Maja Kodani

Comparison of commercial systems for extraction of nucleic acids from DNA/RNA respiratory pathogens.

Abstract This study compared six automated nucleic acid extraction systems and one manual kit for their ability to recover nucleic acids from human nasal wash specimens spiked with five respiratory pathogens, representing Gram-positive bacteria (Streptococcus pyogenes), Gram-negative bacteria (Legionella pneumophila), DNA viruses (adenovirus), segmented RNA viruses (human influenza virus A), and non-segmented RNA viruses (respiratory syncytial virus). The robots and kit evaluated represent major commercially available methods that are capable of simultaneous extraction of DNA and RNA from respiratory specimens, and included platforms based on magnetic-bead technology (KingFisher mL, Biorobot EZ1, easyMAG, KingFisher Flex, and MagNA Pure Compact) or glass fiber filter technology (Biorobot MDX and the manual kit Allprep). All methods yielded extracts free of cross-contamination and RT-PCR inhibition. All automated systems recovered L. pneumophila and adenovirus DNA equivalently. However, the MagNA Pure protocol demonstrated more than 4-fold higher DNA recovery from the S. pyogenes than other methods. The KingFisher mL and easyMAG protocols provided 1- to 3-log wider linearity and extracted 3- to 4-fold more RNA from the human influenza virus and respiratory syncytial virus. These findings suggest that systems differed in nucleic acid recovery, reproducibility, and linearity in a pathogen specific manner.

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.

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.

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.

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.

Delta Hepatitis: Towards Improved Diagnostics

Hepatitis D virus (HDV), the etiologic agent of hepatitis D or delta hepatitis, is a unique human virus that requires the hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV) for its replication and to establish infection. HDV, the only member of its own separate genus, Deltavirus, is the smallest known infectious viral agent of humans. The HDV virion, which measures 35-37 nm in diameter, contains a single-stranded circular 1,700-nucleotide RNA genome of negative polarity, which encloses only a single functional open reading frame encoding the sole viral protein, the hepatitis delta antigen (HDAg). The HDAg is associated with the genomic RNA to form a ribonucleoprotein complex which is encapsulated by a lipid envelope containing the small, middle, and large HBsAg proteins. HDV exhibits a high degree of genetic heterogeneity, with estimated mutation rates between 3 3 10 and 3 3 10 base substitutions per genomic site per year. The virus has a wide geographic distribution with eight distinct genotypes. HDV genotype 1 is the most common genotype, being prevalent in North America, Europe, the Middle East, and North Africa; genotypes 2 and 4 are found predominantly in East Asia; genotype 3 is found exclusively in the Amazon basin in South America; and genotypes 5-8, also known as African genotypes, are predominantly found in West and central Africa. HDV is a highly pathogenic virus, and clinical presentation of hepatitis D ranges from fulminant hepatitis, exacerbation of the course of underlying HBV infection, and acceleration of progression to cirrhosis, leading to early decompensation of liver function and hepatocellular carcinoma in the majority of patients. However, a benign course of HDV infection has also been observed in Greece, Samoa, and the Far East; whether this is related to various viral characteristics such as the infecting genotype or host genetics remains to be determined. Outbreaks of severe and fulminant hepatitis D have been reported from Brazil, Russia, Greenland, and Mongolia. The laboratory diagnosis of coinfection or superinfection with HDV is based on simultaneous detection of various serologic markers of HBV and HDV infection. The markers of acute HDV infection include, along with the markers of HBV infection, HDAg, HDV RNA, and immunoglobulins M and G (IgM and IgG, respectively) antibodies to HDV (anti-HDV). These markers of HDV infection are present only transiently and disappear during early convalescence; IgM anti-HDV and even IgG antiHDV also disappear with time in acute resolving HDV infection. Superinfection of HBsAg carriers, which almost always leads to chronic hepatitis D, is marked by absence of IgM antibody to hepatitis B core antigen and presence of all the other markers of HBV and HDV infections (Fig. 1). However, markers of HBV replication, especially HBV DNA, may be suppressed during the acute phase of HDV infection and remain undetectable. Chronicity of HDV infection is associated with persistence of HDAg, HDV RNA, and IgM and IgG anti-HDV. HDV RNA is the gold standard for diagnosis of current HDV infection because assays for detection of HDAg are fraught with sensitivity and specificity issues. Quantitative assays of HDV RNA are useful for monitoring response to antiviral therapy, and the recent availability of a World Abbreviations: anti-HDV, antibody to HDV; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus; HDAg, hepatitis delta antigen; HDV, hepatitis D virus; IgG/IgM, immunoglobulins G/M; Q-MAC, quantitative microarray antibody capture.

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.

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.

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

Abstract 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.

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.