Annemiek A. van der Eijk

Diagnostic performance of selected commercial HEV IgM and IgG ELISAs for immunocompromised and immunocompetent patients

BACKGROUND Hepatitis E virus (HEV) genotype 3 is recognised as an emerging pathogen in industrialised countries. The currently commercially available HEV-specific enzyme linked immunosorbent assays (ELISAs) are primarily designed for the detection of antibodies against genotypes 1 (Burma) and 2 (Mexico) and may not sensitively detect HEV genotypes 3 or 4. OBJECTIVES This study aimed to evaluate the analytical and clinical performances of eight commercially available HEV serum antibody immunoglobulin M (IgM)- and immunoglobulin G (IgG)-specific ELISAs for genotype 1 and 3 HEV infections in a clinical setting and to study the antibody responses against HEV of immunocompromised versus immunocompetent patient groups. STUDY DESIGN Analytical performance and diagnostic sensitivity and specificity were assessed using well-defined reference samples and samples from patients with polymerase chain reaction (PCR)-confirmed HEV infection (n=88) and a specificity panel (n=98). RESULTS Limiting dilutions indicated that the highest analytical sensitivity in head-to-head comparison was measured for the Mikrogen_new IgG assay. Taking the serum working dilutions of each assay into account, the Wantai IgG assay was the most sensitive assay. Receiver operator curve (ROC) analysis showed area under the curve (AUC) values of 0.943, 0.964, 0.969, 0.971, 0.974 and 0.994 for the DSI, Mikrogen_old, MP Diagnostics, Mikrogen_new, Wantai and DiaPro anti-HEV IgM assays, respectively. The highest specificity of currently available assays was found for the IgM Wantai assay (>99%). If anti-HEV IgM and IgG results from each supplier were combined, DSI and Wantai assays were able to detect the highest number of (passed) HEV infections. CONCLUSIONS Our study showed that current commercial HEV ELISAs could be used to diagnose HEV genotype 3 infection adequately in a clinical setting.

Guillain-Barré syndrome associated with preceding hepatitis E virus infection

Objective: The aim of the study was to determine whether Guillain-Barré syndrome (GBS) is associated with preceding hepatitis E virus infection. Methods: The frequency of hepatitis E virus (HEV) infections was determined by anti-HEV serology in a cohort of 201 patients with GBS and 201 healthy controls with a similar distribution in age, sex, and year of sampling. Blood samples from patients with GBS were obtained in the acute phase before treatment. In a subgroup of patients with GBS, blood, stool, and CSF samples were tested for HEV RNA. Results: An increased ratio of anti-HEV immunoglobulin (Ig) M antibodies was found in 10 patients with GBS (5.0%) compared with 1 healthy control (0.5%, odds ratio 10.5, 95% confidence interval 1.3–82.6, p = 0.026). HEV RNA was detected in blood from 3 of these patients and additionally in feces from 1 patient. Seventy percent of anti-HEV IgM-positive patients had mildly increased liver function tests. All CSF samples tested negative for HEV RNA. The presence of anti-HEV IgM in patients with GBS was not related to age, sex, disease severity, or clinical outcome after 6 months. Conclusions: In the Netherlands, 5% of patients with GBS have an associated acute HEV infection. Further research is required to determine whether HEV infections also precede GBS in other geographical areas.

Hepatitis E virus infection among solid organ transplant recipients, the Netherlands

We screened 1,200 living heart, lung, liver, and kidney transplant recipients for hepatitis E virus infection by reverse transcription PCR. In 12 (1%) patients, hepatitis E virus infection was identified; in 11 patients, chronic infection developed. This immunocompromised population is at risk for hepatitis E virus infection.

Hepatitis E virus: an underestimated opportunistic pathogen in recipients of allogeneic hematopoietic stem cell transplantation

Hepatitis E virus (HEV) is increasingly acknowledged as a cause of hepatitis in healthy individuals as well as immunocompromised patients. Little is known of HEV infection in recipients of allogeneic hematopoietic stem cell transplantation (alloHSCT). Therefore, we set out to study the incidence and sequelae of HEV as a cause of hepatitis in a recent cohort of 328 alloHSCT recipients. HEV RNA was tested in episodes of liver enzyme abnormalities. In addition, HEV RNA and HEV serology were assessed pre- and post-alloHSCT. We found 8 cases (2.4%) of HEV infection, of which 5 had developed chronic HEV infection. Seroprevalence pre-alloHSCT was 13%. Four patients died with HEV viremia, with signs of ongoing hepatitis, having a median time of infection of 4.1 months. The 4 surviving patients cleared HEV after a median period of 6.3 months. One patient was diagnosed with HEV reactivation after a preceding infection prior to alloHSCT. Although the incidence of developing acute HEV post-alloHSCT is relatively low, the probability of developing chronic hepatitis in severely immunocompromised patients is high. Therefore, alloHSCT recipients should be screened pretransplantation by HEV serology and RNA. Furthermore, a differential diagnosis including hepatitis E is mandatory in all alloHSCT patients with severe liver enzyme abnormalities.

Miscarriage Associated with Zika Virus Infection

A 31-year-old woman who was 10 weeks pregnant contracted Zika virus infection in Suriname; this led to fetal loss. ZIKV was detected in the womans blood for at least 21 days.

Exploring the Potential of Next-Generation Sequencing in Detection of Respiratory Viruses

ABSTRACT Efficient detection of human respiratory viral pathogens is crucial in the management of patients with acute respiratory tract infection. Sequence-independent amplification of nucleic acids combined with next-generation sequencing technology and bioinformatics analyses is a promising strategy for identifying pathogens in clinical and public health settings. It allows the characterization of hundreds of different known pathogens simultaneously and of novel pathogens that elude conventional testing. However, major hurdles for its routine use exist, including cost, turnaround time, and especially sensitivity of the assay, as the detection limit is dependent on viral load, host genetic material, and sequencing depth. To obtain insights into these aspects, we analyzed nasopharyngeal aspirates from a cohort of 81 Thai children with respiratory disease for the presence of respiratory viruses using a sequence-independent next-generation sequencing approach and routinely used diagnostic real-time reverse transcriptase PCR (real-time RT-PCR) assays. With respect to the detection of rhinovirus and human metapneumovirus, the next-generation sequencing approach was at least as sensitive as diagnostic real-time RT-PCR in this small cohort, whereas for bocavirus and enterovirus, next-generation sequencing was less sensitive than real-time RT-PCR. The advantage of the sequencing approach over real-time RT-PCR was the immediate availability of virus-typing information. Considering the development of platforms capable of generating more output data at declining costs, next-generation sequencing remains of interest for future virus diagnosis in clinical and public health settings and certainly as an additional tool when screening results from real-time RT-PCR are negative.

Paired measurements of quantitative hepatitis B virus DNA in saliva and serum of chronic hepatitis B patients: implications for saliva as infectious agent

BACKGROUND After intensive source and contact tracing 20 % of acute Hepatitis B virus (HBV) infections remain unexplained. Saliva may be an unexpected vehicle of HBV DNA transmission. OBJECTIVE To further explore this hypothesis we evaluated the quantitative levels of HBV DNA in saliva and compared these with the HBV DNA levels measured in serum. STUDY DESIGN Serum and saliva were collected from 27 chronic HBV patients attending our outpatient clinic. RESULTS There were 16 men and 11 women; 15 patients were HBeAg positive, anti-HBe negative and 11 patients were HBeAg negative, anti-HBe positive. One patient was HBeAg and anti-HBe negative. Samples of serum and saliva were collected on the same day. All saliva specimens were clear on inspection. HBV DNA in serum was measured by the Digene Hybrid Capture II microplate assay (Digene Diagnostics), the HBV Monitor assay (Roche Diagnostics) as well as an in-house developed HBV DNA TaqMan assay. The HBV DNA TaqMan assay was used for the quantitative measurement of HBV DNA in saliva. Median HBV DNA levels in serum were 2.10 x 10(5) geq/ml and ranged from 373 genome equivalents per ml (geq/ml) to 4.13 x 10(9) geq/ml; median HBV DNA levels in saliva were 2.27 x 10(4) geq/ml and ranged from 373 geq/ml to 9.25 x 10(6) geq/ml. A clear correlation was shown between HBV DNA in serum and saliva; log HBV DNA in saliva=1.01 + 0.56 x (log HBV DNA in serum). CONCLUSIONS this is the first report of precise quantitative measurements of HBV DNA levels in saliva and the relationship with HBV DNA levels in serum. Our findings show that saliva is a source of HBV DNA. This finding may have implications in selected patients for the infectivity of saliva and offer further insight in the routes of transmission of HBV infection.

Clinical implications of chronic hepatitis E virus infection in heart transplant recipients

BACKGROUND Recent reports have shown that hepatitis E virus (HEV) infection can become chronic in solid-organ transplant recipients, but few studies have systematically investigated the clinical consequences of this chronic HEV infection in solid-organ transplant (SOT) recipients. METHODS We have undertaken an in-depth study of 6 chronic HEV-infected heart transplant recipients to gain further insight into the clinical, biochemical and virologic presentation of this disorder. RESULTS In 6 patients (2.3%) chronic HEV infection, genotype 3, was identified. Immunosuppression in these patients was tacrolimus-based, combined with either everolimus or prednisolone and/or mycophenolate mofetil. Median follow-up after case detection was 26 months (range 21 to 40 months). All chronic HEV cases had elevated liver enzyme values. IgM antibodies at presentation were positive in 2 of 6 (33%) patients. Liver histology in 4 of 6 (67%) patients showed advanced fibrosis within 2 years after infection. One patient spontaneously cleared the HEV infection: 1 after dose reduction of immunosuppressive therapy and 3 during ribavirin therapy. One patient has yet to clear the virus and remains on ribavirin therapy. CONCLUSIONS Chronic HEV infection in heart transplant (HTx) recipients may lead to rapid fibrosis of the liver. We advise additional HEV RNA screening in solid-organ transplant recipients with elevated liver enzymes, because antibody production is often delayed, as demonstrated in these patients. Dose reduction of immunosuppressive therapy should be the first intervention strategy to achieve viral clearance in chronic HEV-infected immunocompromised patients. Ribavirin treatment should be considered in cases of chronic HEV.

Modeling rotavirus infection and antiviral therapy using primary intestinal organoids.

Despite the introduction of oral vaccines, rotavirus still kills over 450,000 children under five years of age annually. The absence of specific treatment prompts research aiming at further understanding of pathogenesis and the development of effective antiviral therapy, which in turn requires advanced experimental models. Given the intrinsic limitations of the classical rotavirus models using immortalized cell lines infected with laboratory-adapted strains in two dimensional cultures, our study aimed to model infection and antiviral therapy of both experimental and patient-derived rotavirus strains using three dimensional cultures of primary intestinal organoids. Intestinal epithelial organoids were successfully cultured from mouse or human gut tissues. These organoids recapitulate essential features of the in vivo tissue architecture, and are susceptible to rotavirus. Human organoids are more permissive to rotavirus infection, displaying an over 10,000-fold increase in genomic RNA following 24h of viral replication. Furthermore, infected organoids are capable of producing infectious rotavirus particles. Treatment of interferon-alpha or ribavirin inhibited viral replication in organoids of both species. Importantly, human organoids efficiently support the infection of patient-derived rotavirus strains and can be potentially harnessed for personalized evaluation of the efficacy of antiviral medications. Therefore, organoids provide a robust model system for studying rotavirus-host interactions and assessing antiviral medications.

Hepatitis B Surface Antigen Declines and Clearance During Long-Term Tenofovir Therapy in Patients Coinfected With HBV and HIV

BACKGROUND The kinetics of hepatitis B surface antigen (HBsAg) are predictive in HBV-infected patients treated with pegylated interferon. Knowledge about the value of HBsAg levels in patients coinfected with HBV and human immunodeficiency virus (HIV) is lacking. METHODS We quantified serum HBsAg in a Dutch multicenter cohort of 104 patients coinfected with HIV and HBV who were treated with tenofovir disoproxil fumarate (TDF) as part of highly active antiretroviral therapy. The median duration of therapy was 57 months (interquartile range, 34-72 months). RESULTS Hepatitis B e antigen (HBeAg)-positive patients achieved a decline of 2.2 log IU/mL in HBsAg, whereas HBeAg-negative patients only achieved a decline of 0.6 log IU/mL during 6 years of TDF therapy. Declines in HBsAg at months 6 and 12 correlated with CD4 cell count for HBeAg-positive patients. Five HBeAg-positive patients (8%) and 3 HBeAg-negative patients (8%) cleared HBsAg. HBeAg-negative patients who cleared HBsAg had lower baseline HBsAg as compared to patients who remained HBsAg positive. The majority of patients who cleared HBsAg achieved this end point within the first year. In HBeAg-positive patients, decline in HBsAg at month 6 was predictive of achieving HBsAg seroclearance. CONCLUSIONS Receipt of TDF therapy by HIV/HBV-coinfected patients for up to 6 years led to a significant decrease in HBsAg in the HBeAg-positive population. HBsAg kinetics early during treatment were predictive of HBsAg seroclearance and correlated with an increased CD4 cell count, underlining the importance of immune restoration in HBV clearance.