John R. Ticehurst

Human Immunodeficiency Virus Seroconversion and Evolution of the Hepatitis C Virus Quasispecies

ABSTRACT When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (dN ) to synonymous (dS ) substitutions, because dN may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower dN/dS after HIV seroconversion in 7 of 10 subjects and lowerdN/dS in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.

Population Seroprevalence of Hepatitis E Virus Antibodies in Rural Bangladesh

Hepatitis E virus (HEV) causes a substantial burden of sporadic and epidemic disease worldwide. HEV infections result in serious morbidity and mortality, especially among pregnant women, and have significant economic costs. Few population-based studies have characterized the epidemiology of HEV. A rural Bangladeshi population was studied to determine the age- and gender- specific population seroprevalence of antibodies to HEV. Of 1,134 specimens tested from a representative, random population sample, 255 (22.5%) were anti-HEV IgG seropositive. Seroprevalence was lower among women (19.7%) than among men (25.8%). We found anti-HBc (hepatitis B core) in 380 of 1080(35.2%) tested, anti-HCV (hepatitis C) in 14 of 917(1.5%) tested, and anti-HAV (hepatitis A) in 116 of 124(93.5%) tested individuals. Our data suggest that viral hepatitis, especially HEV, remains an under-recognized and significant public health problem in rural Bangladeshi populations, warranting further attention.

Factors Affecting Serum Concentrations of Hepatitis C Virus (HCV) RNA in HCV Genotype 1-Infected Patients with Chronic Hepatitis

ABSTRACT The serum concentration of hepatitis C virus (HCV) RNA is usually stable (4 to 8 log10 IU/ml) in untreated patients with chronic hepatitis C. While this baseline HCV RNA concentration ([HCV RNA]BL) is predictive of a sustained virologic response to treatment, its determinants are only partially identified. We therefore analyzed the baseline characteristics of 2,472 HCV genotype 1-infected patients to identify correlations with gender, age, race, weight, body mass index (BMI), HCV acquisition mode, HCV subtype, alanine aminotransferase concentration, or histopathologic changes in the liver. After separation of the data according to four [HCV RNA]BL groups (≤5.0, >5.0 to 5.6, >5.6 to 5.9, and >5.9 log10 IU/ml), we determined that increasing [HCV RNA]BL correlated (P < 0.05) with increasing proportions of patients who were male, >40 years of age, or heavier (a weight of >85 kg or a BMI of >27 kg/m2). Histologic activity index (HAI) data were available for 1,304 of these patients: increasing [HCV RNA]BL correlated with higher fibrosis and necrosis-inflammation scores. As a continuous variable, [HCV RNA]BL correlated with age, gender, weight (continuous or ≤85 versus >85 kg), BMI (continuous or ≤27 versus >27 kg/m2), subtype, fibrosis score, and necrosis-inflammation score; however, multiple-regression analysis yielded P values of <0.1 only for age, gender, BMI (≤27 versus >27 kg/m2), and fibrosis score. While our findings are suggestive of a role for these factors in maintenance of the pretreatment state of HCV infection, the multiple-regression model accounted for only ≤4.6% of the [HCV RNA]BL differences between individuals (R2 = 0.046 for 1,304 patients with HAI scores; 0.043 for all 2,472 patients).

Accurate Representation of the Hepatitis C Virus Quasispecies in 5.2-Kilobase Amplicons

ABSTRACT Hepatitis C virus (HCV) exists as a swarm of genetically distinct but related variants, or a quasispecies, whose complexity and sequence evolution are critical to studies of viral pathogenesis. Because most studies of the HCV quasispecies have focused on a relatively small genomic segment, the first hypervariable region of the E2 gene, it is possible that viral complexity is occasionally underestimated (due to primer mismatch) and that sequence evolution is misperceived due to unrecognized covariation. This report describes a sensitive and reproducible method to amplify most of the HCV genome as a single 5.2-kb amplicon by using primers directed at relatively conserved genomic segments. Using 52 specimens obtained during acute infection over a range of viral RNA concentrations, the overall rate of successful amplification was 94% and varied in a concentration-dependent manner, with successful amplification in 26 of 26 (100%) specimens at greater than 105 IU/ml, 15 of 16 (94%) at 104 to 105 IU/ml, 6 of 7 (86%) at 103 to 104 IU/ml, and 2 of 3 (67%) at less than 103 IU/ml. Quasispecies complexity, determined by using this novel long-amplicon method followed by heteroduplex mobility assay combined with single-stranded conformational polymorphism (HDA+SSCP) analysis, was very high, even during acute HCV infection, when 10 to 21 (median, 16) different HDA+SSCP patterns were detected among 33 cDNA clones examined. Replicate analyses indicate that this diversity is not dominated by random errors generated during amplification. Therefore, the HCV quasispecies is highly complex even during acute infection and is accurately represented in amplicons representing more than half of the viral genome.

Hepatitis E Seroprevalence and Seroconversion among US Military Service Members Deployed to Afghanistan

BACKGROUND Hepatitis E virus (HEV) has been recognized as a threat to military forces since its discovery. Although HEV seroprevalence in Afghanistan is not known, HEV infection is thought to be highly endemic in that country. This study determined the incidence of HEV seroconversion among United States (US) service members who were deployed to Afghanistan, as well as the prevalence of antibodies to HEV prior to the deployment. METHODS A random sample of 1500 subjects was selected from the cohort of service members who were deployed to Afghanistan between 2002 and 2006. Predeployment and postdeployment serum samples from these subjects were tested by enzyme immunoassay for total antibodies to HEV. Results.  The seroprevalence of antibodies to HEV in US service members prior to deployment was 1.1%. The seroconversion rate among service members deployed to Afghanistan was 0.13%. CONCLUSIONS Although subpopulations may be at higher risk for HEV exposure during deployment, the risk among US service members deployed to Afghanistan in this study was low. Previously implemented and current preventive measures in theater appear to have been adequate. With future deployments to new areas or changes in military operations in areas of risk, continued surveillance for HEV infection in the military will be warranted.

An exploratory case control study of risk factors for hepatitis E in rural Bangladesh.

Hepatitis E virus (HEV) is the major cause of epidemic and sporadic hepatitis globally. Outbreaks are associated with fecal contamination of drinking water, yet the environmental reservoir of HEV between epidemics remains unclear. In contrast to neighboring countries, where epidemics and sporadic disease co-occur, HEV-endemic communities in rural Bangladesh seldom report outbreaks; sporadic hepatitis E is reported from urban and rural areas of the country. Besides typical enteric risk factors, other routes for HEV infection and disease are unclear. We conducted monthly household surveillance of a southern Bangladeshi community of 23,500 people to find incident cases of acute hepatitis E over a 22 month period. An algorithm was used to capture 279 candidate cases, of which 46 were confirmed acute HEV infections. An exploratory case-control study was conducted to identify putative risk factors for disease. Nearly 70% of cases were over 15 years old. Female gender seemed protective (OR:0.34) against hepatitis E in this conservative setting, as was the use of sanitary latrines (OR:0.28). Socioeconomic status or animal exposures were not significant predictors of disease, although outdoor employment and recent urban travel were. Unexpectedly, recent contact with a “jaundiced” patient and a history of injection exposure in the 3 months prior to disease (OR:15.50) were significant. Susceptible individuals from “endemic” communities share similar enteric exposure risks to those commonly associated with tourists from non-endemic countries. This study also raises the novel possibility of parenteral and person-to-person transmission of HEV in non-epidemic, sporadic disease settings.

Hepatitis C Virus (HCV) Core Antigen Assay To Detect Ongoing HCV Infection in Thai Injection Drug Users

ABSTRACT We evaluated a quantitative enzyme immunoassay (trak-C) for hepatitis C virus core antigen (HCV core Ag) by testing serum specimens from 820 injection drug users in Thailand with anti-HCV antibodies. The HCV genotypes in this population include genotypes 3 and 6, which have not been extensively tested with this assay. Among these specimens, 629 (76.7%) yielded positive results, with HCV core Ag concentrations predominantly spanning (35.7%) or above (58.2%) the measurable range of 1.5 to 100 pg/ml. To assess reproducibility, we retested 30 specimens representing six core Ag ranges; the mean coefficient of variation for each range was ≤9.7% (highest for 1.5 to 25 pg/ml). We also tested 204 specimens of the 820-specimen set for HCV RNA: while 146 (71.6%) were core Ag positive, 168 (82.4%) had detectable HCV RNA, of which 96% were typeable as genotype 3 (39%), 1 (31%), or 6 (26%) by nested reverse transcription-PCR. Among RNA-positive specimens, 86.9% had core Ag; 94% of the RNA negatives were core Ag negative. While there was no apparent bias for detecting core Ag representing the tested genotypes, median quantified results were higher for types 1a and 6 than for genotype 3 (P = 0.01); similarly, the median core Ag concentration was higher in HCV-human immunodeficiency virus-coinfected subjects than in HCV-monoinfected subjects. Our results demonstrated a good correlation between core Ag and HCV RNA in this population with high frequencies of genotypes 3 and 6. Because most core Ag concentrations were greater than those in the measurable range, we recommend a 10-fold dilution of the specimen before quantification. Reproducibility, low technical requirements, and high throughput should make this assay useful for clinical or research monitoring of HCV levels during active infection.

Two Generations of “Gold Standards”: The Impact of a Decade in Hepatitis E Virus Testing Innovation on Population Seroprevalence

Hepatitis E virus (HEV) is a global pathogen responsible for approximately 20 million infections every year in developing countries, yet remains under-recognized. In this population-based cohort study, 1,025 randomly selected participants were enrolled from Matlab, Bangladesh (2004–2005). All participants were tested for HEV antibodies and total immunoglobulin (Ig), using an in-house enzyme immunoassay developed by Walter Reed Army Institute of Research (WRAIR). In 2014, we retested the banked sera of 1,009 of those participants using the Wantai anti-HEV IgG enzyme-linked immunosorbent assay (ELISA). The WRAIR assay estimated the overall population seroprevalence as 26.6% (95% confidence interval [CI]: 24.0, 29.5), whereas the Wantai assay produced significantly higher estimated seroprevalence, 46.7% (95% CI: 43.5–49.8) (P < 0.001). However, the two tests give nearly identical findings in those 5 years and under (N = 94) with a 98% agreement between the tests. Retesting populations with modern assays is necessary to establish better population-level estimates of disease burden.

Hepatitis E Vaccine to Prevent Morbidity and Mortality During Epidemics

Recurrent, large, waterborne epidemics of hepatitis E virus (HEV) occur regularly after monsoon rains contaminate water supplies in Asia or during humanitarian crises in Africa. These epidemics commonly affect thousands of persons, and it has a high mortality in pregnant women who become infected. Although a subunit HEV vaccine has been developed by Chinese investigators and was found to be highly effective and safe in a large clinical trial, this vaccine is only available in China. Until it is prequalified by the World Health Organization, the vaccine may not be available for use outside of China in low-income countries that lack national vaccine regulatory agencies. In this manuscript, we explore possible strategies for providing access to this potentially important vaccine for international use in responding to epidemics of HEV in low-resource countries.

Hepatitis E Virus Infection Among Solid Organ Transplant Recipients at a North American Transplant Center

Background. Autochthonous hepatitis E virus (HEV) infection has been reported in over 200 solid organ transplant (SOT) recipients since 2006, yet little is known about the burden of HEV among SOT recipients in North America. We performed a retrospective, cross-sectional study to investigate the prevalence and risk factors associated with HEV infection among SOT recipients at our institution. Methods. Children and adults (n = 311) who received allografts between 1988 and 2012 at the Johns Hopkins Hospital were assessed for evidence of HEV infection by testing posttransplantation serum samples for HEV antibody by enzyme immunoassay and HEV RNA by reverse transcription quantitative polymerase chain reaction. Individuals with evidence of posttransplant HEV infection (presence of anti-HEV immunoglobulin [Ig]M antibody, anti-HEV IgG seroconversion, or HEV RNA) were compared with individuals without evidence of infection and assessed for risk factors associated with infection. Results. Twelve individuals (4%) developed posttransplant HEV infection. Posttransplant HEV infection was associated with an increased risk for graft rejection (odds ratio, 14.2; P = .03). No individuals developed chronic infection. Conclusions. Solid organ transplant recipients in the United States are at risk for posttransplant HEV infection. Further studies are needed to characterize environmental risk factors and the risk of HEV infection after SOT in North America.