In a marked departure, researchers have used a rapid blood test that relies on two Mycobacterium tuberculosis-specific peptide fragments for diagnosis of TB disease and monitoring treatment. Currently, sputum samples are used for diagnosing TB disease in the case of pulmonary TB and tissue samples in the case of extra-pulmonary TB. The blood test accurately detects minute levels of two biomarkers — CFP-10 and ESAT-6 — that are “actively secreted” by the bacteria when it causes TB disease. Currently, the assay costs less than $10.
In a pilot study, the new blood test was able to diagnose active TB cases with “high sensitivity and specificity”. It was able to diagnose active TB even in people who were coinfected with HIV. The results were published in a paper published today (March 28) in the Proceedings of the National Academy of Sciences (PNAS) by Chang Liu from Houston Methodist Research Institute. Dr. Liu is currently at Arizona State University.
The blood-based assay was able to provide quantitative results that will help in knowing the severity of active TB and in monitoring treatment outcomes. Unlike Xpert, it cannot detect rifampicin resistance.
The blood-based assay was able to diagnose both pulmonary and extra-pulmonary TB cases with high sensitivity — over 91% in the case of culture-positive pulmonary TB (PTB) and above 92% extra-pulmonary TB (EPTB), and 82% in culture-negative PTB and 75% in EPTB in HIV-positive patients. In the case of HIV coinfected cases, the sensitivity was 87.5% for PTB and 85.7% for EPTB cases. It also had high specificity (87-100%) in both healthy and high-risk groups.
“We want to detect only active TB, but not latent TB, so we selected CFP-10 and ESAT-6. However, we believe these two biomarkers are capable of detecting early activation of latent TB, but we are conducting more experiments to confirm that,” says Dr. Liu in response to a question on the choice of the two peptide fragments.
According to a 2014 WHO report, there is a need for a “rapid biomarker based non-sputum-based diagnostic test that uses an easily accessible sample and is able to accurately diagnose pulmonary TB (and ideally also extrapulmonary TB)”.
Obtaining sputum samples is not always easy and is particularly difficult in the case of little children and people who are HIV positive. About 15-25% of all TB cases are extrapulmonary and biopsy samples are needed in such cases. Even Gene Xpert, introduced a few years ago to improve sensitivity and specificity, relies on sputum samples, and as per a 2014 WHO update, Xpert has “very low quality evidence” for EPTB diagnosis. Also, cerebrospinal fluid or other samples are needed for diagnosing EPTB using Xpert.
The blood-based TB diagnostic assay will be go a long way in the war against TB, particularly in diagnosing TB in little children, people with HIV and extra-pulmonary TB cases.
The blood sample is first microwave irradiated for about 20 minutes, and the target peptides are enriched using a nanoparticle enrichment platform and a high-throughput mass spectroscopy for enhancing the detection of Mycobacterium-specific biomarkers. It take some about four hours to prepare a serum sample and 10 minutes to know if the two peptides are present in the blood.
“CFP-10 and ESAT-6 are also expressed by some other mycobacteria, they cause NTM infection, not TB. We discovered peptides in CFP-10 and ESAT-6 that are specific to TB. So we digest the two proteins [using microwave irradiation] before diagnosis,” Dr. Liu, who is the first author of the paper, says in an email. “NanoDisk is functionalized with antibody. Their capture and isolate the peptide targets from patient serum sample. In addition, due to their special material and nanostructure, they can enhance the signal of mass spectrometry during detection.”
“We are particularly excited about the ability of our high-throughput assay to provide rapid quantitative results that can be used to monitor treatment effects, which will give physicians the ability to better treat worldwide TB infections,” said Prof. Ye Hu from the Houston Methodist Research Institute and the Corresponding author of the paper said in a release. Prof. Hu is currently at Arizona State University. “Furthermore, our technology can be used with standard clinical instruments found in hospitals worldwide.”
According to the authors, the NanoDisk-MS assay meets several of the WHO criteria for a noninvasive TB assay — “it uses a small, non-invasive specimen; does not require bacterial isolation; has high sensitivity and specificity for active TB cases in extrapulmonary, culture-negative, and HIV-infected TB patients; and directly quantifies Mtb antigens for rapid monitoring of anti-TB therapy effects”.
The assay was able to detect marked reduction in the peptides levels in both HIV-positive and HIV-negative TB cases that were started on TB treatment. But more studies need to be carried out to evaluate the performance of NanoDisk-MS assay in treatment monitoring as the pilot study was not designed to measure the rate of decline of TB bacteria with treatment.
“We have tested 21 patients (HIV- and HIV+) with multiple longitudinal samples, and were able to see biomarker decrease correlated to treatment in 19 of them. Larger clinical validation is underway [to know treatment resistance and therapeutic efficacy],” he says.
The researchers note that larger, randomised studies are needed to confirm the results of the pilot study.
“Any blood-based, rapid TB diagnostic assay is ideal and has huge potential as it does not depend on sputum samples [for pulmonary TB], and tissue samples in the case of extra-pulmonary TB. But many studies based on blood-based assays have not been successful earlier,” says Dr. Soumya Swaminathan, Director-General of ICMR, Delhi. “High specificity [correctly identify those with the disease] is very important and so large-scale tests have to be carried out in countries like India where a large population has latent TB infection.”