Molecular respiratory viral diagnostics are rapidly becoming the new gold standard in respiratory viral detection.
Molecular respiratory viral diagnostics are rapidly becoming the new gold standard in respiratory viral detection, and are the tests of choice in immunocompromised individuals. During her presentation at the 48th annual meeting of the IDSA, Kimberly Hanson, MD, MHS, University of Utah ARUP Laboratories, reviewed several of these diagnostic tests, using the example of a case study of a woman with aplastic anemia to highlight several points.
The patient, a 58-year-old woman with aplastic anemia who had undergone allogeneic-stem cell transplant (SCT), developed fever and hypoxia on post-transplant day 16. These developed in the setting of negative surveillance for cytomegalovirus CMV, and negative sequential biomarkers. "The differential diagnosis certainly looked like a respiratory virus at this point," Hanson said.
The patient underwent a bronchoscopy on the day of symptom presentation; multiple-specimen testing, including Directigen Flu A B Enzyme Immunoassay (DFA) and cultures, were negative. She also had a plasma adenovirus (ADV) PCR, which was negative. She was admitted to intensive care and intubated. On day 28 her bronchoscopy was repeated; her DFA was negative. "Despite maximal support, she expired on day 37. Just two days after her death, her cell line became positive for ADV. An autopsy confirmed that adenovirus pneumonia was likely contributory to her death, but she was never treated for ADV,” Hanson said.
The case highlights the main limitations of using culture to test for ADV. DFA and RNA have reduced sensitivity for ADV specifically, and conventional cell culture, while useful, is not clinically helpful in real time because results are often not available to support treatment decisions. For these reasons, she suggested that the new gold standard diagnostic in the clinical laboratory is molecular-based testing. "The go-to method for us is real-time (RT) PCR," Hanson said. The assays can be designed to detect almost all known viruses, regardless of whether they grow in cell culture. They are highly sensitive and specific, and less influenced by specimen quality. Importantly, the results can be available within 2-3 hours.
However, there are limitations for RT-PCR in the context of respiratory viruses in their inability to do heavy multiplexing, or detect multiple viruses simultaneously in one reaction. This can be problematic in detecting a disease that could result from a variety of pathogens. Also, there are few FDA-cleared RT-PCR assays for respiratory viruses and the panels are expensive.
Nevertheless, RT-PCR has demonstrated remarkable efficacy, and Hanson cited data from a study in 131 post-stem cell transplant (SCT) patients who provided a total of 688 specimens collected for the first 100 days post-transplant over four seasons.
The study compared DNA testing with traditional testing and RT-PCR. The results show that the diagnostic yield was vastly superior with RT-PCR. "The main gain was in parainfluenza virus (PIV1-3) and human metapneumovirus (hMPV)," Hanson said. She also noted that “PCR increased the diagnostic yield approximately five times compared with DFA and two times compared with culture.” Of note, the 13 specimens that were PCR positive only came from asymptomatic patients or patients with very low viral loads. “This really illustrates the sensitivity of this test,” she said.
Hanson said that, although this technology has dramatically changed clinical practice, future diagnostic tools are even more robust. By way of example, she mentioned a film array technology currently under review for approval by the FDA that could analyze up to 18 viral and three bacterial targets at the same time, with results available in 60 minutes.