Prevalence and determinants of hepatitis delta virus infection among HIV/hepatitis B-coinfected adults in care in the United States.

Hepatitis delta virus (HDV) infection increases the risk of liver complications compared to hepatitis B virus (HBV) alone, particularly among persons with human immunodeficiency virus (HIV). However, no studies have evaluated the prevalence or determinants of HDV infection among people with HIV/HBV in the US. We performed a cross-sectional study among adults with HIV/HBV coinfection receiving care at eight sites within the Center for AIDS Research Network of Integrated Clinical Systems (CNICS) between 1996 and 2019. Among patients with available serum/plasma specimens, we selected the first specimen on or after their initial HBV qualifying test. All samples were tested for HDV IgG antibody and HDV RNA. Multivariable log-binomial generalized linear models were used to estimate prevalence ratios (PRs) with 95% CIs of HDV IgG antibody-positivity associated with determinants of interest (age, injection drug use [IDU], high-risk sexual behaviour). Among 597 adults with HIV/HBV coinfection in CNICS and available serum/plasma samples (median age, 43 years; 89.9% male; 52.8% Black; 42.4% White), 24/597 (4.0%; 95% CI, 2.4%-5.6%) were HDV IgG antibody-positive, and 10/596 (1.7%; 95% CI, 0.6%-2.7%) had detectable HDV RNA. In multivariable analysis, IDU was associated with exposure to HDV infection (adjusted PR = 2.50; 95% CI, 1.09-5.74). In conclusion, among a sample of adults with HIV/HBV coinfection in care in the US, 4.0% were HDV IgG antibody-positive, among whom 41.7% had detectable HDV RNA. History of IDU was associated with exposure to HDV infection. These findings emphasize the importance of HDV testing among persons with HIV/HBV coinfection, especially those with a history of IDU.

Development of a Bead-Based Multiplex Assay for Use in Multianalyte Screening and Surveillance of HIV, Viral Hepatitis, Syphilis, and Herpes.

Diagnostic assays that can simultaneously determine the presence of infection with multiple pathogens are key for diagnosis and surveillance. Current multiplex diagnostic assays are complex and often have limited availability. We developed a simple, multianalyte, pathogen detection assay for screening and serosurveillance using the Luminex Magpix platform that is high throughput and can be helpful in monitoring multiple diseases. The Luminex bead-based 10-plex immunoassay for the detection of HIV-1, HIV-2, Treponema pallidum, hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus 1 (HSV-1), and HSV-2 infections was accomplished by coupling beads with specific antigens to detect IgG antibodies in plasma or serum samples. Each coupled antigen was systematically optimized, and the performance was evaluated using a panel of well-characterized specimens (n = 417) that contained antibodies to HIV-1, HIV-2, T. pallidum, HBV, HCV, HSV-1, and HSV-2. The multiplex assay had a sensitivity of 92.2% (95% Clopper-Pearson confidence interval [CI], 90.2 to 94.0%) and a specificity of 98.1% (95% CI, 97.6 to 98.7%). The sensitivities and specificities for disease-specific biomarker detection ranged from 68.7 to 100% and 95.6 to 100%, respectively. The results showed that the 10-plex immunoassay had an overall agreement of 96.7% (95% CI, 96.7 to 97.3%) with reference tests and a corresponding kappa value of 0.91 (95% CI, 0.90 to 0.93). Kappa values for the individual pathogens ranged from 0.69 to 1.00. The assay is robust and allows the simultaneous detection of antibodies to multiple antigens using a small sample volume in a high-throughput format. This assay has the potential to simplify disease surveillance by providing an alternative to expensive and highly specialized individual tests.

Determination of potential biotin interference on accuracy of results of serologic assays for various viral hepatitis markers.

Biotin taken orally can interfere with some diagnostic immunoassays, including those for thyroid hormones, ferritin, and markers of infectious disease. Assays affected are ones that use streptavidin-biotin in their design. The goal of our study was to examine the effect of biotin concentrations of up to 1200 ng/mL on three serological assays performed on VITROS 3600 system, Immunoglobulin M (IgM) antibodies to hepatitis A virus (anti-HAV), total anti-HAV, and IgM antibodies to hepatitis B virus core antigen (anti-HBc), by spiking serum samples with variable amounts of biotin. No false-negative results were generated with either concentration of biotin for total anti-HAV (65/65). Likewise, biotin caused no false-positive IgM anti-HAV results (59/59) with either concentration of biotin; however, 6.7% false negativity was found for IgM anti-HAV when samples were spiked with 1200 ng/mL of biotin. Conversely, 100% false positivity (30/30) was produced by biotin interference in total anti-HAV negative specimens with both concentrations of biotin. False negativity rate was 87.5% in IgM anti-HBc positive samples when biotin levels were at 1200 ng/mL. These data show that individuals taking biotin-containing supplements may test false-positive in some serologic assays using streptavidin-biotin chemistries. Further studies are warranted to determine the extent of biotin interference resulting in false-positive and negative results and their impact, if any, on surveillance and diagnostic settings.

Feasibility of Hepatitis B Vaccination by Microneedle Patch: Cellular and Humoral Immunity Studies in Rhesus Macaques.

BACKGROUND: This study evaluated dissolvable microneedle patch (dMNP) delivery of hepatitis B vaccine in rhesus macaques and provides evidence that dMNP delivery elicits seroprotective anti-HBs levels comparable with human seroprotection, potentially useful for hepatitis B birth dose vaccination in resource-constrained regions. METHODS: Sixteen macaques were each vaccinated twice; they were treated in 4 groups, with dMNP delivery of AFV at 24 ± 8 µg (n = 4) or 48 ± 14 µg (n = 4), intramuscular injection of AFV (10 µg; n = 4), or intramuscular injection of AAV (10 µg; n = 4). Levels of antibody to hepatitis B surface antigen (HBsAg) (anti-HBs) and HBsAg-specific T-cell responses were analyzed. RESULTS: Six of 8 animals with dMNP delivery of AFV had anti-HBs levels ≥10 mIU/mL after the first vaccine dose. After dMNP delivery of AFV, interferon γ, interleukin 2, and interleukin 4 production by HBsAg-specific T cells was detected. A statistically significant positive correlation was detected between anti-HBs levels and cells producing HBsAg-specific interferon γ and interleukin 2 (T-helper 1-type cytokine) and interleukin 4 (T-helper 2-type cytokine) in all anti-HBs-positive animals. CONCLUSIONS: dMNP delivery of AFV can elicit seroprotective anti-HBs levels in rhesus macaques that are correlated with human seroprotection, and it could be particularly promising for birth dose delivery of hepatitis B vaccine in resource-constrained regions.

An Automated Immunoblot Method for Detection of IgG Antibodies to Hepatitis C Virus: a Potential Supplemental Antibody Confirmatory Assay.

An estimated 41,200 people were newly infected with hepatitis C virus (HCV) in 2016 in the United States. Screening tests for antibodies to HCV may generate up to 32% false positivity in low-risk populations. Current Centers for Disease Control and Prevention (CDC) screening recommendations do not require confirmatory testing of a screening anti-HCV-positive test; however, confirmation is valuable for surveillance in the absence of HCV RNA testing. A recombinant immunoblot assay (RIBA) was used as a confirmatory assay for anti-HCV-reactive samples but was discontinued in 2013. Another anti-HCV confirmatory assay, INNO-LIA, is commercially available in Europe but is not approved by the Food and Drug Administration (FDA) in the United States. We report the development of an anti-HCV assay that was performed on an automated immunoblot platform using a fourth-generation HCV recombinant fusion protein. Based on testing of 70 well-characterized samples, of which 40 were HCV RNA and anti-HCV positive, 15 were HCV RNA positive/anti-HCV negative, and 15 were HCV RNA and anti-HCV negative, the specificity and sensitivity of the HCV-WES assay were 100% and 95%, respectively. Concordance between INNO-LIA and HCV-WES was determined by testing 205 HCV RNA-negative/anti-HCV-positive samples, of which 149 (72.7%) were positive by HCV-WES, while 146 (71.2%) were positive by INNO-LIA. We have shown proof of concept for the use of this test for confirmation of screened anti-HCV results. The HCV-WES assay has advantages over manual Western blot assays and INNO-LIA, including ease of use, lower cost, and reduced hands-on time.

Hepatitis B vaccination using a dissolvable microneedle patch is immunogenic in mice and rhesus macaques.

Chronic Hepatitis B virus infection remains a major global public health problem, accounting for about 887,000 deaths in 2015. Perinatal and early childhood infections are strongly associated with developing chronic hepatitis B. Adding a birth dose of the hepatitis B vaccine (HepB BD) to routine childhood vaccination can prevent over 85% of these infections. However, HepB BD coverage remains low in many global regions, with shortages of birth attendants trained to vaccinate and limited HepB BD supply at birth. To address the challenges, we developed coated metal microneedle patches (cMNPs) and dissolvable microneedle patches (dMNPs) that deliver adjuvant-free hepatitis B vaccine to the skin in a simple-to-administer manner. The dMNP contains micron-scale, solid needles encapsulating vaccine antigen and dissolve in the skin, generating no sharps waste. We delivered HepB BD via cMNP to BALB/c mice and via dMNP to both mice and rhesus macaques. Both cMNP and dMNP were immunogenic, generating hepatitis B surface antibody levels similar to human seroprotection. Biomechanical analysis showed that at high forces the microneedles failed mechanically by yielding but microneedles partially blunted by axial compression were still able to penetrate skin. Overall, this study indicates that with further development, dMNPs could offer a method of vaccination to increase HepB BD access and reduce needle waste in developing countries.

Characterization of real-time microarrays for simultaneous detection of HIV-1, HIV-2, and hepatitis viruses.

Real-time PCR assays for nucleic acid testing (NAT) of hepatitis viruses A-E and for HIV-1 and HIV-2 have been developed; however, a multiplex assay that can simultaneously detect all of these agents is not yet available. Standardized TaqMan assays for detection of hepatitis viruses A-E have been described and applied to TaqMan Array Cards (TAC) which are capable of multiple pathogen detection using a single set of optimized PCR conditions. Assays for three gene regions of HIV-1 (long-terminal repeat (LTR), gag, and polymerase) and HIV-2 (overlap of LTR and gag, protease and integrase) were designed using the hepatitis assay conditions. Nucleic acid extracts of HIV-1-infected samples (44 plasma, 41 whole blood, 20 HIV-1 viral stocks) were tested on the TAC cards; 98 were reactive (92%) with 70 in multiple gene regions. Twenty-four of the 27 (89%) HIV-2 specimens (10 plasma, 1 PBMC lysate, 6 whole blood and 10 plasmids containing HIV-2 polymerase) were detected on TAC. No HIV or hepatitis virus sequences were detected in 30 HIV-negative samples (specificity 100%). Three HBV and 18 HCV co-infections were identified in the HIV-1-infected specimens. Multi-pathogen detection using TAC could provide a rapid, sensitive and more efficient method of surveying for a variety of infectious disease nucleic acids.

The Prevalence of Hepatitis C Virus Antibody in HIV-Negative Persons in Kenya, 2007.

The prevalence of hepatitis C virus (HCV) infection in the Kenyan population has not been previously determined. We estimated the Kenyan HCV prevalence in HIV-negative persons aged 15-64 years. This is a retrospective cross-sectional study using data from the 2007 Kenya AIDS Indicator Survey-a nationally representative sample of 15,853 persons aged 15-64 years who completed a health interview and provided a blood specimen. Of the 1,091 randomly selected participants, 50 tested positive for HCV antibody using the automated chemiluminescence immunoassay, corresponding to a weighted HCV antibody positivity rate of 4.4% (95% confidence interval: 3.3-5.9%) or 848,000 (range: 634,000-1,100,000) persons. Hepatitis C virus RNA, a marker for current infection, was not detected in any of the tested antibody-positive specimens. The high HCV antibody prevalence together with no current infection suggests that some HCV antibody serologic testing in Kenya may result in false positives whereas others may be because of spontaneous viral clearance.

Stability of hepatitis C virus RNA and anti-HCV antibody in air-dried and freeze-dried human plasma samples.

Diagnosis of hepatitis C virus (HCV) infection is based on testing for antibodies to HCV (anti-HCV), hepatitis C core antigen (HCV cAg) and HCV RNA. To ensure quality control (QC) and quality assurance (QA), proficiency panels are provided by reference laboratories and various international organizations, requiring costly dry ice shipments to maintain specimen integrity. Alternative methods of specimen preservation and transport can save on shipping and handling and help in improving diagnostics by facilitating QA/QC of various laboratories especially in resource limited countries. Plasma samples positive for anti-HCV and HCV RNA were either dried using dried tube specimens (DTS) method or lyophilization for varying durations of time and temperature. Preservation of samples using DTS method resulted in loss of anti-HCV reactivity for low-positive samples and did not generate enough volume for HCV RNA testing. Lyophilized samples tested positive for anti-HCV even after storage at 4 °C and 25 °C for 12 weeks. Further, HCV RNA was detectable in 5 of 5 (100%) samples over the course of 12 week storage at 4, 25, 37 and 45 °C. In conclusion, lyophilization of specimens maintains integrity of plasma samples for testing for markers of HCV infection and can be a potent mode of sharing proficiency samples without incurring huge shipping costs and avoids challenges with dry ice shipments between donor and recipient laboratories.

Variability in the performance characteristics of IgG anti-HEV assays and its impact on reliability of seroprevalence rates of hepatitis E.

Hepatitis E is a major public health problem in developing countries and is increasingly being recognized as a cause of substantial sporadic viral hepatitis infections in industrialized countries. Variable rates of hepatitis E seroprevalence have been reported from the same geographic regions depending on the assay used. In this study, we evaluated the performance characteristics of four assays which included two commercial assays, Wantai HEV-IgG ELISA kit (Wantai, China), and DS-EIA-ANTI-HEV-G kit (DSI, Italy), one NIH-developed immunoassay (NIH-55 K, Kuniholm et al. [2009] Journal of Infectious Diseases 200:48-56), previously used in several major seroprevalence studies and one in-house Western blot assay (CDC-WB). The limit of detection of IgG anti-HEV is 100 mIU/mL for Wantai assay, 200 mIU/mL for CDC-WB assay, 1000 mIU/mL for DSI assay, and 40 mIU/mL for NIH-55 K assay. Pairwise concordance between the four assays ranged from 56% to 87%. The concordance among all four assays was observed in 52% of the samples, while the concordance among three assays was observed in 37% of the samples. These data show a wide discordance between various IgG anti-HEV assays and warrant a comprehensive evaluation of all the assays using well characterized global serum reference panels.

Evaluation of fast-track diagnostics and TaqMan array card real-time PCR assays for the detection of respiratory pathogens.

Several commercial assays are now available to detect the nucleic acid of multiple respiratory pathogens from a single specimen. Head-to-head comparisons of such assays using a single set of standard specimens provide additional information about key assay parameters such as sensitivity, specificity and lower limits of detection, and help to inform the decision regarding which method to use. We evaluated two real-time PCR platforms: the Fast-track Diagnostics® (FTD) multiplex respiratory panel and a TaqMan array card (TAC) for simultaneous uniplex detection of multiple respiratory pathogens. Two sets of samples were used to evaluate the assays. One set was created by spiking pooled nasal wash or phosphate buffered saline with specified volumes of known concentrations of virus and/or bacteria. Clinical nasal wash specimens from children with lower respiratory tract illness comprised the other set. Thirteen pathogen targets were compared between the two platforms. Testing with a validation panel of spiked samples revealed a sensitivity of 96.1% and 92.9% for the FTD and TAC assays, respectively. Specificity could not be reliably calculated due to a suspected contamination of the sample substrate. Inter-assay agreement was high (> 95%) for most targets. Previously untested clinical specimens tested by both assays revealed a high percent agreement (> 95%) for all except rhinovirus, enterovirus and Streptococcus pneumoniae. Limitations of this evaluation included extraction of the validation samples by two different methods and the evaluation of the assays in different laboratories. However, neither of these factors significantly impacted inter-assay agreement for these sets of samples, and it was demonstrated that both assays could reliably detect clinically relevant concentrations of bacterial and viral pathogens.

Rapid and sensitive approach to simultaneous detection of genomes of hepatitis A, B, C, D and E viruses.

BACKGROUND: Five viruses have been etiologically associated with viral hepatitis. Nucleic acid testing (NAT) remains the gold standard for diagnosis of viremic stages of infection. NAT methodologies have been developed for all hepatitis viruses; however, a NAT-based assay that can simultaneously detect all five viruses is not available. OBJECTIVES: We designed TaqMan card-based assays for detection of HAV RNA, HBV DNA, HCV RNA, HDV RNA and HEV RNA. STUDY DESIGN: The performances of individual assays were evaluated on TaqMan Array Cards (TAC) for detecting five viral genomes simultaneously. Sensitivity and specificity were determined by testing 329 NAT-tested clinical specimens. RESULTS: All NAT-positive samples for HCV (n = 32), HDV (n = 28) and HEV (n = 14) were also found positive in TAC (sensitivity, 100%). Forty-three of 46 HAV-NAT positive samples were also positive in TAC (sensitivity, 94%), while 36 of 39 HBV-NAT positive samples were positive (sensitivity, 92%). No false-positives were detected for HBV (n = 32), HCV (n = 36), HDV (n = 30), and HEV (n = 31) NAT-negative samples (specificity 100%), while 38 of 41 HAV-NAT negative samples were negative by TAC (specificity 93%). CONCLUSIONS: TAC assay was concordant with corresponding individual NATs for hepatitis A-E viral genomes and can be used for their detection simultaneously. The TAC assay has potential for use in hepatitis surveillance, for screening of donor specimens and in outbreak situations. Wider availability of TAC-ready assays may allow for customized assays, for improving acute jaundice surveillance and for other purposes for which there is need to identify multiple pathogens rapidly.

Wnt signaling in triple negative breast cancer is associated with metastasis.

BACKGROUND: Triple Negative subset of (TN) Breast Cancers (BC), a close associate of the basal-like subtype (with limited discordance) is an aggressive form of the disease which convey unpredictable, and poor prognosis due to limited treatment options and lack of proven effective targeted therapies. METHODS: We conducted an expression study of 240 formalin-fixed, paraffin-embedded (FFPE) primary biopsies from two cohorts, including 130 TN tumors, to identify molecular mechanisms of TN disease. RESULTS: The annotation of differentially expressed genes in TN tumors contained an overrepresentation of canonical Wnt signaling components in our cohort and others. These observations were supported by upregulation of experimentally induced oncogenic Wnt/ß-catenin genes in TN tumors, recapitulated using targets induced by Wnt3A. A functional blockade of Wnt/ß-catenin pathway by either a pharmacological Wnt-antagonist, WntC59, sulidac sulfide, or ß-catenin (functional read out of Wnt/ß-catenin pathway) SiRNA mediated genetic manipulation demonstrated that a functional perturbation of the pathway is causal to the metastasis- associated phenotypes including fibronectin-directed migration, F-actin organization, and invasion in TNBC cells. A classifier, trained on microarray data from ß-catenin transfected mammary cells, identified a disproportionate number of TNBC breast tumors as compared to other breast cancer subtypes in a meta-analysis of 11 studies and 1,878 breast cancer patients, including the two cohorts published here. Patients identified by the Wnt/ß-catenin classifier had a greater risk of lung and brain, but not bone metastases. CONCLUSION: These data implicate transcriptional Wnt signaling as a hallmark of TNBC disease associated with specific metastatic pathways.

One-step real-time PCR assay for detection and quantitation of hepatitis D virus RNA.

Hepatitis D virus (HDV) is a defective virus which requires hepatitis B virus (HBV) surface antigen (HBsAg) for its assembly. Hepatitis B infected individuals co-infected or superinfected with HDV often present with more severe hepatitis, progress faster to liver disease, and have a higher mortality rate than individuals infected with HBV alone. Currently, there are no commercially available clinical tests for the detection and quantitation of HDV RNA in the United States. A one-step TaqMan quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for detection of HDV RNA, designing primers located in the region just downstream from the HDV antigen gene. The assay has the potential to detect all eight HDV genotypes. A quantifiable synthetic RNA control was also developed for use in the determination of HDV RNA titers in clinical samples. The limit of detection of this assay is 7.5×10(2) HDV RNA copies/ml with a dynamic range of six logs. Most clinical specimens tested (40/41) fell within the linear range of the assay. The median HDV RNA titer of the tested specimens was 6.24×10(6) copies/ml, with a range of 8.52×10(3)-1.79×10(9) copies/ml. Out of 132 anti-HDV-positive specimens 41 (31.1%) were positive for HDV RNA.

Streptococcus pneumoniae serotype 15A in psychiatric unit, Rhode Island, USA, 2010-2011.

During a pneumococcal disease outbreak in a pediatric psychiatric unit in a hospital in Rhode Island, USA, 6 (30%) of 20 patients and staff were colonized with Streptococcus pneumoniae serotype 15A, which is not included in pneumococcal vaccines. The outbreak subsided after implementation of antimicrobial drug prophylaxis and enhanced infection control measures.

Outbreak of lower respiratory tract illness associated with human enterovirus 68 among American Indian children.

Human enterovirus 68 (EV68) infections are rarely reported. We describe a respiratory outbreak associated with EV68 among 18 children admitted to a remote Indian Health Service facility during August 11, 2010 through September 14, 2010. Clinical illness was characterized by pneumonia and wheezing. EV68 should be considered as an etiology in outbreaks of lower respiratory tract illness.

Survey of legionella species found in thai soil.

Members of the Gram-negative genus Legionella are typically found in freshwater environments, with the exception of L. longbeachae, which is present in composts and potting mixes. When contaminated aerosols are inhaled, legionellosis may result, typically as either the more serious pneumonia Legionnaires' disease or the less severe flu-like illness Pontiac fever. It is presumed that all species of the genus Legionella are capable of causing disease in humans. As a followup to a prior clinical study of legionellosis in rural Thailand, indigenous soil samples were collected proximal to cases' homes and workplaces and tested for the presence of legionellae by culture. We obtained 115 isolates from 22/39 soil samples and used sequence-based methods to identify 12 known species of Legionella represented by 87 isolates.

Engineered combined-positive-control template for real-time reverse transcription-PCR in multiple-pathogen-detection assays.

Recently we evaluated a custom TaqMan array card (TAC) detection system, formerly known as a TaqMan low-density array (TLDA) card, for simultaneous real-time PCR identification of 21 pathogens and three control targets in duplicate from respiratory specimens (M. Kodani et al., J. Clin. Microbiol. 49:2175-2182, 2011). We engineered an adaptable and expandable system of in vitro RNA transcripts to serve as a combined positive control for both DNA and RNA targets in multiple-pathogen-detection technologies based on real-time reverse transcription-PCR.

Air1 zinc knuckles 4 and 5 and a conserved IWRXY motif are critical for the function and integrity of the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) RNA quality control complex.

In Saccharomyces cerevisiae, non-coding RNAs, including cryptic unstable transcripts (CUTs), are subject to degradation by the exosome. The Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex in S. cerevisiae is a nuclear exosome cofactor that recruits the exosome to degrade RNAs. Trf4/5 are poly(A) polymerases, Mtr4 is an RNA helicase, and Air1/2 are putative RNA-binding proteins that contain five CCHC zinc knuckles (ZnKs). One central question is how the TRAMP complex, especially the Air1/2 protein, recognizes its RNA substrates. To characterize the function of the Air1/2 protein, we used random mutagenesis of the AIR1/2 gene to identify residues critical for Air protein function. We identified air1-C178R and air2-C167R alleles encoding air1/2 mutant proteins with a substitution in the second cysteine of ZnK5. Mutagenesis of the second cysteine in AIR1/2 ZnK1-5 reveals that Air1/2 ZnK4 and -5 are critical for Air protein function in vivo. In addition, we find that the level of CUT, NEL025c, in air1 ZnK1-5 mutants is stabilized, particularly in air1 ZnK4, suggesting a role for Air1 ZnK4 in the degradation of CUTs. We also find that Air1/2 ZnK4 and -5 are critical for Trf4 interaction and that the Air1-Trf4 interaction and Air1 level are critical for TRAMP complex integrity. We identify a conserved IWRXY motif in the Air1 ZnK4-5 linker that is important for Trf4 interaction. We also find that hZCCHC7, a putative human orthologue of Air1 that contains the IWRXY motif, localizes to the nucleolus in human cells and interacts with both mammalian Trf4 orthologues, PAPD5 and PAPD7 (PAP-associated domain containing 5 and 7), suggesting that hZCCHC7 is the Air component of a human TRAMP complex.