Exploring Diagnostic Methods for Drug-Resistant Tuberculosis

Sanchini, A., Lanni, A., Giannoni, F. and Mustazzolu, A., 2024. Exploring Diagnostic Methods for Drug-Resistant Tuberculosis: A Comprehensive Overview. Tuberculosis, p.102522.

Laboratories are well-equipped to test resistance to established drugs like fluoroquinolones (FQs), linezolid (LZD), and second-line injectable drugs (SLIDs) such as amikacin (AMK) and kanamycin (KAN).

However, they are less prepared for novel drugs, like bedaquiline (BDQ), which are crucial for treating multidrug-resistant tuberculosis (MDR-TB).

Phenotypic drug susceptibility tests (pDSTs) classify bacteria as resistant if they grow in the presence of a drug, indicating the drug is ineffective.

Some pDSTs can determine the minimum inhibitory concentration (MIC) of a drug, while others only test for critical concentration (CC), determining whether a bacteria is susceptible or resistant.

pDSTs require a positive culture of Mycobacterium tuberculosis (MTB), which takes 2-3 weeks, and the tests need to be conducted in a biosafety level 3 laboratory.

Obtaining drug susceptibility results can take an additional 2-3 weeks, totaling 4-6 weeks from sample collection to pDST results.

The gold standards for pDST are the agar proportion method (on solid medium like Lowenstein-Jensen or 7H11 Middlebrook) or the Mycobacterium Growth Indicator Tube (MGIT) liquid medium, with MGIT being faster and more common.

The BACTEC MGIT 960 is a fully automated system that detects mycobacterial growth using fluorescence, testing only one drug concentration (CC) and categorizing isolates as susceptible or resistant.

The Sensititre system can determine the MIC of 12 anti-TB drugs using the microdilution method on a 96-well plate.

A colorimetric assay, such as the Crystal Violet Decolorization Assay, can detect resistance to rifampicin (RIF) and isoniazid (INH) with high sensitivity and specificity.

The microplate Alamar Blue Assay tests for BDQ resistance using resazurin dye, which turns pink and fluorescent when reduced by cellular metabolism. This assay found BDQ-resistant isolates without plausible resistance mutations that would have been misclassified by gene sequencing alone.

The Thin-Layer Agar (TLA) method for resistance detection involves inoculating a thin layer of agar with clinical samples, where growth in drug-containing sections indicates resistance. This method is promising for low-resource settings or field conditions.

Molecular drug susceptibility tests (DSTs) for detecting drug resistance in Mycobacterium tuberculosis (MTB) are based on identifying mutations in resistance-associated genes.

Molecular DSTs are simpler and faster than phenotypic DSTs (pDSTs) and can be applied directly to clinical samples without needing prior culture or a biosafety level 3 laboratory.

The main limitation of molecular DSTs is that they can only detect known mutations, potentially missing new, unknown, or rare mutations that confer drug resistance. Additionally, molecular DSTs cannot determine minimum inhibitory concentrations (MICs).

The gold standard molecular DSTs for detecting drug resistance in MTB are the PCR-based Xpert MTB/RIF and Xpert MTB/RIF Ultra (for detecting TB and rifampicin [RIF] resistance), endorsed by the World Health Organization (WHO).

Line probe assays (LPAs) such as GenoType MTBDRplus (Hain LifeScience) and the Nipro NTM + MDRTB detection kit (Nipro Corp.) are used for testing resistance to RIF and isoniazid (INH). The GenoType MTBDRsl (Hain LifeScience) is used for testing fluoroquinolones (FQs) and aminoglycosides.

The Xpert MTB/RIF system, developed by Cepheid, can identify MTB and its resistance to RIF from respiratory samples in approximately 90 minutes. This system uses automated nested real-time PCR, targeting mutations in the rpoB gene.

Xpert MTB/RIF Ultra improves sensitivity for RIF resistance detection by incorporating additional targets and lowering the detection limit compared to the original Xpert MTB/RIF.

The Xpert MTB/XDR system is approved for testing INH, FQs, amikacin (AMK), kanamycin (KAN), and ethionamide (ETH) after a positive result from Xpert MTB/RIF or Xpert MTB/RIF Ultra.

Xpert MTB/RIF used on bronchoalveolar lavage (BAL) samples shows a sensitivity of 87.0% and specificity of 92.0%. BAL samples are a useful alternative to sputum, especially in cases of sputum smear-negative results or when sputum production is insufficient.

The Xpert MTB/XDR assay can detect heteroresistance and serves as a useful follow-up test for MTB-positive samples to diagnose primary resistance in MDR-TB isolates.

The GeneXpert Omni system is a portable, point-of-care (POC) molecular diagnostic tool designed for field settings and remote areas. It is battery-powered, cloud-connected, and performs well under stressful field conditions.

The GenoType MTBDRplus system, developed by Hain Lifescience, identifies MTB complex and its resistance to RIF and INH directly from both pulmonary samples and isolated cultures using LPA, PCR amplification, and reverse hybridization. Turnaround time (TAT) is approximately five hours.

The GenoType MTBDRsl (Hain Lifescience) detects resistance to second-line drugs (SLDs) such as FQs (e.g., ofloxacin [OFL] and moxifloxacin [MOX]) and second-line injectable drugs (SLIDs) like KAN, AMK, and capreomycin (CAP).

The BD MAX™ MDR-TB test is an automated system that extracts DNA and performs real-time PCR to detect MTB and resistance to RIF and INH from respiratory samples.

BD MAX showed a sensitivity of 90.0% and specificity of 95.0% for RIF resistance, and sensitivity of 81.5% and specificity of 100% for INH resistance, compared to pDST.

The BD MAX system processes up to 24 specimens in one round, with a TAT of approximately four hours from start to results, making it a promising tool for drug-resistance testing. 

Whole-genome sequencing (WGS) analyzes all genes and compensatory mutations, and sequencing data can be analyzed retrospectively if new resistance genes or mutations are discovered.

Disadvantages of WGS include the need for a positive Mycobacterium tuberculosis (MTB) culture (typically a flagged positive MGIT culture), the requirement for complex bioinformatics expertise, high costs (around 100 EUR per sample), and labor-intensive laboratory work.

To reduce turnaround time (TAT), sequencing can be performed directly on sputum samples, but this may lower sensitivity due to contamination or low bacterial load.

Next-generation sequencing (NGS) approaches, such as targeted sequencing of specific DNA regions, provide higher depth of coverage.

Several platforms are available for WGS, including Illumina, Ion Torrent, PacBio SMRT RSII, and Oxford Nanopore MinION.

Various bioinformatic tools are available to predict drug resistance profiles from raw WGS data, such as KvarQ, TBProfiler, CASTB, Mykrobe Predictor, PhyResSE, Resistance Sniffer, and Treesist-TB.

Benefits of replacing routine phenotypic DST (pDST) with WGS include reduced TAT, shorter time on inappropriate treatment, reduced treatment duration, fewer patients in treatment, and cost savings (up to £7.2 million in 10 years).

Both the MiSeq and MinION platforms have similar TAT (approximately three days), but MinION is more flexible as sequencing can be terminated once sufficient data is collected. MinION has a lower cost, at 57 USD per sample compared to 130 USD with MiSeq.

MinION's portability, flexibility, and cost-effectiveness make it valuable for clinical settings.

The QuantaMatrix Multiplexed Assay Platform (QMAP) is an assay based on reverse hybridization, using magnetic microparticles to identify resistance to RIF, INH, EMB, FQs, and SLIDs within six hours from a positive MTB culture.

MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) uses a laser to ionize molecules and analyze them by mass, useful for detecting resistance mutations. The MassARRAY system identifies resistance mutations for streptomycin (SM), RIF, INH, EMB, and FQs.

Key factors for choosing a drug-resistance test for MTB include rapid TAT, simplicity of procedure, cost, equipment requirements, and test sensitivity and specificity.

BACTEC MGIT 960 remains a widely used pDST method, offering susceptibility testing for a full panel of drugs, though it requires significant investment in installation, maintenance, and ongoing costs, which can be a barrier in low-resource settings.

MGIT supports WGS by helping interpret results and identifying new resistance mutations, though there is contamination risk during sample handling in culture-based methods.

Common molecular tests include the Hain MTBDRplus, MTBDRsl, and GenXpert systems, but WGS offers the most comprehensive and reliable resistance profiling, along with molecular typing for identifying clones and clusters.

Regional centers for WGS could support peripheral hospitals and labs by allowing them to send strains or extracted DNA for sequencing.

MALDI-TOF is a valid competitor to WGS, offering similar information but with limitations related to instrument costs and the need for skilled personnel to interpret results.