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

Disclosures

Lab Med. 2020;51(6):606-613. 

In This Article

Abstract and Introduction

Abstract

Rapid and accurate diagnosis of multidrug-resistant tuberculosis (MDR-TB) is important for timely and appropriate therapy. In this study, a rapid and easy-to-perform molecular test that integrated polymerase chain reaction (PCR) amplification and a specific 96-well microplate hybridization assay, called PCR-ELISA (enzyme-linked immunosorbent assay), were developed for detection of mutations in rpoB, katG, and inhA genes responsible for rifampin (RIF) and isoniazid (INH) resistance and prediction of drug susceptibility in Mycobacterium tuberculosis clinical isolates. We evaluated the utility of this method by using 32 multidrug-resistent (MDR) isolates and 22 susceptible isolates; subsequently, we compared the results with data obtained by conventional drug susceptibility testing and DNA sequencing. The sensitivity and specificity of the PCR-ELISA test were 93.7% and 100% for detecting RIF resistance, and 87.5% and 100% for detecting INH resistance, respectively. These results were comparable to those yielded by commercially available molecular tests such as the GenoType MTBDRplus assay. Based on the aforementioned results, we conclude that the PCR-ELISA microplate hybridization assay is a rapid, inexpensive, convenient, and reliable test that will be useful for rapid diagnosis of MDR-TB, for improved clinical care.

Introduction

Tuberculosis (TB) remains the most prevalent cause worldwide of death from an infectious disease. In 2017, there were an estimated 10 million new cases of TB and 1.3 million deaths from TB.[1] Multidrug-resistant tuberculosis (MDR-TB), which was caused by Mycobacterium tuberculosis strains resistant to at least the drugs isoniazid (INH) and rifampin (RIF), has contributed to several fatal outbreaks worldwide and is an increasing threat to global TB-control programs, particularly in developing nations.[2]

Due to the emergence of MDR-TB, a great deal of attention has been paid to exploring the molecular mechanisms of drug resistance in M. tuberculosis and developing more rapid molecular tests for detection of drug-resistant TB.[3,4] The results of molecular genetic studies[5,6] have revealed that nearly 95% of RIF-resistant strains have mutations within the 81-bp determining rifampin resistance-determining region (RRDR) in the rpoB gene, encoding the β-subunit of the RNA polymerase. In contrast, mutations in the katG gene (60–70% of INH-resistant strains) encoding the catalase-peroxide involved in INH activation,[7–9] and the regulatory region of the inhA gene (20–35% of INH-resistant strains) encoding an enzyme involved in mycolic acid biosynthesis,[10] are mainly responsible for INH resistance. Therefore, mutations in these genes associated with RIF and INH resistance can serve as a useful marker for the diagnosis of MDR-TB. Development of rapid, reliable, and inexpensive diagnostic methods based on this knowledge is urgently needed.

The need to minimize the transmission of multidrug-resistent (MDR) strains requires rapid identification procedures. Conventional drug susceptibility testing (DST) using culture techniques requires a minimum of 7 to 10 days, and sometimes as many as 8 to 12 weeks for a conclusive result,[11] delaying the delivery of chemotherapy to patients.

With the advent of modern molecular biology tools, many molecular methods have been applied to identification of gene mutations in relation to drug resistance, which include restriction fragment length polymorphism (RFLP),[12,13] single-strand conformation polymorphism (SSCP),[14,15] DNA sequencing,[16] DNA microarrays,[17] real-time polymerase chain reaction (PCR),[18] and line probe assays such as the commercially available GenoType MTBDRplus assay (Hain Lifescience GMBH)[19] for rapid detection of MDR-TB. Among these methods, RFLP or SSCP analysis of PCR amplicons could only determine whether mutations exist; these methods do not have the ability to detect the location and the types of mutation. Use of additional expensive equipment, cumbersome procedures, or highly expensive measures, such as DNA sequencing, real-time PCR, and DNA microarrays, are impractical in the laboratory. Although the GenoType MTBDRplus assay has reasonable sensitivity and specificity for detecting MDR-TB, it is expensive and requires special instrumentation.

Several specific genes, where many mutations conferring RIF and INH resistance have occurred, have been chosen for rapid detection of MDR-TB; those genes are rpoB, katG, and the inhA regulatory region. PCR-microplate hybridization assay has been demonstrated[20] to be an effective technique that provides sensitive and specific and rapid detection for viral DNA sequence variations. This technique involves labeling of amplicons during PCR, hybridization with probes specific for the target, and capture of labeled hybrids onto microplates and detection by immunoassay. Although the PCR-microplate hybridization assay for detection of RIF resistance in M. tuberculosis was reported by Garcia et al,[21] probes for screening the mutations of genes related to INH resistance in M. tuberculosis were not included. In this study, we optimized the conditions of the PCR-ELISA microplate hybridization assay and used this assay for detection of MDR-TB, compared with DNA sequencing and conventional DST for detection of MDR-TB strains isolated from patients now living in China.

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