Scientists from a number of Chinese research institutions and government agencies have published the first peer-reviewed paper describing the clinical validation of a tuberculosis drug-resistance assay developed by Xiamen-based molecular diagnostics firm Zeesan Biotech.
The assay, based on a real-time PCR method called multicolor melting curve analysis (MMCA) developed by Xiamen University and licensed to Zeesan, demonstrated a clinical sensitivity and specificity of 90.8 percent and 96.4 percent, respectively, compared to bacterial cultivation in a study of more than 1,000 clinical isolates from three geographically distinct tuberculosis centers.
The assay is one of two MeltPro drug-resistance tests recently approved by the Chinese State Food and Drug Administration, and three additional Zeesan tests for different drug resistances are currently under review at the agency, Qingge Li, a professor of molecular diagnostics at Xiamen University and corresponding author on the new study, told PCR Insider in an email.
In addition, Li said, Zeesan also earlier this year released its own brand of real-time PCR instruments with dedicated software to perform the assays, and is moving toward commercializing an integrated molecular diagnostic system containing a package of rapid molecular TB assays.
Zeesan's SFDA-approved assays — MeltPro TB/INH and MeltPro TB/RIF for detecting isoniazid and rifampin resistance mutations, respectively — as well as its pending assays for ethambutol, streptomycin, and fluoroquinolones all leverage the MMCA method, first described in 2011 in a paper published in PLOS One.
The technique is an extension of melt curve analysis, a well-known and highly sensitive technique for analyzing the melting characteristics of different PCR products in order to determine the presence of mutations. However, unlike standard melt curve analysis, which uses adjacent probes that need dedicated Förster resonance energy transfer detection channels, the MMCA technique uses dual-labeled, self-quenched probes in the form of either a hairpin or linear probes, which allows for the use of common real-time PCR instruments.
Xiamen's Li, who works closely with Zeesan on commercial projects, told PCR Insider last year that the tuberculosis tests were just a few of a stable of assays that the company has been developing for things like beta-thalassemia mutation detection and human papillomavirus genotyping.
However, the company's tuberculosis work seems to have progressed the farthest to this point, and the new study, published this month in the Journal of Clinical Microbiology, presents data from a study carried out in 2010 and 2011 specifically on the MeltPro TB/INH assay, Li said.
In the study, researchers from Xiamen University, the Shenzhen Center for Chronic Disease Control, the Xiamen Center for Disease Control and Prevention, and other institutions enrolled 1,096 clinical isolates collected from tuberculosis centers in southern, central, and northern China, including 437 INH-resistant and 659 INH-susceptible isolates characterized by traditional drug susceptibility testing.
Besides demonstrating that the assay had a sensitivity of 90.8 percent and 96.4 percent, the researchers also used DNA sequencing to show that, except for five mutants outside the detection range of the MeltPro assay, there was 99.1 percent concordance between MeltPro TB/INH and drug susceptibility testing.
In addition to demonstrating the assay's clinical utility, the researchers also showed that it could be run on five different types of real-time PCR machines, with the shortest running time — 105 minutes — obtained using Roche's LightCycler 480 II.
"Based on this paper, several significant features of MeltPro assays can be seen, such as the wide coverage of drug-resistance mutations, heteroresistance mutation detectability, and cross-platform compatibility, which are commonly unmet by existing assays," Li said in his email.
The study also revealed several limitations of MeltPro TB/INH that deserve improvement, the authors noted. First, the researchers did not find an isolate harboring a specific mutation, inhA S94A, among the 437 INH-resistant isolates, raising the idea that it might not be necessary to include this mutation in the assay. This is a particularly important point considering the mutation necessitated a dedicated probe and fluorescence detection channel.
In addition, the two regions of the ahpC promoter detected by two FAM-labeled probes led to a complex merged melting curve that was difficult to analyze when more than one mutation occurred — a situation that was exacerbated in the case of heteroresistant mutations.
And finally, the researchers noted, the evaluation was restricted to clinical isolates of bacteria. The performance of the assay with real clinical samples, such as sputum, remains to be validated. However, they added, current analysis of drug resistance in Mycobacterium tuberculosis is routinely conducted following the cultivation step to confirm TB infection. "Therefore, we can at least recommend that the MeltPro TB/INH assay be used for rapid detection of INH resistance status according to the current protocol," the authors wrote.
Li and colleagues have already begun addressing many of these possible shortcomings, including recently finishing another on-site validation study that recruited from more than 10 hospitals, Li noted.
"This study evaluated different MeltPro assays using mainly sputum samples together with [a] cross-reaction study with a large number of non-tuberculosis Mycobacteria," Li said. "In addition, the MelPro XDR TB assays are now under evaluation in three major TB centers of China sponsored by the National Center for Tuberculosis Control and Prevention of China CDC and under the support of the China-Gates Foundation TB Cooperation Program."
Li said that this evaluation study includes a Zeesan-made automatic DNA extraction machine that allows 20 sputum samples to be treated within one hour. By the end of the evaluation, the researchers will have tested nearly 2,000 sputum samples using MeltPro and compared the results with culture methods.
Finally, Zeesan is currently conducting official clinical validation of the TB DNA assay, as well as an assay for resistance mutations to injectable second-line drugs.
"Moreover, through collaboration with [the] Engineering Research Center of Molecular Diagnostics, Ministry of Education — an affiliate [of] Xiamen University — a new program for the development of a new generation of TB typing assay is to be launched within this month under the support of the National High Technology Research and Development Program," Li said.