The story has been updated to correct the name of the company, which was previously identified as NanoTin. 360Dx regrets the error and apologizes.
NEW YORK (360Dx) – A team led by researchers at Arizona State University's Biodesign Institute has demonstrated the ability of a mass spec-based tuberculosis test to detect active disease in patients who are culture negative.
Detailed in a paper published this month in Clinical Chemistry, the blood-based test could be useful for identifying patients who might be missed by conventional sputum tests, as well as for monitoring response to treat, said ASU researcher Ye Hu, one of the developers of the test and senior author on the study.
Hu and his colleagues have founded a company, called NanoPin, to commercialize the TB test as well as tests for other mycobacterial infections. The tests are based on a target enrichment device, called the NanoDisk, developed by Hu that enables more sensitive detection of target analytes by MALDI mass spec.
NanoDisks feature antibodies to target analytes, providing an upfront enrichment step to improve sensitivity. The structure of the NanoDisks further increases assay performance by boosting the MALDI signal, leading to what Hu said is a sensitivity increase of up to a hundredfold.
This allows the researchers to measure antigens from TB and other mycobacteria that are often present in patient blood samples in low concentrations, Hu noted. In the case of TB, the test measures the levels of two peptides from the established mycobacteria markers culture filtrate protein (CFP) and early secretory antigenic target (ESAT). The researchers selected peptides from the proteins that they established as specific to M. tuberculosis.
Hu and his colleagues introduced the test in a paper published last year in Proceedings of the National Academy of Sciences in which they showed it could identify active TB cases with specificity of 87.1 percent and sensitivity of 90.6 percent in a cohort of 27 active pulmonary tuberculosis cases, 31 latent cases (LTBI), 32 non-tuberculosis mycobacterial infections (NTM), and 21 healthy controls.
In a cohort of HIV/tuberculosis co-infected patients, the test identified 91.3 percent and 82.4 percent, respectively, of culture-positive and culture-negative pulmonary TB cases, and 92.3 percent and 75 percent, respectively, of culture-positive and culture-negative extrapulmonary TB cases.
Based on these findings, Hu and his colleagues set out to investigate the test's performance in a larger group of culture-negative patients, as this group is especially difficult to diagnose and represents a particular challenge for clinicians in developing countries. Culturing from sputum samples is commonly used for TB diagnosis, Hu said, but a significant portion of patients are culture negative despite having active disease, due, in some cases, to the fact that their disease is extrapulmonary.
"No matter how many times you repeat the culture test, [these patients] are always negative, but they [have TB], so the bacteria is somewhere, but it is hard to detect," Hu said. "We believed that as long as you have the bacteria and it is active and secreting antigens, those antigens have a chance to get into the blood circulation."
To look further at the test's performance in this culture-negative population, the researchers collaborated with clinicians at China's Beijing Chest Hospital, looking at serum from 294 prospectively enrolled subjects, 96 without active TB, and 198 with active TB. Of those 198 with active TB, culture tests were positive in just 48 percent of patients, demonstrating, Hu said, the high incidence of culture-negative TB cases.
The clinicians taking the samples and doing the culturing were well trained, he said, "so there is no doubt they are getting reliable results from the culturing."
"But the [high rate of culture-negative cases] is the fact of [TB] in China, and also for many other developing countries," he said. Past reports from the American Thoracic Society and the Centers for Disease Control have put the percentage of culture-negative cases at around 17 percent, though that figure was for pulmonary TB only. A PLOS ONE study looking at data from New York City TB patients found that around 20 percent were culture negative.
In their analysis of the 294 patients published in Clinical Chemistry, the researchers found that the test diagnosed active cases with 88.3 percent sensitivity and 95.8 percent specificity. The test was 91.6 percent sensitive for culture-positive cases and 85.3 percent sensitive for culture-negative cases and performed with 88 percent and 90 percent sensitivity for pulmonary and extrapulmonary cases, respectively.
Hu said he and his colleagues are now planning larger trials they will use to collect data for a regulatory submission. He said they hope to submit the test to the US Food and Drug Administration and China FDA simultaneously, adding that he expects they will be able to take the test to market in China first, hopefully in around three years.
Commercialization of the test will run through NanoPin, Hu said, noting that the company is currently in talks with potential investors to line up initial funding and that he expects to complete a deal in February.
NanoPin will position the test in China and other developing countries primarily for detecting active TB cases. In the US, the company expects the test will be most useful for monitoring patient response to treatment. Hu said that initial results show clear changes in the CFP and ESAT peptides upon treatment, and that the test could be particularly useful for identifying patients who are not responding due to antibiotic resistance, which is a common issue in TB.
NanoPin is also working to develop a broader mycobacterial test based on research published this month by Hu and collaborators in Clinical Proteomics. In that study, they identified peptides from the mycobacterial protein Ag85B that Hu said could be used to detect infections by seven major mycobacteria, covering around 85 percent of the infected patient population.
In addition to development of this pan-mycobacteria test and validating the TB test in larger cohorts, the researchers are refining the NanoDisk system to make it more amenable to clinical implementation, Hu said.
One concern they had, particularly with regard to the developing countries they are targeting, was around access to the MALDI mass spec instrumentation needed to run the test. In the US and Europe, the commercial success of Bruker's MALDI Biotyper clinical microbiology platform (and BioMérieux's Vitek MS) means many hospitals and reference labs already have the instrumentation needed to run such a test.
However, Hu said, it was unclear if that would be the case in countries like China and India. Looking into this question he said he found that "most of the hospitals [in these countries] have a Biotyper, and they also have more advanced MALDI-TOF or LC-MS/MS instruments."
"They do have sufficient funds to purchase the new [instrumentation] as long as it really addresses a fundamental problem in the clinic," he said.