be HCV PCR-positive. Phylogenetic analysis of the HCV NS5b sequences showed that the HCV variants belong to genotype 1 (HCV1) (n = 12, 67%) and genotype 2 (HCV2) (n = 6, 33%), with a maximum genetic diversity among HCV variants in each genotype being 20.7% and 24.0%, respectively. Although all HCV2 variants were genetically distant from each other, six HCV1 variants formed two tight sub-clusters belonging to HCV1a and HCV1b. Analysis of molecular variance (AMOVA) showed that the genetic structure of HCV isolates from West Africa with Côte d'Ivoire included were significantly different from Central African strains (P = 0.0001). Examination of intra-host viral populations using next-generation sequencing of the HCV HVR1 showed a significant variation in intra-host genetic diversity among infected individuals, with some strains composed of sub-populations as distant from each other as viral populations from different hosts. Collectively, the results indicate a complex HCV evolution in Côte d'Ivoire, similar to the rest of West Africa, and suggest a unique HCV epidemic history in the country.

HCV transmission in high-risk communities in Bulgaria.

BACKGROUND: The rate of HIV infection in Bulgaria is low. However, the rate of HCV-HIV-coinfection and HCV infection is high, especially among high-risk communities. The molecular epidemiology of those infections has not been studied before. METHODS: Consensus Sanger sequences of HVR1 and NS5B from 125 cases of HIV/HCV coinfections, collected during 2010-2014 in 15 different Bulgarian cities, were used for preliminary phylogenetic evaluation. Next-generation sequencing (NGS) data of the hypervariable region 1 (HVR1) analyzed via the Global Hepatitis Outbreak and Surveillance Technology (GHOST) were used to evaluate genetic heterogeneity and possible transmission linkages. Links between pairs that were below and above the established genetic distance threshold, indicative of transmission, were further examined by generating k-step networks. RESULTS: Preliminary genetic analyses showed predominance of HCV genotype 1a (54%), followed by 1b (20.8%), 2a (1.4%), 3a (22.3%) and 4a (1.4%), indicating ongoing transmission of many HCV strains of different genotypes. NGS of HVR1 from 72 cases showed significant genetic heterogeneity of intra-host HCV populations, with 5 cases being infected with 2 different genotypes or subtypes and 6 cases being infected with 2 strains of same subtype. GHOST revealed 8 transmission clusters involving 30 cases (41.7%), indicating a high rate of transmission. Four transmission clusters were found in Sofia, three in Plovdiv, and one in Peshtera. The main risk factor for the clusters was injection drug use. Close genetic proximity among HCV strains from the 3 Sofia clusters, and between HCV strains from Peshtera and one of the two Plovdiv clusters confirms a long and extensive transmission history of these strains in Bulgaria. CONCLUSIONS: Identification of several HCV genotypes and many HCV strains suggests a frequent introduction of HCV to the studied high-risk communities. GHOST detected a broad transmission network, which sustains circulation of several HCV strains since their early introduction in the 3 cities. This is the first report on the molecular epidemiology of HIV/HCV coinfections in Bulgaria.

Reactivation of a Vaccine Escape Hepatitis B Virus Mutant in a Cambodian Patient During Anti-Hepatitis C Virus Therapy.

A 76-year-old Cambodian man co-infected with hepatitis B virus (HBV) and hepatitis C virus (HCV) 6c-1 presented for care. HBV DNA was intermittently detectable despite anti-HBs levels being above the protective threshold. During treatment for HCV, HBV DNA levels increased. Sequencing revealed multiple mutations including vaccine escape mutation and mutations predicted to enhance fitness. This case represents exacerbation of an HBV vaccine escape mutant during a direct-acting antiviral therapy.

A large HCV transmission network enabled a fast-growing HIV outbreak in rural Indiana, 2015.

BACKGROUND: A high prevalence (92.3%) of hepatitis C virus (HCV) co-infection among HIV patients identified during a large HIV outbreak associated with injection of oxymorphone in Indiana prompted genetic analysis of HCV strains. METHODS: Molecular epidemiological analysis of HCV-positive samples included genotyping, sampling intra-host HVR1 variants by next-generation sequencing (NGS) and constructing transmission networks using Global Hepatitis Outbreak and Surveillance Technology (GHOST). FINDINGS: Results from the 492 samples indicate predominance of HCV genotypes 1a (72.2%) and 3a (20.4%), and existence of 2 major endemic NS5B clusters involving 49.8% of the sequenced strains. Among 76 HIV co-infected patients, 60.5% segregated into 2 endemic clusters. NGS analyses of 281 cases identified 826,917 unique HVR1 sequences and 51 cases of mixed subtype/genotype infections. GHOST mapped 23 transmission clusters. One large cluster (n = 130) included 50 cases infected with ≥2 subtypes/genotypes and 43 cases co-infected with HIV. Rapid strain replacement and superinfection with different strains were found among 7 of 12 cases who were followed up. INTERPRETATION: GHOST enabled mapping of HCV transmission networks among persons who inject drugs (PWID). Findings of numerous transmission clusters, mixed-genotype infections and rapid succession of infections with different HCV strains indicate a high rate of HCV spread. Co-localization of HIV co-infected patients in the major HCV clusters suggests that HIV dissemination was enabled by existing HCV transmission networks that likely perpetuated HCV in the community for years. Identification of transmission networks is an important step to guiding efficient public health interventions for preventing and interrupting HCV and HIV transmission among PWID. FUND: US Centers for Disease Control and Prevention, and US state and local public health departments.

Next-Generation Sequencing Reveals Frequent Opportunities for Exposure to Hepatitis C Virus in Ghana.

Globally, hepatitis C Virus (HCV) infection is responsible for a large proportion of persons with liver disease, including cancer. The infection is highly prevalent in sub-Saharan Africa. West Africa was identified as a geographic origin of two HCV genotypes. However, little is known about the genetic composition of HCV populations in many countries of the region. Using conventional and next-generation sequencing (NGS), we identified and genetically characterized 65 HCV strains circulating among HCV-positive blood donors in Kumasi, Ghana. Phylogenetic analysis using consensus sequences derived from 3 genomic regions of the HCV genome, 5'-untranslated region, hypervariable region 1 (HVR1) and NS5B gene, consistently classified the HCV variants (n = 65) into genotypes 1 (HCV-1, 15%) and genotype 2 (HCV-2, 85%). The Ghanaian and West African HCV-2 NS5B sequences were found completely intermixed in the phylogenetic tree, indicating a substantial genetic heterogeneity of HCV-2 in Ghana. Analysis of HVR1 sequences from intra-host HCV variants obtained by NGS showed that three donors were infected with >1 HCV strain, including infections with 2 genotypes. Two other donors share an HCV strain, indicating HCV transmission between them. The HCV-2 strain sampled from one donor was replaced with another HCV-2 strain after only 2 months of observation, indicating rapid strain switching. Bayesian analysis estimated that the HCV-2 strains in Ghana were expanding since the 16th century. The blood donors in Kumasi, Ghana, are infected with a very heterogeneous HCV population of HCV-1 and HCV-2, with HCV-2 being prevalent. The detection of three cases of co- or super-infections and transmission linkage between 2 cases suggests frequent opportunities for HCV exposure among the blood donors and is consistent with the reported high HCV prevalence. The conditions for effective HCV-2 transmission existed for ~ 3-4 centuries, indicating a long epidemic history of HCV-2 in Ghana.