INTRODUCTION: ultimately detected in 2016, wild poliovirus (WPV) transmission continued undetected after 2011 in Northeast Nigeria Borno and Yobe States in security-compromised areas, inaccessible due to armed insurgency. Varying inaccessibility prevented children aged <5 years in these areas from polio vaccination interventions and surveillance, while massive population displacements occurred. We examined progress in access over time to provide data supporting a very low probability of undetected WPV circulation within remaining trapped populations after 2016. METHODS: to assess the extent of inaccessibility in security-compromised areas, we obtained empirical historical data in 2020 on a quarterly and annual basis from relevant polio eradication staff for the period 2010-2020. The extent of access to areas for immunization by recall was compared to geospatial data from vaccinator tracking. Population estimates over time in security-compromised areas were extracted from satellite imagery. We compared the historical access data from staff with tracking and population esimates. RESULTS: access varied during 2010-2020, with inaccessibility peaking during 2014-2016. We observed concurrent patterns between historical recalled data on inaccessibility and contemporaneous satellite imagery on population displacements, which increased confidence in the quality of recalled data. CONCLUSION: staff-recalled access was consistent with vaccinator tracking and satellite imagery of population displacments. Despite variability in inaccessibility over time, innovative immunization initiatives were implemented as access allowed and surveillance initiatives were initiated to search for poliovirus transmission. Along with escape and liberation of residents by the military in some geographic areas, these initiatives resulted in a massive reduction in the size of the unvaccinated population remaining resident.

Machine-learning methods in the form of Bayesian networks (BN), linear projection (LP) and self-organizing tree (SOT) models were used to explore association among polymorphic sites within the HVR1 and NS5a regions of the HCV genome, host demographic factors (ethnicity, gender and age) and response to the combined interferon (IFN) and ribavirin (RBV) therapy. The BN models predicted therapy outcomes, gender and ethnicity with accuracy of 90%, 90% and 88.9%, respectively. The LP and SOT models strongly confirmed associations of the HVR1 and NS5A structures with response to therapy and demographic host factors identified by BN. The data indicate host specificity of HCV evolution and suggest the application of these models to predict outcomes of IFN/RBV therapy.

Hepatitis C Virus sequence studies mainly focus on the viral amplicon containing the Hypervariable region 1 (HVR1) to obtain a sample of sequences from which several population genetics parameters can be calculated. Recent advances in sequencing methods allow for analyzing an unprecedented number of viral variants from infected patients and present a novel opportunity for understanding viral evolution, drug resistance and immune escape. In the present paper, we compared three recent technologies for amplicon analysis: (i) Next-Generation Sequencing; (ii) Clonal sequencing using End-point Limiting-dilution for isolation of individual sequence variants followed by Real-Time PCR and sequencing; and (iii) Mass spectrometry of base-specific cleavage reactions of a target sequence. These three technologies were used to assess intra-host diversity and inter-host genetic relatedness in HVR1 amplicons obtained from 38 patients (subgenotypes 1a and 1b). Assessments of intra-host diversity varied greatly between sequence-based and mass-spectrometry-based data. However, assessments of inter-host variability by all three technologies were equally accurate in identification of genetic relatedness among viral strains. These results support the application of all three technologies for molecular epidemiology and population genetics studies. Mass spectrometry is especially promising given its high throughput, low cost and comparable results with sequence-based methods.

After the 2015 documentation of global eradication of wild poliovirus type 2,* Sabin type 2 oral poliovirus vaccine (OPV) was withdrawn from routine immunization (RI) in all OPV-using countries in 2016, in a global synchronized switch from trivalent OPV (containing vaccine virus serotypes 1, 2, and 3) to bivalent OPV (containing serotypes 1 and 3), to reduce the rare risks for type 2 vaccine-associated paralytic poliomyelitis. Concurrently, the Global Polio Eradication Initiative (GPEI) recommended that all OPV-using countries introduce ≥1 dose of inactivated poliovirus vaccine (IPV) into RI programs; IPV protects against paralysis caused by all three serotypes but cannot be transmitted from person to person or cause paralysis. Use of OPV, especially in areas with low vaccination coverage, is associated with low risk of emergence of vaccine-derived polioviruses (VDPVs). As susceptible persons in new birth cohorts accumulated after withdrawal of OPV type 2, population immunity against infection with serotype 2 declined (1), facilitating the emergence of circulating VDPV type 2 (cVDPV2). During the previous 7 years, cVDPV2 outbreaks required response supplementary immunization activities (SIAs) with monovalent type 2 OPV (mOPV2); however, if SIAs were not of sufficiently high quality and did not achieve high enough coverage, new emergences of cVDPV2 occurred.

This study examined the impact of armed conflict on public health surveillance systems, the limitations of traditional surveillance in this context, and innovative strategies to overcome these limitations. A qualitative case study was conducted to examine the factors affecting the functioning of poliovirus surveillance in conflict-affected areas of Borno state, Nigeria using semi-structured interviews of a purposeful sample of participants. The main inhibitors of surveillance were inaccessibility, the destroyed health infrastructure, and the destroyed communication network. These three challenges created a situation in which the traditional polio surveillance system could not function. Three strategies to overcome these challenges were viewed by respondents as the most impactful. First, local community informants were recruited to conduct surveillance for acute flaccid paralysis in children in the inaccessible areas. Second, the informants engaged in local-level negotiation with the insurgency groups to bring children with paralysis to accessible areas for investigation and sample collection. Third, GIS technology was used to track the places reached for surveillance and vaccination and to estimate the size and location of the inaccessible population. A modified monitoring system tracked tailored indicators including the number of places reached for surveillance and the number of acute flaccid paralysis cases detected and investigated, and utilized GIS technology to map the reach of the program. The surveillance strategies used in Borno were successful in increasing surveillance sensitivity in an area of protracted conflict and inaccessibility. This approach and some of the specific strategies may be useful in other areas of armed conflict.

OBJECTIVE: To stop transmission of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections in association with myocardial perfusion imaging (MPI) at a cardiology clinic. DESIGN: Outbreak investigation and quasispecies analysis of HCV hypervariable region 1 genome. SETTING: Outpatient cardiology clinic. PATIENTS: Patients undergoing MPI. METHODS: Case patients met definitions for HBV or HCV infection. Cases were identified through surveillance registry cross-matching against clinic records and serological screening. Observations of clinic practices were performed. RESULTS: During 2012-2014, 7 cases of HCV and 4 cases of HBV occurred in 4 distinct clusters among patients at a cardiology clinic. Among 3 case patients with HCV infection who had MPI on June 25, 2014, 2 had 98.48% genetic identity of HCV RNA. Among 4 case patients with HCV infection who had MPI on March 13, 2014, 3 had 96.96%-99.24% molecular identity of HCV RNA. Also, 2 clusters of 2 patients each with HBV infection had MPI on March 7, 2012, and December 4, 2014. Clinic staff reused saline vials for >1 patient. No infection control breaches were identified at the compounding pharmacy that supplied the clinic. Patients seen in clinic through March 27, 2015, were encouraged to seek testing for HBV, HCV, and human immunodeficiency virus. The clinic switched to all single-dose medications and single-use intravenous flushes on March 27, 2015, and no further cases were identified. CONCLUSIONS: This prolonged healthcare-associated outbreak of HBV and HCV was most likely related to breaches in injection safety. Providers should follow injection safety guidelines in all practice settings.

In this study, the prevalence rate, associated risk factors and genetic diversity of hepatitis C virus (HCV) infection were determined among people who use crack from an international drug trafficking route in Central-West, Brazil. Blood samples were collected from 700 users of crack from Campo Grande and two border cities of Mato Grosso do Sul State and tested for HCV infection using serological and molecular testing methodologies. Anti-HCV was detected in 31/700 (4.5%, 95% CI: 2.9-6.0%) and HCV RNA in 26/31 (83.9%) of anti-HCV positive samples. Phylogenetic analysis of three HCV sub-genomic regions (5'UTR, NS5B and HVR-1) revealed the circulation of 1a (73.9%), 1b (8.7%) and 3a (17.4%) genotypes. Next-generation sequencing and phylogenetic analysis of intra-host viral populations of HCV HVR-1 showed a significant variation in intra-host genetic diversity among infected individuals, with 58.8% composed of more than one sub-population. Bayesian analysis estimated that the most recent common HCV ancestor for strains identified here was introduced to this region after 1975 following expansion of intravenous drug use in Brazil. Multivariate analyses showed that only 'ever having injected drugs' was independently associated with HCV infection. These results indicate an increasing spread of multiple HCV strains requiring public health intervention, such as harm reduction, testing services and treatment among crack users in this important border region of Central Brazil.

Beginning in 2013, multiple local government areas (LGAs) in Borno and Yobe in northeast Nigeria and other parts of the Lake Chad basin experienced a violent insurgency that resulted in substantial numbers of isolated and displaced people. Northeast Nigeria represents the last known reservoir country of wild poliovirus (WPV) transmission in Africa, with detection of paralytic cases caused by serotype 1 WPV in 2016 in Borno and serotype 3 WPV in late 2012. Parts of Borno and Yobe are also problematic areas for transmission of serotype 2 circulating vaccine-derived polioviruses, and they continue to face challenges associated with conflict and inadequate health services in security-compromised areas that limit both immunization and surveillance activities. We model poliovirus transmission of all three serotypes for Borno and Yobe using a deterministic differential equation-based model that includes four subpopulations to account for limitations in access to immunization services and dynamic restrictions in population mixing. We find that accessibility issues and insufficient immunization allow for prolonged poliovirus transmission and potential undetected paralytic cases, although as of the end of 2019, including responsive program activities in the modeling suggest die out of indigenous serotypes 1 and 3 WPVs prior to 2020. Specifically, recent and current efforts to access isolated populations and provide oral poliovirus vaccine continue to reduce the risks of sustained and undetected transmission, although some uncertainty remains. Continued improvement in immunization and surveillance in the isolated subpopulations should minimize these risks. Stochastic modeling can build on this analysis to characterize the implications for undetected transmission and confidence about no circulation.

The number of wild poliovirus (WPV) cases in Nigeria decreased from 1,122 in 2006 to six WPV type 1 (WPV1) in 2014 (1). During August 2014-July 2016, no WPV cases were detected; during August-September 2016, four cases were reported in Borno State. An insurgency in northeastern Nigeria had resulted in 468,800 children aged <5 years deprived of health services in Borno by 2016. Military activities in mid-2016 freed isolated families to travel to camps, where the four WPV1 cases were detected. Oral poliovirus vaccine (OPV) campaigns were intensified during August 2016-December 2017; since October 2016, no WPV has been detected (2). Vaccination activities in insurgent-held areas are conducted by security forces; however, 60,000 unvaccinated children remain in unreached settlements. Since 2018, circulating vaccine-derived poliovirus type 2 (cVDPV2) has emerged and spread from Nigeria to Niger and Cameroon; outbreak responses to date have not interrupted transmission. This report describes progress in Nigeria polio eradication activities during January 2018-May 2019 and updates the previous report (2). Interruption of cVDPV2 transmission in Nigeria will need increased efforts to improve campaign quality and include insurgent-held areas. Progress in surveillance and immunization activities will continue to be reviewed, potentially allowing certification of interruption of WPV transmission in Africa in 2020.

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.

BACKGROUND: Hepatitis B e antigen (HBeAg) is considered an indicator of high hepatitis B virus (HBV) replication. Performance characteristics of commercially available HBeAg assays have not been determined, thus it is unknown whether lack of HBeAg detection is because of test sensitivity or HBV basal core promoter and precore mutations. OBJECTIVES: We studied the correlation between HBeAg reactivity with HBV DNA levels in three commercially available HBeAg assays using 335 HBsAg and HBV DNA positive serum/plasma samples. STUDY DESIGN: Diagnostic sensitivity was determined by serial dilutions of a WHO HBeAg standard. The limit of HBeAg detection estimated through regression was 1 IU/mL (Centaur), 97 IU/mL (DiaSorin) and 129 IU/mL (Vitros). Of these 335 samples, enough sample volume remained in 253 samples for head-to-head comparison of the assays. RESULTS: 81 (32%), 41 (16%) and 36 (14%) of the samples were HBeAg positive by the Centaur, DiaSorin and Vitros assays, respectively. Compared to the FDA-approved Centaur assay the specificity of the other two assays was 98%, while sensitivity was 47% for the DiaSorin assay and 41% for the Vitros assay. Significant association was found between HBeAg positive samples and HBV DNA levels >20,000 IU/mL; 31% of HBeAg negative samples (Centaur) had HBV DNA levels >20,000 IU/mL, 26% of HBeAg positive samples had HBV DNA levels <20,000 IU/mL and 5 HBeAg positive samples had HBV DNA levels <2000 IU/mL. CONCLUSION: Discordance was seen between these HBeAg assays, indicating reliance on HBeAg alone as a marker of high HBV replication can be misleading. Detection and quantification of HBV DNA remains the accurate and reliable marker of HBV replication.

Nearly three decades after the World Health Assembly launched the Global Polio Eradication Initiative in 1988, four of the six World Health Organization (WHO) regions have been certified polio-free (1). Nigeria is one of three countries, including Pakistan and Afghanistan, where wild poliovirus (WPV) transmission has never been interrupted. In September 2015, after >1 year without any reported WPV cases, Nigeria was removed from WHO's list of countries with endemic WPV transmission (2); however, during August and September 2016, four type 1 WPV (WPV1) cases were reported from Borno State, a state in northeastern Nigeria experiencing a violent insurgency (3). The Nigerian government, in collaboration with partners, launched a large-scale coordinated response to the outbreak (3). This report describes progress in polio eradication activities in Nigeria during January-December 2017 and updates previous reports (3-5). No WPV cases have been reported in Nigeria since September 2016; the latest case had onset of paralysis on August 21, 2016 (3). However, polio surveillance has not been feasible in insurgent-controlled areas of Borno State. Implementation of new strategies has helped mitigate the challenges of reaching and vaccinating children living in security-compromised areas, and other strategies are planned. Despite these initiatives, however, approximately 130,000-210,000 (28%-45%) of the estimated 469,000 eligible children living in inaccessible areas in 2016 have not been vaccinated. Sustained efforts to optimize surveillance and improve immunization coverage, especially among children in inaccessible areas, are needed.

Despite the significant public health problems associated with hepatitis B virus (HBV) in sub-Saharan Africa, many countries in this region do not have systematic HBV surveillance or genetic information on HBV circulating locally. Here, we report on the genetic characterization of 772 HBV strains from Tanzania. Phylogenetic analysis of the S-gene sequences showed prevalence of HBV genotype A (HBV/A, n=671, 86.9 %), followed by genotypes D (HBV/D, n=95, 12.3 %) and E (HBV/E, n=6, 0.8 %). All HBV/A sequences were further classified into subtype A1, while the HBV/D sequences were assigned to a new cluster. Among the Tanzanian sequences, 84 % of HBV/A1 and 94 % of HBV/D were unique. The Tanzanian and global HBV/A1 sequences were compared and were completely intermixed in the phylogenetic tree, with the Tanzanian sequences frequently generating long terminal branches, indicating a long history of HBV/A1 infections in the country. The time to the most recent common ancestor was estimated to be 188 years ago [95 % highest posterior density (HPD): 132 to 265 years] for HBV/A1 and 127 years ago (95 % HPD: 79 to 192 years) for HBV/D. The Bayesian skyline plot showed that the number of transmissions 'exploded' exponentially between 1960-1970 for HBV/A1 and 1970-1990 for HBV/D, with the effective population of HBV/A1 having expanded twice as much as that of HBV/D. The data suggest that Tanzania is at least a part of the geographic origin of the HBV/A1 subtype. A recent increase in the transmission rate and significant HBV genetic diversity should be taken into consideration when devising public health interventions to control HBV infections in Tanzania.

A cloned stable cell line, HepG2-HBVE6, was established following transfection of HepG2 cells with a retroviral plasmid into which a 1.1-fold genomic construct of hepatitis B virus (HBV) belonging to genotype E (HBV/E) was inserted. The cell line retains the entire HBV/E insert, and produces episomal HBV DNA. It expresses HBV pregenomic, preS1 and preS2/S transcripts, and sheds hepatitis B surface and e antigens as well as structures resembling HBV-subviral and Dane particles. The HepG2-HBVE6 cell line, in permitting recapitulation of the HBV life cycle, may be used for studying viral characteristics, therapeutic and preventative outcomes and for preparing reagents specific to HBV genotype E.

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.

Outbreaks of hepatitis C virus (HCV) infections can occur among hemodialysis patients when recommended infection control practices are not followed (1). On January 30, 2014, a dialysis clinic in Tennessee identified acute HCV in a patient (patient A) during routine screening and reported it to the Tennessee Department of Health. Patient A had enrolled in the dialysis clinic in March 2010 and had annually tested negative for HCV (including a last HCV test on December 19, 2012), until testing positive for HCV antibodies (anti-HCV) on December 18, 2013 (confirmed by a positive HCV nucleic acid amplification test). Patient A reported no behavioral risk factors, but did have multiple health care exposures.

BACKGROUND: In November and December 2012, 6 patients at a hemodialysis clinic were given a diagnosis of new hepatitis C virus (HCV) infection. OBJECTIVE: To investigate the outbreak to identify risk factors for transmission. METHODS: A case patient was defined as a patient who was HCV-antibody negative on clinic admission but subsequently was found to be HCV-antibody positive from January 1, 2008, through April 30, 2013. Patient charts were reviewed to identify and describe case patients. The hypervariable region 1 of HCV from infected patients was tested to assess viral genetic relatedness. Infection control practices were evaluated via observations. A forensic chemiluminescent agent was used to identify blood contamination on environmental surfaces after cleaning. RESULTS: Eighteen case patients were identified at the clinic from January 1, 2008, through April 30, 2013, resulting in an estimated 16.7% attack rate. Analysis of HCV quasispecies identified 4 separate clusters of transmission involving 11 case patients. The case patients and previously infected patients in each cluster were treated in neighboring dialysis stations during the same shift, or at the same dialysis station on 2 consecutive shifts. Lapses in infection control were identified. Visible and invisible blood was identified on multiple surfaces at the clinic. CONCLUSIONS: Epidemiologic and laboratory data confirmed transmission of HCV among numerous patients at the dialysis clinic over 6 years. Infection control breaches were likely responsible. This outbreak highlights the importance of rigorous adherence to recommended infection control practices in dialysis 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, being 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) 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 from 239 individuals obtained using next-generation sequencing, showing the same accuracy as EPLD. In average, nucleotide diversity of the intra-host population was 6.2-times greater in the source 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 for detecting HCV transmissions developed here streamlines molecular investigation of outbreaks, thus improving the public health capacity for rapid and effective control of hepatitis C.

Nucleic acid testing (NAT) for hepatitis C virus (HCV) is recommended for screening of organ donors, yet not all donor infections may be detected. We describe three US clusters of HCV transmission from donors at increased risk for HCV infection. Donor's and recipients' medical records were reviewed. Newly infected recipients were interviewed. Donor-derived HCV infection was considered when infection was newly detected after transplantation in recipients of organs from increased risk donors. Stored donor sera and tissue samples were tested for HCV RNA with high-sensitivity quantitative PCR. Posttransplant and pretransplant recipient sera were tested for HCV RNA. Quasispecies analysis of hypervariable region-1 was used to establish genetic relatedness of recipient HCV variants. Each donor had evidence of injection drug use preceding death. Of 12 recipients, 8 were HCV-infected-6 were newly diagnosed posttransplant. HCV RNA was retrospectively detected in stored samples from donor immunologic tissue collected at organ procurement. Phylogenetic analysis showed two clusters of closely related HCV variants from recipients. These investigations identified the first known HCV transmissions from increased risk organ donors with negative NAT screening, indicating very recent donor infection. Recipient informed consent and posttransplant screening for blood-borne pathogens are essential when considering increased risk donors.

Hepatitis C virus (HCV) is classified into 7 genotypes based on genetic diversity and most genotypes have been found in Africa. Infections with HCV genotype 2 (HCV2) are most prevalent in West Africa, and it was suggested that HCV2 originated in West Africa. To better understand the evolutionary epidemiology of HCV2 in Africa, we examined new NS5b sequences of HCV2 strains obtained from Cote d'Ivoire, Ghana and Nigeria sequenced in this laboratory with those available from West, North and Central Africa. Bayesian phylogeographic analysis using a discrete trait model showed that Ghana is the most likely geographic region for origin of HCV2. Spread of HCV2 from Ghana does not appear to be through diffusion to adjacent countries along the coast. Rather, it was transmitted from Ghana to many distant countries in Africa, suggesting that certain routes of geographic dissemination were historically more efficient than mere proximity and that the HCV2 epidemic history in West Africa is extremely complex.

BACKGROUND: Sub-Saharan Africa (SSA) has among the highest global Hepatitis C Virus (HCV) prevalence estimates. However, reports suggesting high rates of serologic false positives and low levels of viremia have led to uncertainty regarding the burden of active infection in this region. Additionally, little is known about the predominant transmission risk factors in SSA. METHODS: We prospectively recalled 363 past blood donors (180 who were rapid screen assay (RSA) positive and 183 that were RSA negative at time of donation) to identify the level of active infection and risk factors for infection at a teaching hospital in Kumasi, Ghana. Participants had repeat blood testing and were administered a questionnaire on risk factors. RESULTS: The frequency of HCV active infection ranged from 74.4% to 88% depending on the criteria used to define serologically positive cases. Individuals with active disease had biochemical evidence of liver inflammation and median viral loads of 5.7 log copies/ml. Individuals from the northern and upper regions of Ghana had greater risks of infection compared to participants from other areas. Additional risk factors included traditional circumcision, home birth, tribal scarring and HBV co-infection. CONCLUSIONS: Viremic infection was common among serologically confirmed cases. Attention to testing algorithms is needed to define the true HCV burden in SSA. These data also suggest that several transmission modes are likely contributing to the current HCV epidemic in Ghana, and the distribution of these practices may result in substantial regional variation in prevalence.

Dried blood spots (DBS) expedite the collection, storage and shipping of blood samples, thereby facilitating large-scale serologic studies. We evaluated the sensitivity of anti-HCV IgG testing and HCV-RNA quantitation using freshly prepared and stored DBS derived from HCV-infected patients. Protocols for elution were optimized using DBS prepared from plasma of 52 HCV-infected persons and 51 uninfected persons (control DBS), then applied to DBS from 33 chronic hepatitis C patients that had been stored at -20 degrees C for 5 years (stored DBS). Control and stored DBS, and their corresponding plasma, were processed for anti-HCV IgG testing using the VITROS chemiluminescence assay (CIA) and the HCV 3.0 enzyme immunoassay (EIA) (Ortho-Clinical Diagnostics), and for HCV RNA quantitation by quantitative (q) RT-PCR. HCV genotyping was conducted by nucleotide sequencing. The sensitivity of CIA and EIA in control DBS was 92% and 90%, respectively, compared to 100% and 97%, respectively, in stored DBS. The sensitivity of HCV RNA detection was 88% in control DBS, compared to 36% in stored DBS. Specificity was 100% for all the assays in both control and stored DBS. Genotypes 1, 2 and 3 were detected in 16 (62%), 6 (23.1%), and 4 (15.3%) samples, respectively. Sequences generated from DBS and their corresponding plasma samples were identical. Whereas the sensitivity of anti-HCV IgG detection in stored DBS was equivalent to that in recently prepared DBS, the sensitivity of HCV RNA detection was markedly lower in stored DBS compared to recently prepared DBS. Stored DBS may be reliably used for anti-HCV detection but for HCV-RNA-based testing freshly prepared DBS is preferable to stored DBS.

Hepatitis C virus (HCV) genotype 3a accounts for approximately 80% of HCV infections in Pakistan, where approximately 10 million people are HCV-infected. Here, we report analysis of the genetic heterogeneity of HCV NS3 and NS5b subgenomic regions from genotype 3a variants obtained from Pakistan. Phylogenetic analyses showed that Pakistani genotype 3a variants were as genetically diverse as global variants, with extensive intermixing. Bayesian estimates showed that the most recent ancestor for genotype 3a in Pakistan was last extant in approximately 1896-1914 C.E. (range: 1851-1932). This genotype experienced a population expansion starting from approximately 1905 to approximately 1970 after which the effective population leveled. Death/birth models suggest that HCV 3a has reached saturating diversity with decreasing turnover rate and positive extinction. Taken together, these observations are consistent with a long and complex history of HCV 3a infection in Pakistan.

BACKGROUND: Next-generation sequencing (NGS) allows for sampling numerous viral variants from infected patients. This provides a novel opportunity to represent and study the mutational landscape of Hepatitis C Virus (HCV) within a single host. RESULTS: Intra-host variants of the HCV E1/E2 region were extensively sampled from 58 chronically infected patients. After NGS error correction, the average number of reads and variants obtained from each sample were 3202 and 464, respectively. The distance between each pair of variants was calculated and networks were created for each patient, where each node is a variant and two nodes are connected by a link if the nucleotide distance between them is 1. The work focused on large components having > 5% of all reads, which in average account for 93.7% of all reads found in a patient. CONCLUSIONS: Most intra-host variants are organized into distinct single-mutation components that are: well separated from each other, represent genetic distances between viral variants, robust to sampling, reproducible and likely seeded during transmission events. Facilitated by NGS, large components offer a novel evolutionary framework for genetic analysis of intra-host viral populations and understanding transmission, immune escape and drug resistance.

BACKGROUND: Four patients were hospitalized July 2011 with Pseudomonas aeruginosa bloodstream infection (BSI), 2 of whom also had Klebsiella pneumoniae BSI. All 4 patients had an indwelling port and received infusion services at the same outpatient oncology center. METHODS: Cases were defined by blood or port cultures positive for K pneumoniae or P aeruginosa among patients receiving infusion services at the oncology clinic during July 5-20, 2011. Pulsed-field gel electrophoresis (PFGE) was performed on available isolates. Interviews with staff and onsite investigations identified lapses of infection control practices. Owing to concerns over long-standing deficits, living patients who had been seen at the clinic between January 2008 and July 2011 were notified for viral blood-borne pathogen (BBP) testing; genetic relatedness was determined by molecular testing. RESULTS: Fourteen cases (17%) were identified among 84 active clinic patients, 12 of which involved symptoms of a BSI. One other patient had a respiratory culture positive for P aeruginosa but died before blood cultures were obtained. Available isolates were indistinguishable by PFGE. Multiple injection safety lapses were identified, including overt syringe reuse among patients and reuse of syringes to access shared medications. Available BBP results did not demonstrate iatrogenic viral infection in 331 of 623 notified patients (53%). CONCLUSIONS: Improper preparation and handling of injectable medications likely caused the outbreak. Increased infection control oversight of oncology clinics is critical to prevent similar outbreaks.

Hepatitis C virus (HCV) infection presents an important, but underappreciated public health problem in Africa. In Cote d'Ivoire, very little is known about the molecular dynamics of HCV infection. Plasma samples (n = 608) from pregnant women collected in 1995 from Cote d'Ivoire were analyzed in this study. Only 18 specimens ( approximately 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 Cote 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 Cote d'Ivoire, similar to the rest of West Africa, and suggest a unique HCV epidemic history in the country.

Using HCV and IFN-resistance as a proof of concept, we have devised a new methodology for calculating the effect of a drug over a viral population and the resistance of its individual intra-host variants. By means of next-generation sequencing, HCV variants were obtained from sera collected at 9 time-points from 16 patients during the first 48 hours after injection of IFN-a. IFN-resistance coefficients were calculated for individual variants using changes in their relative frequencies, and for the entire intra-host viral population using changes in viral titer during the initial 48 hours. Population-wide resistance and presence of IFN-resistant variants were highly associated with pegIFN-a2a/RBV treatment outcome at week 12 (p = 3.78x10-5 and 0.0114, respectively). This new method allows an accurate measurement of resistance based solely on changes in viral titer or the relative frequency of intra-host viral variants during a short observation time.

Clinical infection by hepatitis A virus (HAV) is generally self-limited but in some cases can progress to liver failure. Here, an HAV outbreak investigation among children with acute liver failure in a highly endemic country is presented. In addition, a sensitive method for HAV whole genome amplification and sequencing suitable for analysis of clinical samples is described. In this setting, two fatal cases attributed to acute liver failure and two asymptomatic cases living in the same household were identified. In a second household, one HAV case was observed with jaundice which resolved spontaneously. Partial molecular characterization showed that both households were infected by HAV subtype IA; however, the infecting strains in the two households were different. The HAV outbreak strains recovered from all cases grouped together within cluster IA1, which contains closely related HAV strains from the United States commonly associated with international travelers. Full-genome HAV sequences obtained from the household with the acute liver failure cases were related (genetic distances ranging from 0.01% to 0.04%), indicating a common-source infection. Interestingly, the strain recovered from the asymptomatic household contact was nearly identical to the strain causing acute liver failure. The whole genome sequence from the case in the second household was distinctly different from the strains associated with acute liver failure. Thus, infection with almost identical HAV strains resulted in drastically different clinical outcomes.

The genetic characterization of hepatitis A virus (HAV) strains is commonly accomplished by sequencing subgenomic regions, such as the VP1/P2B junction. HAV genome is not extensively variable, thus presenting opportunity for sharing sequences of subgenomic regions among genetically unrelated isolates. The degree of misrepresentation of phylogenetic relationships by subgenomic regions is especially important for tracking transmissions. Here, we analyzed whole-genome (WG) sequences of 101 HAV strains identified from 4 major multi-state, food-borne outbreaks of hepatitis A in the Unites States and from 14 non-outbreak-related HAV strains that shared identical VP1/P2B sequences with the outbreak strains. Although HAV strains with an identical VP1/P2B sequence were specific to each outbreak, WG were different, with genetic diversity reaching 0.31% (mean 0.09%). Evaluation of different subgenomic regions did not identify any other section of the HAV genome that could accurately represent phylogenetic relationships observed using WG sequences. The identification of 2-3 dominant HAV strains in 3 out of 4 outbreaks indicates contamination of the implicated food items with a heterogeneous HAV population. However, analysis of intra-host HAV variants from eight patients involved in one outbreak showed that only a single sequence variant established infection in each patient. Four non-outbreak strains were found closely related to strains from 2 outbreaks, whereas ten were genetically different from the outbreak strains. Thus, accurate tracking of HAV strains can be accomplished using HAV WG sequences, while short subgenomic regions are useful for identification of transmissions only among cases with known epidemiological association.

Infection with hepatitis A virus (HAV) is the commonest viral cause of liver disease and presents an important public health problem worldwide. Several unique HAV properties and molecular mechanisms of its interaction with host were recently discovered and should aid in clarifying the pathogenesis of hepatitis A. Genetic characterization of HAV strains have resulted in the identification of different genotypes and subtypes, which exhibit a characteristic worldwide distribution. Shifts in HAV endemicity occurring in different parts of the world, introduction of genetically diverse strains from geographically distant regions, genotype displacement observed in some countries and population expansion detected in the last decades of the 20th century using phylogenetic analysis are important factors contributing to the complex dynamics of HAV infections worldwide. Strong selection pressures, some of which, like usage of deoptimized codons, are unique to HAV, limit genetic variability of the virus. Analysis of subgenomic regions has been proven useful for outbreak investigations. However, sharing short sequences among epidemiologically unrelated strains indicates that specific identification of HAV strains for molecular surveillance can be achieved only using whole-genome sequences. Here, we present up-to-date information on the HAV molecular epidemiology and evolution, and highlight the most relevant features of the HAV-host interactions.