Development of simple, rapid, and sensitive methods for detection of hepatitis C virus RNA from whole blood using reverse transcription loop-mediated isothermal amplification.

Hepatitis C virus (HCV) infection is an underdiagnosed global health problem. Diagnosis of current HCV infections typically requires testing for HCV RNA using high-complexity laboratory tests. Methods for the detection of HCV RNA that are simple, inexpensive, rapid, and compatible with use outside of a laboratory setting are very important in order to improve access to hepatitis C diagnostic testing and facilitate accelerated linkage to care. We developed and evaluated three simple workflows for extracting HCV RNA from small volumes of whole blood for use in a sensitive, pan-genotypic RT-LAMP assay. The water workflow uses osmotic stress to release HCV RNA and has a limit of detection of 4.3 log10(IU/mL) (95% CI 4.0-4.9). The heat workflow uses a heating step to release HCV RNA and has a limit of detection of 4.2 log10(IU/mL) (95% CI 3.8-5.1). The bead workflow, which uses chemical lysis of the sample and a streamlined paramagnetic solid phase reversible immobilization bead procedure for nucleic acid purification, has a limit of detection of 2.8 log10(IU/mL) (95% CI 2.5-3.4). When used to test whole blood spiked with HCV RNA-positive plasma samples in which most HCV levels were below 5.0 log10(IU/mL), the water, heat, and bead workflows detected HCV RNA in 69%, 75%, and 94% of samples, respectively. These workflows are compatible with visual lateral flow dipsticks, and each takes less than 60 min from sample to result. Each workflow can be performed with minimal and inexpensive equipment. With further procedural simplifications, these workflows may form the basis of assays for the point-of-care diagnosis of HCV infections.

Long-term virological and adherence outcomes to antiviral treatment in a 4-year cohort chronic HBV study.

BACKGROUND: Chronic hepatitis B (CHB) treatment adherence has been poorly studied worldwide. We evaluated long-term virological and adherence outcomes to antiviral treatment in CHB patients. METHODS: A prospective 183 Brazilian CHB patient cohort treated with monotherapy or combination adefovir dipivoxil, entecavir, lamivudine and/or tenofovir disoproxil fumarate was studied in a reference tertiary centre. Treatment adherence was evaluated by a validated questionnaire named 'Assessment of Adherence to Antiviral Therapy Questionnaire' (CEAT-HBV) within three yearly periods (2010/2011, 2013/2014 and 2014/2015). RESULTS: CEAT-HBV identified 43% (79/183) patients with non-adherence to antiviral treatment and among them, 67% (53/79) were viral load positive. The main causes associated with non-response to antiviral treatment were drug resistance variants followed by non-adherence, insufficient treatment duration and other causes. Single-dose pharmacokinetics demonstrated 35% (23/65) antiviral non-adherence. 2 years after the first assessment, the CEAT-HBV indicated that 71% (101/143) of subjects adhered to treatment (per-protocol population). However, 21% (40/183) of the patients could not be evaluated and were excluded. The main reasons for exclusion were death (20/183), 11 out 20 deaths due to hepatocellular carcinoma. HBV booklet was used for medical education. The third CEAT-HBV assessment (2014/2015) showed that 83% (112/135) patients were compliant with treatment adherence (per-protocol population). Long-term evaluation showed that adherence rate based on CEAT-HBV continue to increase after 4-years (P<0.001). CONCLUSIONS: The results highlight the importance of CHB therapy adherence assessment monitoring. Long-term adherence outcomes were dynamic and it is possible to increase the migration rate to adherence/HBV-DNA-negative group.

Molecular epidemiology of hepatitis B virus infection in Tanzania.

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.

Characteristics of US-Born Versus Foreign-Born Americans of African Descent With Chronic Hepatitis B.

Hepatitis B virus (HBV) infection is more common in African Americans than in white Americans. We compared the epidemiologic, clinical, and virological characteristics of US-born African Americans (USAAs) to those of foreign-born African Americans (FBAAs) with chronic hepatitis B. The adult cohort study of the Hepatitis B Research Network enrolls patients with HBV infection from 21 clinical sites in the United States and Canada. A total of 237 (15%) of the adult participants with chronic HBV infection that were enrolled from January 20, 2011, to October 2, 2013, were of African descent, including 57 USAAs and 180 FBAAs (76%). Compared with FBAAs, USAAs were older and more likely to have acquired HBV through sexual exposure, to be HBeAg-positive, to have higher HBV DNA levels, and to be infected with HBV genotype A2. FBAAs from West Africa were more likely to have elevated serum alanine aminotransferase (72% vs. 50%; P < 0.01) and higher HBV DNA levels (median, 3.2 log10 IU/mL vs. 2.8 log10 IU/mL; P = 0.03) compared with East African FBAAs. The predominant HBV genotype among West African FBAAs was E (67%), whereas genotypes A (78%) and D (16%) were common in East African FBAAs. Significant differences were found between USAAs and FBAAs, highlighting the need for tailored strategies for prevention and management of chronic HBV infection for African Americans.

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.

Application of mass spectrometry to molecular diagnostics of viral infections.

Mass spectrometry (MS) has found numerous applications in life sciences. It has high accuracy, sensitivity and wide dynamic range in addition to medium- to high-throughput capabilities. These features make MS a superior platform for analysis of various biomolecules including proteins, lipids, nucleic acids and carbohydrates. Until recently, MS was applied for protein detection and characterization. During the last decade, however, MS has successfully been used for molecular diagnostics of microbial and viral infections with the most notable applications being identification of pathogens, genomic sequencing, mutation detection, DNA methylation analysis, tracking of transmissions, and characterization of genetic heterogeneity. These new developments vastly expand the MS application from experimental research to public health and clinical fields. Matching of molecular techniques with specific requirements of the major MS platforms has produced powerful technologies for molecular diagnostics, which will further benefit from coupling with computational tools for extracting clinical information from MS-derived data.

Detection of hepatitis C virus transmission by use of DNA mass spectrometry.

The molecular detection of transmission of rapidly mutating pathogens such as hepatitis C virus (HCV) is commonly achieved by assessing the genetic relatedness of strains among infected patients. We describe the development of a novel mass spectrometry (MS)-based approach to identify HCV transmission. MS was used to detect products of base-specific cleavage of RNA molecules obtained from HCV polymerase chain reaction fragments. The MS-peak profiles were found to reflect variation in the HCV genomic sequence and the intrahost composition of the HCV population. Serum specimens originating from 60 case patients from 14 epidemiologically confirmed outbreaks and 25 unrelated controls were tested. Neighbor-joining trees constructed using MS-peak profile-based Hamming distances showed 100% accuracy, and linkage networks constructed using a threshold established from the Hamming distances between epidemiologically unrelated cases showed 100% sensitivity and 99.93% specificity in transmission detection. This MS-based approach is rapid, robust, reproducible, cost-effective, and applicable to investigating transmissions of other pathogens.

Epidemic history of hepatitis C virus infection in two remote communities in Nigeria, West Africa.

We investigated the molecular epidemiology and population dynamics of HCV infection among indigenes of two semi-isolated communities in North-Central Nigeria. Despite remoteness and isolation, ~15% of the population had serological or molecular markers of hepatitis C virus (HCV) infection. Phylogenetic analysis of the NS5b sequences obtained from 60 HCV-infected residents showed that HCV variants belonged to genotype 1 (n=51; 85%) and genotype 2 (n=9; 15%). All sequences were unique and intermixed in the phylogenetic tree with HCV sequences from people infected from other West African countries. The high-throughput 454 pyrosequencing of the HCV hypervariable region 1 and an empirical threshold error correction algorithm were used to evaluate intra-host heterogeneity of HCV strains of genotype 1 (n=43) and genotype 2 (n=6) from residents of the communities. Analysis revealed a rare detectable intermixing of HCV intra-host variants among residents. Identification of genetically close HCV variants among all known groups of relatives suggests a common intra-familial HCV transmission in the communities. Applying Bayesian coalescent analysis to the NS5b sequences, the most recent common ancestors for genotype 1 and 2 variants were estimated to have existed 675 and 286 years ago, respectively. Bayesian skyline plots suggest that HCV lineages of both genotypes identified in the Nigerian communities experienced epidemic growth for 200-300 years until the mid-20th century. The data suggest a massive introduction of numerous HCV variants to the communities during the 20th century in the background of a dynamic evolutionary history of the hepatitis C epidemic in Nigeria over the past three centuries.

Application of mass spectrometry to molecular surveillance of hepatitis B and C viral infections.

Detection of genotypes and drug resistance mutations are important molecular tools assisting in clinical management of patients with chronic hepatitis B and C. Together with methods for assessment of genetic heterogeneity and relatedness of viral strains, they form the foundation of molecular surveillance. Currently, all these methods are based mainly on DNA sequencing followed by phylogenetic analysis. Mass spectrometry (MS) emerged recently as a rapid, cost-effective, reproducible and accurate alternative approach. MS-based molecular assays are highly amenable to automation and provide a suitable platform for routine application to the surveillance of HBV and HCV infections.

Evaluation of intra-host variants of the entire hepatitis B virus genome.

Genetic analysis of hepatitis B virus (HBV) frequently involves study of intra-host variants, identification of which is commonly achieved using short regions of the HBV genome. However, the use of short sequences significantly limits evaluation of genetic relatedness among HBV strains. Although analysis of HBV complete genomes using genetic cloning has been developed, its application is highly labor intensive and practiced only infrequently. We describe here a novel approach to whole genome (WG) HBV quasispecies analysis based on end-point, limiting-dilution real-time PCR (EPLD-PCR) for amplification of single HBV genome variants, and their subsequent sequencing. EPLD-PCR was used to analyze WG quasispecies from serum samples of patients (n = 38) infected with HBV genotypes A, B, C, D, E and G. Phylogenetic analysis of the EPLD-isolated HBV-WG quasispecies showed the presence of mixed genotypes, recombinant variants and sub-populations of the virus. A critical observation was that HBV-WG consensus sequences obtained by direct sequencing of PCR fragments without EPLD are genetically close, but not always identical to the major HBV variants in the intra-host population, thus indicating that consensus sequences should be judiciously used in genetic analysis. Sequence-based studies of HBV WG quasispecies should afford a more accurate assessment of HBV evolution in various clinical and epidemiological settings.

Epidemic history and evolutionary dynamics of hepatitis B virus infection in two remote communities in rural Nigeria.

BACKGROUND: In Nigeria, hepatitis B virus (HBV) infection has reached hyperendemic levels and its nature and origin have been described as a puzzle. In this study, we investigated the molecular epidemiology and epidemic history of HBV infection in two semi-isolated rural communities in North/Central Nigeria. It was expected that only a few, if any, HBV strains could have been introduced and effectively transmitted among these residents, reflecting limited contacts of these communities with the general population in the country. METHODS AND FINDINGS: Despite remoteness and isolation, approximately 11% of the entire population in these communities was HBV-DNA seropositive. Analyses of the S-gene sequences obtained from 55 HBV-seropositive individuals showed the circulation of 37 distinct HBV variants. These HBV isolates belong predominantly to genotype E (HBV/E) (n=53, 96.4%), with only 2 classified as sub-genotype A3 (HBV/A3). Phylogenetic analysis showed extensive intermixing between HBV/E variants identified in these communities and different countries in Africa. Quasispecies analysis of 22 HBV/E strains using end-point limiting-dilution real-time PCR, sequencing and median joining networks showed extensive intra-host heterogeneity and inter-host variant sharing. To investigate events that resulted in such remarkable HBV/E diversity, HBV full-size genome sequences were obtained from 47 HBV/E infected persons and P gene was subjected to Bayesian coalescent analysis. The time to the most recent common ancestor (tMRCA) for these HBV/E variants was estimated to be year 1952 (95% highest posterior density (95% HPD): 1927-1970). Using additional HBV/E sequences from other African countries, the tMRCA was estimated to be year 1948 (95% HPD: 1924-1966), indicating that HBV/E in these remote communities has a similar time of origin with multiple HBV/E variants broadly circulating in West/Central Africa. Phylogenetic analysis and statistical neutrality tests suggested rapid HBV/E population expansion. Additionally, skyline plot analysis showed an increase in the size of the HBV/E-infected population over the last approximately 30-40 years. CONCLUSIONS: Our data suggest a massive introduction and relatively recent HBV/E expansion in the human population in Africa. Collectively, these data show a significant shift in the HBV/E epidemic dynamics in Africa over the last century.

End-point limiting-dilution real-time PCR assay for evaluation of hepatitis C virus quasispecies in serum: performance under optimal and suboptimal conditions.

An approach for determination of hepatitis C virus (HCV) quasispecies by end-point limiting-dilution real-time PCR (EPLD-PCR) is described. It involves isolation of individual coexisting sequence variants of the hypervariable region 1 (HVR1) of the HCV genome from serum specimens using a limiting-dilution protocol. EPLD-PCR applied to an HCV outbreak study provided insights into the epidemiological relationships between incident and chronic cases. When applied to samples from a longitudinal study of infected patients, HVR1 sequences from each sampling time-point were observed to group as distinct phylogenetic clusters. Melting peak analysis conducted on EPLD-PCR products generated from these patients could be used for evaluation of HVR1 sequence heterogeneity without recourse to clonal sequencing. Further, to better understand the mechanism of single-molecule PCR, experiments were conducted under optimal and suboptimal annealing temperatures. Under all temperature conditions tested, HVR1 variants from the major phylogenetic clusters of quasispecies could be amplified, revealing that successful HVR1 quasispecies analysis is not contingent to dilution of starting cDNA preparations to a single-molecule state. It was found that EPLD-PCR conducted at suboptimal annealing temperatures generated distributions of unique-sequence variants slightly different from the distribution obtained by PCR conducted at the optimal temperature. Hence, EPLD-PCR conditions can be manipulated to access different subpopulations of HCV HVR1 quasispecies, thus, improving the range of the quasispecies detection. Although EPLD-PCR conducted at different conditions detect slightly different quasispecies populations, as was shown in this study, the resulted samples of quasispecies are completely suitable for molecular epidemiological investigation in different clinical and epidemiological settings.

Hepatitis C virus infections from a contaminated radiopharmaceutical used in myocardial perfusion studies.

CONTEXT: Nuclear pharmacies prepare radiopharmaceutical products for use in common diagnostic procedures, including myocardial perfusion studies. Hepatitis C virus (HCV) transmission has not been reported previously in the setting of nuclear imaging studies. OBJECTIVE: To investigate an outbreak of acute HCV infection identified among patients who underwent myocardial perfusion studies on October 15, 2004, using an injected radiopharmaceutical. DESIGN, SETTING, AND PATIENTS: Outbreak investigation including molecular epidemiology and pharmacy site investigation at outpatient cardiology clinics and a nuclear pharmacy in Maryland. Ninety patients who received injections drawn from select radiopharmaceutical vials prepared on October 14-15, 2004, at a single nuclear pharmacy were offered testing for bloodborne pathogens. Pharmacy procedures were reviewed and HCV quasi species analysis was performed. MAIN OUTCOME MEASURES: Hepatitis C virus infection and quasispecies sequence similarity. RESULTS: Sixteen patients with acute HCV infection were identified from 3 separate clinics. All patients received radiopharmaceutical injections drawn from a single pharmacy vial (vial 1). None of the 59 tested patients who received doses from 6 other vials had acute HCV infection. Blood from a potential source patient with HCV and human immunodeficiency virus (HIV) infection was processed for a radiolabeled white blood cell study in the pharmacy 12 hours before vial 1 was prepared. The HCV quasispecies sequences from this potential source patient were nearly identical to those from cases (97.8%-98.5% similarity). No acute HIV infections were identified. Pharmacy practices that could have led to blood cross-contamination included reuse of needles and syringes during dilutions and use of common flow hoods for some steps in the preparation of sterile and blood-derived products. CONCLUSIONS: Sixteen persons acquired HCV infection from a blood-contaminated radiopharmaceutical. The source and practices that could have facilitated breaks in aseptic technique were identified at the pharmacy. Nuclear pharmacies that handle biological products should follow appropriate aseptic technique to prevent contamination of sterile radiopharmaceuticals.