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

Results

Comparison of PCR-microplate Hybridization Assay With Phenotypic Drug-Susceptibility Testing

The performance of PCR-microplate hybridization assay was compared with phenotypic drug-susceptibility testing using the clinical strains for which RIF or INH drug susceptibility testing results are available (Table 1). Five wild-type (wt) probes and 3 mutant probes were used to determine resistance to RIF. Of the 32 total MDR strains, 30 containing rpoB mutations in the RRDR (approximately 93.7%) were identified by the PCR-microplate hybridization assay. In all, 24 isolates (75.0%) showed a clear hybridization signal with the corresponding mutant probes but lack of hybridization with the respective wt probe; therefore, their mutation genotypes could be correctly identified. Hybridization results of 5 MDR isolates (WH32, WH33, WH35, WH64, and WH73) were negative. Only 1 isolate, WH64, presented a simultaneous absence of hybridization with wt and mutant probes that targeted the specific region, thereby indicating a mutation within that region. A specific capture probe for this mutation was not tiled on the array.

Two MDR strains, WH39 and WH60, had no mutations in rpoB confirmed by DNA sequencing (Table 1), although these strains were phenotypically resistant to RIF. The most frequently encountered mutation of amino-acid position was at 531 (19 isolates; 59.4%), followed by 526 (3 isolates; 9.4%) and 516 (2 isolates; 6.2%). Regarding the relative frequencies of mutation genotype in these codons, S531L (TCG→TTG) is the predominant pattern.

None of the isolates in this study were found to have a mutation in the region of rpoB covered by RW1 and RW3 probes. All of the 22 susceptible isolates hybridized with the corresponding wt probes, and none showed any signals with the mutation-specific probes, thereby demonstrating that no mutation was found in those isolates. The sensitivity and specificity of the PCR-microplate hybridization assay were 93.7% and 100%, respectively, for detecting RIF resistance, compared with conventional DST.

For analysis of resistance to INH, 32 MDR strains were investigated by a PCR-microplate hybridization test of a 160-bp katG fragment covering codon 315; in total, 26 strains (81.2%) had mutations at that codon. The wild-type codon AGC (Ser) was altered to ACC (Thr) or AAC (Asn) in 24 strains (75.0%). Two strains, WH50 and WH83, which showed mutation S315T (AGC→ACA) and mutation S315G (AGC→GGC), respectively, via sequencing analysis, displayed a simultaneous absence of hybridization with KW and KM probes, indicating the presence of mutation within the region corresponding to the probe. Due to the specific capture probe for this mutation not being designated on the array, we were unable to determine the mutant genotype using PCR-microplate hybridization assay.

Also, of the 4 INH-resistant strains (WH32, WH54, WH72, and WH73) identified with mutation at the position −15C→T in the inhA regulatory region, 2 strains (WH54 and WH72) had a mutation accompanying the katG mutation at codon 315. In 4 strains (WH23, WH27, WH63, and WH70), the INH resistance mechanism was not due to a mutation in katG codon 315 or inhA-15 (Table 1). Therefore, these strains were not detected by PCR-microplate hybridization assay as being INH-resistant.

In analysis of the PCR-microplate hybridization assay, 87.5% (28/32) of INH-resistant isolates harbored at least 1 mutation at katG codon 315 or inhA -15; no such mutations were detected in the 22 susceptible strains. Compared with phenotypic DST, the sensitivity and specificity of PCR-microplate hybridization assay were 87.5% and 100%, respectively, for detecting INH resistance.

Comparison of PCR-microplate Hybridization Assay With DNA Sequencing

DNA sequencing data, combined with the results of PCR-microplate hybridization assay for detecting mutations conferring RIF and INH resistance, are shown in Table 1. Due to the virtually binary responses of the probes to mutations, visual discrimination is straightforward. Among 31 of 32 MDR strains, including 5 strains that had simultaneous absence of hybridization with wt and mutant probes targeting the RRDR, mutations in the RRDR of rpoB, as detected by PCR-microplate hybridization assay, were in accordance with sequencing results. One strain, WH54, which showed wild-type genotype at rpoB codon 526, as determined by sequencing analysis, displayed hybridization with RW4 and RM4 probes (Table 1, Figure 1). Also, the genotype at the rpoB codon 526 was not identified using the PCR-microplate hybridization assay because multiple hybridization signals were observed.

Figure 1.

The design of microtiter plate format and detection of mutation using polymerase chain reaction–enzyme-linked immunosorbent assay (PCR-ELISA). WH1, WH36, WH25, and WH54 are specimen numbers. A, Using specimen WH1, No 1 WH clincal isolate. B, Using specimen WH1, No 1 WH clincal isolate. C, Using specimen WH36, No 36 WH clincal isolate. D, Using specimen WH36, No 36 WH clincal isolate. E, Using specimen WH25, No 25 WH clincal isolate. F, Using specimen WH25, No 25 WH clincal isolate. G, Using specimen WH54, No 54 WH clincal isolate. H, Using specimen WH54, No 54 WH clincal isolate. RW1-5 represents wild-type probes of the rpoB gene; RM2,4,5, mutant-type probes of rpoB; KW, wild-type probe of katG; RM, mutant-type probe of katG. IW, wild-type probe of inhA; IM, mutant-type probe of inhA.

For the analysis of mutation responsible for INH resistance, 32 MDR-TB strains with a variety of hybridization patterns, including 2 strains with lack of hybridization with KW and KM probes, were sequenced. In addition, the sequencing results were in line with the data obtained via PCR-microplate hybridization assay. No mutations in the rpoB, katG, and inhA regulatory regions were detected in the 22 susceptible strains, via PCR-microplate hybridization assay and sequencing.

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