A Rapid and Accurate Detection Approach for Multidrug-Resistant Tuberculosis Based on PCR-ELISA Microplate Hybridization Assay

Ye-Cheng Zhou, MS; Shu-Mei He, MS; Zi-Lu Wen, MS; Jun-Wei Zhao, PhD; Yan-Zheng Song, PhD; Ying Zhang, PhD; Shu-Lin Zhang, PhD


Lab Med. 2020;51(6):606-613. 

In This Article


According to WHO statistics,[26] TB is the most common worldwide cause of death due to a single infectious agent, with one-third of the population infected and 9 million active cases. Anti-TB drugs have substantially reduced the mortality of tuberculosis in the world since their invention during the previous century. However, these gains are seriously jeopardized by the emergence and spread of MDR strains, which pose a significant challenge for effective TB control. Thus, development of low-cost and rapid diagnostic methods for the effective control and prevention of MDR-TB is urgent, particularly for developing countries.[27]

In the present study, we have developed and evaluated a PCR-microplate hybridization assay for rapid and accurate detection of mutations responsible for RIF or INH resistance and prediction of drug susceptibility of MDR M. tuberculosis. Conventional DST and DNA sequencing were used as criterion-standard tests for analyzing the diagnostic performance of the PCR-microplate hybridization assay. Among all 54 isolates, RIF and INH resistances were detected by PCR-microplate hybridization assay, with sensitivity of 93.7% and 87.5%, respectively, and both had specificity of 100%, compared with phenotypic DST. Also, the concordance between PCR-microplate hybridization assay and DNA sequencing for the detection of RIF and INH resistance in MDR isolates was 96.9%.

The results obtained from all susceptible strains by PCR-microplate hybridization assay were 100%; this value was concordant with the sequencing data. It was not possible to identify the genotype at rpoB codon 526 in 1 strain (Table 1, Figure 1) because it displayed hybridization with wt and mutant probes (PW4 and PM4). This ambiguous result occurred probably because the strain is a heterogeneous or mixed population, containing wild-type sequence and mutation at rpoB codon 526.

To our knowledge, this article gives the first description in the literature of PCR-microplate hybridization assay for the identification of MDR-TB isolates from patients. As described in this study report, the screening of mutations in MDR isolates has successfully demonstrated the high sensitivity and specificity of this PCR-microplate hybridization assay.

The novel genotypic assay presents several advantages compared with classical and other molecular detection methods. First, besides the high sensitivity and specificity, the distinct advantage of PCR-microplate hybridization assay compared with phenotypic assays is in reduced turnaround time. The entire procedure, including PCR amplification, hybridization, and ELISA detection, requires only 4 to 5 hours, completing the detection of RIF and INH resistance in MDR isolates, compared with the multiple weeks required with phenotypic tests. Therefore, use of this assay allows for the more timely implementation of an appropriate TB treatment regimen.

Second, this assay is easy to perform and allows the simultaneous handling of a large number of specimens. This probe technique is performed using the routine microplate format and colorimetric detection. However, the 96-well plate format facilitates the manipulations and enables increased specimen throughput. In contrast to other molecular tests, PCR-microplate hybridization assay does not require the use of expensive equipment, such as real-time PCR or DNA sequencing; instead, it uses basic, inexpensive instruments and commonly available reagents that are already present in most diagnostic laboratories. It is essential that development of new techniques consider the fact that most drug-resistance cases occur in resource-poor countries—therefore, the methods must not only be inexpensive but also convenient. The simplicity of the assay we describe herein allows the methodology to be used in less-endowed laboratories in less-developed countries.

Third, there are currently 2 products commercially available for laboratory-based RIF and INH resistance screening: the INNO-LiPA Rif. TB assay (Fujirebio Diagnostics Inc.) and the GenoType MTBDRplus assay (Hain Lifescience GmbH). The INNO-LiPA assay (which detects only RIF resistance) provides resistance information within 48 hours, whereas the turnaround time for the MTBDR assays (RIF and INH resistance) is 6 hours to 2 days.[12] However, these assays have limited utility in the developing countries where TB is most prevalent because of their relatively high cost. In other words, the vast majority of settings where drug-resistant TB is emerging will likely not benefit widely from the availability of these tests. Thus, the development of the PCR-microplate hybridization assay offers a viable alternative to the currently available testing options.

In conclusion, the result of this study show that PCR-ELISA is a rapid, inexpensive, convenient, and reliable test for screening potential MDR strains and characterizing mutations in codon 315 of katG, at position -15 of the inhA regulatory region, and in the 81-bp hot spot region of rpoB. The application of this method to rapidly detect MDR-TB will be useful in improving national and global clinical care to ensure stronger control of TB in patients.