NEW YORK – Researchers associated with infectious disease diagnostics firm NanoPin Technologies have published additional data supporting the performance of its blood-based mass spectrometry-based test for tuberculosis.
The data, presented in a study published last week in BMC Medicine, demonstrates that the assay, which NanoPin plans to launch as a laboratory-developed test near the end of 2021, could be useful for the early detection of TB and monitoring of treatment in children.
The study looked at samples from 519 infants enrolled in a trial for the TB drug isoniazid conducted in southern Africa who were followed for up to 192 weeks to determine if they were infected with TB. Among HIV-infected infants (284 total), the test, which measures levels of the Mycobacterium tuberculosis CFP-10 virulence factor, had 100 percent sensitivity for culture-confirmed TB and 84 percent sensitivity for unconfirmed but suspected TB. Among subjects without HIV, the test detected the one culture-confirmed TB case and had 75 percent sensitivity for unconfirmed but suspected cases. It performed with 93 percent specificity in the HIV-positive subjects and 96 percent specificity in HIV-negatives subjects.
Diagnosis of pediatric TB is a particular challenge, said Tony Hu, director of the center of cellular and molecular diagnosis at Tulane University and senior author on the study. Hu founded New Orleans-based NanoPin in 2017 and currently serves as a scientific adviser to the company.
One of the main challenges is that sputum samples, which are required for sputum culture — generally considered the gold standard in TB diagnosis — are difficult to collect in the pediatric population.
Another issue is "the majority of pediatric TB is extra-pulmonary," Hu said, noting that this means that the pathogen may not be detectable in sputum.
At the same time, TB often progresses more quickly in children than adults due to their less-developed immune systems and the extra-pulmonary nature of many pediatric TB cases, making the need for more effective methods of diagnosis especially urgent, Hu said.
Hu initially developed the test for use on a MALDI mass spec platform while he was a researcher at Arizona State University's Biodesign Institute. The test used an enrichment device called the NanoDisk that used antibodies to capture targets prior to mass spec analysis to provide the sensitivity needed to measure antigens from TB and other pathogens in patient blood samples.
NanoPin has since shifted to an LC-MS system for its work, a move that Hu said was necessitated by the fact that the MALDI-based platform (which Hu and his colleagues still used for the BMC Medicine study) did not provide the necessary performance when samples were collected in the plastic tubes commonly used for sample collection.
The earlier phases of assay development were done using samples that had been collected in glass tubes.
"Glass tubes are expensive, but they are super clean, and we could clearly observe the signal in those samples, so they were pretty friendly for MALDI," he said.
When using samples collected in plastic collection tubes, however, the researchers found that "without further separation some of the polymer contamination [from the tubes] would cause huge background on MALDI," Hu said.
This led the researchers and NanoPin to move to LC-MS, where they could use upfront LC separation to lessen the background caused by the plastic collection devices. The assay still uses antibody-based enrichment of the target CFP-10 peptide. It also enriches at the same time spiked in isotope-labeled synthetic CFP-10 peptide standards. Both sets of peptides elute off the LC column at the same time with the synthetic peptide standards producing a high-intensity mass spec signal that can be used to confirm the elution time and to quantify the levels of TB-generated CFP-10, which may be present in very low levels in patient blood.
Hu said the company is working with both Thermo Fisher Scientific and Danaher firm Sciex on implementing high-throughput LC-MS workflows for the assay and that NanoPin was aiming to be able to process around 100 samples per day. Last year, Thermo Fisher and NanoPin announced a collaboration to develop mass spec workflows for infectious disease diagnosis and monitoring.
Given that TB remains an issue primarily in the developing world, access to mass spec technology could prove a challenge to the test's adoption, though Hu said he believed a model where samples are shipped to a central laboratory would prove effective. He added, though, that the company is exploring point-of-care detection approaches including miniaturized mass spectrometry systems and nanopore-based protein sequencing.
"I think the LDT version is very necessary to enable high-throughput screening," he said. "Point-of-care devices could help serve patients in resource-limited areas, but they have limited capabilities to run in a high-throughput mode."
Hu said he envisions a number of uses for the test, perhaps most prominently as a tool for monitoring patients with known latent TB to enable early detection of individuals who are shifting into active infection. Another potential use is monitoring patients being treated for the disease to assess their response to treatment. In the BMC Medicine study, the researchers found that CFP-10 positivity and concentration levels declined in patients following anti-TB therapy.
NanoPin CEO Thomas Tombler said the company aims to launch an LDT version of the test out of its CLIA facility by the end of the year. He said the test will target all patient groups but that the company will emphasize detection of active TB in high-risk populations including children and those with extra-pulmonary forms of the disease.
He added the company is pursuing clinical studies to support a US Food and Drug Administration 510(k) application for a kit version of the test with the goal of launching such a product in 2023.
NanoPin also plans to launch in June a COVID-19 LDT using a CRISPR-based approach licensed from Hu's lab that has demonstrated higher sensitivity than existing RT-PCR assays.
Tombler said the company believes the test could find a place "in clinical applications where patients may be presenting with severe symptoms of COVID-19 but consistently test negative for the disease using nasal swab PCR."