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.

Impact of nucleic acid extraction platforms on hepatitis virus genome detection.

Detection and quantification of viral nucleic acids are important for diagnosing current viral infections and monitoring response to antiviral therapy. Automated nucleic acid extraction and purification platforms are routinely used during the first step in these processes in clinical and research laboratories. Here, we compare the extraction efficiencies of four MagNA Pure magnetic bead-based nucleic acid extraction platforms and associated kits using samples positive for nucleic acids from HAV, HBV, HCV, HDV, and HEV. These five hepatitis viruses are diverse in their virion structures and type of nucleic acid that compose their genomes. We found that the most efficient nucleic acid extraction platform and corresponding kit, when averaged across all tested viruses, was the MagNA Pure 96, which yielded twice as much detectable nucleic acid as the other platforms. However, the relative efficiencies of the different platforms varied by virus type, suggesting that an extraction platform that is more efficient for one virus type will not necessarily function better with a different virus type. Our results show that the choice of a nucleic acid extraction platform influences the sensitivity of the methodology and has the potential to generate false-negative results especially in samples with low levels of viral nucleic acids.

Risk Factors Associated with Blood Exposure for Sporadic Hepatitis E in Dhaka, Bangladesh.

Fecal contamination of drinking water is associated with large hepatitis E virus (HEV) outbreaks of genotypes 1 and 2 in endemic areas. Sporadic transmission of HEV genotypes 3 and 4 in high-income countries has been associated with exposure to blood and animal contact. The objective of the study was to identify the risk factors for hepatitis E and the genotype(s) causing sporadic hepatitis E in Dhaka, Bangladesh. We selected, from a diagnostic center in Dhaka between November 2008 and November 2009, cases presenting with jaundice and anti-HEV IgM antibodies and age-matched controls were defined as those with no history of jaundice and normal blood test results. Serum samples were tested for HEV RNA using real-time reverse transcriptase polymerase chain reaction followed by a sequencing and phylogenetic analysis. A total of 109 cases and 109 controls were enrolled. The cases were more likely to be male (adjusted matched odds ratios [mOR] 2.2, 95% CI: 1.2-3.9; P = 0.01), or have reported contact with another person's blood or blood product, or contact with blood-contaminated sharp instruments (adjusted mOR 2.1, 95% CI: 1.1-4.1; P = 0.03) than controls. There were no significant differences between the cases and the controls in terms of reported high-risk sexual intercourse, consumption of undercooked meat, or contact or drinking fecally-contaminated water. The sera from three cases carried HEV RNA, all belonging to genotype 1. Findings from this study suggest that contact with human blood and sharing sharp instruments may transmit sporadic hepatitis E in Dhaka, Bangladesh. Efforts to prevent the transmission of blood-borne pathogens may also prevent sporadic HEV transmission in this endemic setting.

Laboratory diagnostics for hepatitis C virus infection.

Identification of prevalent infection by hepatitis C virus (HCV) is based serologically on detecting anti-HCV immunoglobulin G, using immunoassays, immunoblot assays, and, more recently, immunochromatography-based rapid tests. None discriminate between active and resolved HCV infection. Tests for detecting HCV RNA identify active HCV infection but are costly. Serologic assays for HCV antigens have been developed and show potential for diagnosis of active HCV infection, and their performance characteristics are undergoing evaluation. The diagnosis of acute HCV infection without the demonstration of seroconversion remains elusive.