NEW YORK (GenomeWeb) – Johns Hopkins University researchers have developed a proof-of-concept platform and assay for use in the clinic to test for the hepatitis C virus. The development comes on the back of an internal study that indicated 60 percent of patients don't return to the clinic to take required confirmatory RNA testing for the hepatitis C virus.
Importantly, the system could enable testing and prescribing treatment for patients during a single office visit, Jeff Tza-Huei Wang, one of the test developers and a researcher at Johns Hopkins, said in an interview.
A point-of-care test used in this manner may help reduce patient attrition due to lack of follow-up while removing the psychological harm that occurs when a person is told infection is possible but not yet confirmed, the researchers said. Additionally, by bringing diagnostics to the point of care, HCV screening could be made more widely available for early detection of the virus before the onset of related and serious medical conditions, such as cirrhosis and hepatocellular carcinoma.
In a study published last week in the journal Scientific Reports, the researchers described the development of a droplet magnetofluidic platform with functionalized magnetic particles that miniaturizes and automates assays for use at the point of care.
The system combines a thermoformed, disposable cartridge and a portable multiaxial magnetofluidic instrument to enable real-time PCR detection of nucleic acids from serum. Instead of employing the complicated micron-size channels, chambers, pumps, and valves of traditional microfluidics, the magnetofluidic instrument uses thermoformed extruded walls to sequester digitized reagent droplets and silicone oil as an immiscible transfer medium.
The thermoforming manufacturing process using acrylic material is scalable and contributes to the platform's suitability for PCR testing in the clinic, said Wang, a professor at the Johns Hopkins Institute of NanoBioTechnology.
The cost to build an instrument is less than $300 and to fabricate a disposable test cartridge with reagents is less than $3. That should enable pricing and adoption in low-resource settings as well as in developed economies, Wang said.
The researchers are exploring options to develop the assay and its magnetofluidic platform for commercial use. They have begun recruiting patients to conduct preclinical trials. Further, they are developing the system to test blood samples extracted by fingerpick, which is more convenient than testing serum.
The Johns Hopkins researchers are in discussions with diagnostic companies in anticipation of commercializing the test for use in the clinic within two years, Wang said. The university is also considering launching a startup to pursue commercialization.
Potential uses for the magnetofluidic platform extend beyond testing for HCV. The cartridge design could enable a variety of other assays involving the capture and transport of nucleic acids. For example, outside of HCV, the group has two separate projects focused on developing assays for urinary tract infections and sexually transmitted infections.
Cost aside, the platform's ease of operation should accelerate its adoption, Wang said. The system enables testing by injecting a sample into the magnetic particle-based cartridge and then loading the cartridge into the instrument.
In their study, the group showed that the platform quantified HCV RNA viral load from blood serum in less than 40 minutes, Wang said. There were no false positives when the investigators processed 18 serum samples in the cartridges.
HCV RNA levels in blood of chronically infected patients are usually between 105 and 107 IU/ml, and levels persist if the condition is left untreated. The Johns Hopkins cartridge has demonstrated sensitivity of around 4,500 IU/ml, "which adequately meets the threshold needed for diagnosis and identification of significant changes in a patient’s viral load," the researchers said.
Wang noted that the instrument is far simpler to operate than either traditional microfluidic or lab-based HCV test systems that require purification, elution, and spiking of the sample into a PCR buffer prior to testing.
Traditional microfluidic approaches have struggled to enable cost-effective, scalable assay integration, because they require complex device fabrication and instrumentation, Wang said. In addition, quantitative nucleic acid tests for diagnosis of HCV are primarily implemented in laboratories and can take a few days to process samples, he noted.
Overcoming the dropouts
Patients presenting to the emergency department are increasingly being tested for HCV on a routine basis. In hospitals, they receive a whole-blood antibody test that takes about two hours to produce a result. In outreach settings established to offer screening, a fingerstick antibody test is done in about 20 minutes, said Yu-Hsiang Hsieh, an emergency department researcher at Johns Hopkins School of Medicine and one of the clinical advisors to the test development team.
In either case, a positive result leads to the requirement for either reflex or confirmatory RNA testing, he said, but only about 40 percent of patients return for confirmatory tests. There are likely many reasons for that, including challenges with transportation, getting time off work, and ignoring the infection because it doesn't initially produce symptoms, Hsieh said.
"At our site, we have a reflex in place for patients whose anti-HCV test are reactive," Hsieh said. "However, many medical settings do not have the reflex in their protocol."
HCV RNA detection is critical for evaluation of a patient’s current state of infection and for monitoring treatment success. Early stages of HCV are often asymptomatic, and patients accordingly do not receive the timely care and treatment needed to prevent complications, including cirrhosis and hepatocellular carcinoma.
Current US guidelines recommend HCV screening of high-risk individuals, a strategy which neglects up to 75 percent of infected individuals who are unaware of their infection, the researchers said.
In Europe, too, clinical guidelines recommend that hepatitis C virus screening targets people at high risk of infection. A study published this week in the Journal of Hepatology noted that according to 2014 public health data in France, 75,000 people aged 18 to 80 were infected by HCV but were unaware of their status. In at least one in 10 cases, they are at an advanced stage of the disease when diagnosed, the researchers said.
Their modeling results indicated that universal screening is associated with better life expectancy, and it is cost-effective if the patients tested for HCV infection are treated rapidly after diagnosis.
In Australia, Jason Grebely, an associate professor at the Kirby Institute, University of New South Wales in Sydney, sees HCV point-of-care testing as holding significant potential for patients.
The ability to take blood using a fingerstick instead of a vein puncture can be an important factor driving uptake, he said, especially for people injecting drugs, among whom HCV prevalence is high.
If the cost can be reduced to suitable levels, an RNA viral load test for HCV at the point of care that is easy to use and shows high sensitivity and specificity "might serve as the only diagnostic test needed for the detection of HCV infection in the future," Hsieh said.