NGS: An innovative approach for detecting, characterizing, and eradicating tuberculosis

Introduction

What is TB?

Tuberculosis (TB) is an infectious disease caused by bacteria in the Mycobacterium tuberculosis complex (MTBC), which includes M. tuberculosis (Mtb). Mtb typically infects the lungs and is spread when infected persons expel bacteria into the air (ie, by coughing). TB can be present as an active infection or as a latent TB infection (LTBI). Nearly 25% of the world’s population is infected with LTBI, during which bacteria lie dormant in the body— unable to cause symptoms or transmit to others— but harbor the potential to awaken and cause TB disease in the future. Patients that develop active TB disease after initial exposure or after latent TB infection experience cough, chest pain, fever, and night sweats. If active TB is left untreated, it can cause serious health problems that can result in death. Risk factors for contracting TB include poverty, undernourishment, HIV infection, diabetes, and living in congregate settings (eg, nursing homes, correctional facilities, and homeless shelters).

Prior to the COVID-19 pandemic, TB was the leading infectious disease killer worldwide. Even still, TB remains the greatest cause of death of HIV-infected persons.

Mycobacterium tuberculosis complex

Mycobacteria are a group of slow-growing, rod-shaped bacteria that can cause a variety of diseases. Certain mycobacteria that cause TB disease in human and animals are grouped into the MTBC. While most human TB cases are caused by Mtb, other MTBC members such as M. bovis, M. africanum, M. canetti, and M. microti can also cause human TB disease. Other mycobacteria, known as nontuberculous mycobacteria (NTM), can cause human pulmonary diseases distinct from TB. While NTM are not usually spread from person to person, the presence of an NTM infection can sometimes complicate the molecular diagnosis of Mtb, given their genetic similarity.

How is TB treated?

Despite being considered a treatable disease, many of the bacterial strains that cause TB have evolved to become resistant to current drug regimens, complicating global eradication efforts. Without treatment, the death rate from active TB disease is about 50% in HIV-negative persons and close to 100% for those living with HIV. However, with combination anti-TB drug treatment regimens, roughly 85% of infected patients can be cured. Available anti-TB drugs can be classified as “first-line” or “second-line” depending on the patient and the profile of TB drug resistance. In general, treatment regimens are predefined; however, drug resistance, contraindication, or intolerance can warrant changes to which drugs are used.

The standard treatment regimen for TB requires a 6-month course of four first-line anti-TB drugs: isoniazid (H), rifampin (R), pyrazinamide, and ethambutol. More recently, the World Health Organization (WHO) has recommended a shorter, 4-month treatment regimen comprised of the first-line drugs rifapentine (a derivative of rifampin), isoniazid (H), pyrazinamide, and moxifloxacin (considered “first-line” in this combinatorial context).

Implications of drug-resistant TB (DR-TB)

What is DRUG-RESISTANT TB?

Drug-resistant TB (DR-TB) refers to a TB infection that is resistant to one or more anti-TB drugs. The ability of TB to resist anti-TB drugs represents a major barrier to TB diagnostic, treatment, and eradication efforts. DR-TB can arise spontaneously over time as Mtb strains acquire random DNA mutations that enable the bacteria to counteract the effects of a given drug. Additionally, DR-TB can be spread from one infected person to another.

The current WHO classifications of DR-TB are determined by the anti-TB drug resistance profiles of Mtb strains. Mono-resistant TB refers to Mtb strains that have gained resistance to a single first-line anti-TB drug, most commonly becoming rifampin-resistant (RR) or isoniazid-resistant (Hr). Poly-resistant TB refers to Mtb that has gained resistance to more than one first-line anti-TB drug, other than both isoniazid and rifampin. Other common types of drug-resistant TB include multidrug-resistant (MDR), pre-extensively drug- resistant (pre-XDR), or extensively drug-resistant (XDR) strains.

WHO-approved definitions for MDR, pre-XDR, and XDR are updated as Mtb gains new mutations and new treatment guidelines are recommended. Currently, MDR-TB refers  to  Mtb that is resistant to both rifampin and isoniazid; pre- XDR-TB refers to Mtb that is resistant to rifampin and at least one fluoroquinolone (either levofloxacin or moxifloxacin); and XDR-TB refers to Mtb that is resistant to rifampin, at least one fluoroquinolone (either levofloxacin or moxifloxacin), and at least one other “Group A” drug (bedaquiline or linezolid)

The standard treatment regimens for the various classifications of DR-TB are updated by the WHO in response to the evolving landscape of TB drug resistance. For example, the anti-TB drug pretomanid was first recommended by the WHO in 2022 as part of a 6-month bedaquiline, pretomanid, linezolid, and moxifloxacin (BPaLM) regimen for MDR/RR-TB and pre-XDR-TB. However, the WHO has acknowledged gaps in the mechanistic understanding of resistance to pretomanid and more research is needed to understand its implications in combatting TB.

Implications of DR-TB on global public health

Historically, Mtb has quickly evolved to be resistant to anti-TB drugs, resulting in increasingly resistant strains of Mtb arising over time. The original main drivers of resistance were low user adherence to treatment regimens, prescription of poor-quality drugs or drugs that didn’t match the TB resistance profile, and prescription of the wrong dosage of antibiotics. These factors contributed to a setting in which Mtb bacteria were exposed to antibiotics at a level that allowed resistance mutations to accumulate but prevented complete clearance of the infection. Today, the incidence of DR-TB is now largely driven by transmission of the resulting DR-TB strains.

Given its ability to rapidly evolve new mutations to resist new treatment regimens, DR-TB is an ever-present and growing threat to public health. DR-TB is much harder and more expensive to treat than drug-sensitive (DS) TB, resulting in an overall potentially more deadly infection  These factors are due in large part to unknown drug resistance profiles limiting informed and accurate treatment regimens.

Traditional and next-generation methods for TB detection and drug resistance profiling

The successful treatment of TB and DR-TB relies on rapid detection and accurate drug resistance profiling. Traditional phenotypic drug susceptibility testing (DST) relies on the slow culture of TB bacteria, which requires several weeks of incubation in Biosafety Level 3 (BSL-3) laboratories. Since many low-income regions lack access to BSL-3 laboratories and resources, culture-free molecular DST represents an attractive TB diagnostic alternative. However, available molecular assays are limited to detecting a restricted panel of specific mutations associated with resistance to only one or a few anti-TB drugs. Since Mtb is continually adapting to new anti-TB drugs and evolving cognate resistance mutations, there is a need for a faster and more accurate method to distinguish drug-resistance properties. Next-generation sequencing (NGS) represents a rapid and precise platform for detecting and combatting DR-TB.

NGS fuels the future of the DR-TB research landscape

The standard NGS workflow

Due to its unique ability to broadly capture genetic regions associated with the presence of Mtb and anti-TB drug resistance, NGS is well-suited for advancing the field of TB research.

NGS involves the sequencing of millions of small fragments of DNA in parallel. These DNA fragments can be from a cultured isolate or directly from the primary sample. Illumina offers several workflows for NGS applications, including whole-genome sequencing (WGS) and targeted amplicon sequencing (tNGS). Illumina WGS and tNGS workflows can be applied to TB genomic investigation, providing crucial and precise TB detection and drug resistance profiling.

A standard workflow for Illumina NGS involves the isolation of genetic material, preparation of sequencing-ready libraries, processing using sequencing instrumentation, and data analysis and interpretation

Applications of NGS towards TB eradication

Illumina WGS and tNGS workflows boast high-quality data accuracy to help researchers gain key insights into public health surveillance (eg, TB outbreak investigation and TB genomic epidemiology) and drug resistance profiling (eg, early emergence of drug resistance).

Illumina recommended workflow: WGS

Sequence-level information on the bacterial genome provides insight into the path of transmission and the antibiotic resistance profile of the pathogen, with the potential to reveal new mechanisms of resistance.

Illumina recommended Workflow: tNGS

Illumina and GenoScreen Deeplex® Myc-TB Combo Kit

Illumina has partnered with GenoScreen to increase the global accessibility of the Deeplex Myc-TB assay, a revolutionary culture-free technology that amplifies 24 TB genomic regions, including 18 drug resistance–associated gene targets, to detect resistance to 15 commonly used anti-TB drugs. This targeted NGS approach provides high sequencing depth for the detection of Mtb, as well as > 100 NTM species, Mtb strain typing, and drug resistance profiling, including highly sensitive detection of hetero- resistant TB subpopulations.

TB research examples: WGS

Public health surveillance	Researchers at the International Institute for Zoonosis Control in Hokkaido, Japan in collaboration with the Zambia National Public Health Institute investigated the recent transmission events of specific MDR-TB strains in Lusaka, Zambia using WGS with the Illumina MiSeq System. From this analysis, they were able to identify in-depth transmission and drug resistance patterns and phylogenetically cluster related resistant strains. Additionally, through WGS, they identified a novel compensatory mutation conferring ethambutol resistance to some strains.
Drug resistance profiling	Microbiologists at the German Center for Infection Research compared the clinical utility of molecular DST and WGS against pDST data. They found that a drug regimen determined by an RT-PCR method (Cepheid GeneXpert), an LPA (Hain’s GenoType MTBDRplus 2.0), and WGS predicted 49%, 63%, and 93% of drug resistances determined by pDST, respectively. Importantly, WGS did not miss any pDST-confirmed resistances and the 7% of cases that did not align between WGS and pDST were cases in which WGS predicted resistance but pDST showed susceptibility. The researchers concluded that this discrepancy was likely due to flaws in the minimum inhibitory concentration (MIC) used for pDST rather than shortcomings in WGS.
Drug resistance profiling	Democratic Republic of the Congo Public health officials conducted the first nationwide survey based on tNGS performed directly on sputum. New and previously treated patients with sputum smear-positive pulmonary TB (n=1661) were tested by the Xpert MTB/RIF assay followed by the Deeplex Myc-TB tNGS assay. The prevalence of rifampicin resistance was low, at 1.8% among new and 17.3% among previously treated patients. Isoniazid resistance prevalence among the RIF- susceptible was 6.6% for new and 8.7% for previously treated patients. Many isoniazid-resistant specimens would be missed by the current algorithm highlighting a potential public health risk.

Choosing the right TB detection method: NGS offers more

When choosing an assay for TB characterization, its important to consider the breadth of coverage provided by each assay and the timeframe for assay completion. Using the Illumina and Genoscreen Deeplex Myc-TB assay, the number of genes detected is maximized while still ensuring rapid time to result