Molecular diagnosis of tuberculosis: we need solutions that span the healthcare value chain

KEYWORDS:

• Tuberculosis
• molecular diagnostics
• Xpert MTB/RIF
• line probe assays
• next-generation sequencing

1. Introduction

With over 10 million new tuberculosis (TB) cases occurring each year, TB continues to be one of the most important infectious threats, primarily affecting low- and middle-income countries (LMICs) [1]. Without early and accurate diagnosis, TB is impossible to control. Historically, TB control programs have relied on sputum smear microscopy as the main diagnostic tool for active TB [2]. Even in 2016, smear microscopy remains the most widely used TB test in LMICs, but unfortunately, smear microscopy is neither sensitive nor able to detect drug resistance. More recently, molecular TB diagnostics are starting to get rolled out, even in LMICs. WHO has endorsed three rapid nucleic acid amplification tests (NAATs) since 2008: line probe assays for detection of resistance to first- and second-line TB drugs, Xpert MTB/RIF, an automated, cartridge-based assay, and a manual TB-LAMP assay, based on isothermal amplification process. However, most patients in LMICs have yet to benefit from their potential. Investment in R&D must be continued to develop and validate more affordable and point-of-care molecular diagnostics at lower tiers of health care, while planning for specialized solutions at the referral level, with efficient systems for sample referral.

2. WHO-endorsed molecular TB tests

While molecular tests are not new, their use in LMICs for TB was limited until the introduction and WHO endorsement of rapid NAATs. In 2008, WHO endorsed the use of rapid LPAs for detection of resistance to isoniazid (INH) and rifampicin, two key first-line anti-TB drugs [3]. LPAs (e.g. Genotype MTBDRplus, Hain Lifesciences, Nehren, Germany, and NTM+MDRTB Detection Kit 2 by Nipro Corporation, Osaka, Japan) identify drug-resistance mutations by detecting the binding of PCR-amplified fragments of M. tuberculosis DNA to probes that target the most common mutation sequences conferring resistance to isoniazid and rifampicin. In 2008, WHO endorsed the use of Version 1 of the Genotype MTBDRplus assay for rapid detection of INH and rifampin resistance on smear-positive samples. In 2015, WHO published an update of the LPA policy where GenoType MTBDRplus Version 2 and the Nipro Corporation NTM+MDRTB Detection Kit 2 were endorsed. Either tool can be used to detect TB and to genotype alleles that confer resistance to rifampicin and INH from either smear positive sputum samples or culture-derived isolates. LPAs are intended to be deployed at tertiary or reference laboratories.

In 2010, WHO endorsed the use of Xpert MTB/RIF (Cepheid Inc, Sunnyvale, CA, USA) [4]. This cartridge-based, automated molecular assay can detect M. tuberculosis in 90 min with high accuracy, and simultaneously detect rifampicin resistance directly from clinical specimens, with minimal operator dependence. Furthermore, the technology was designed to be implemented at the district or subdistrict level. WHO currently recommends that Xpert MTB/RIF should be used rather than conventional microscopy, culture, and drug susceptibility testing (DST) as the initial diagnostic test in adults and children suspected of having MDR-TB or HIV-associated TB. WHO also recommends that Xpert MTB/RIF may be used rather than conventional microscopy and culture as the initial diagnostic test in all adults and children suspected of having TB (conditional recommendation acknowledging resource implications). Xpert MTB/RIF should be used as the initial diagnostic test for cerebrospinal fluid specimens from patients suspected of having TB meningitis. Xpert MTB/RIF may also be used as a replacement for standard practice for testing of lymph nodes and other tissues from patients suspected of having extrapulmonary TB.

Since the endorsement of Xpert MTB/RIF by WHO, over 17 million cartridges have been procured under subsidized pricing agreements by high TB burden countries [5]. Thanks to Xpert, the detection of MDR-TB has increased three- to eight-fold compared to conventional testing [5]. The rollout has galvanized stakeholders, from donors to civil society, paving the way for universal DST, and attracting new product developers to TB, resulting in a revitalized molecular diagnostics pipeline.

However, as reviewed by Albert and colleagues, the rollout has also highlighted gaps that have constrained scale-up and limited impact on patient outcomes [5]. Relatively high cost for under-funded national TB programs, unavailability of a complete solution package (training, maintenance, quality assurance, etc.), and lack of impact assessment have also hampered its rollout. Insufficient focus has been afforded to effective linkage to care of diagnosed patients, and clinical impact has been blunted by weak health-care delivery systems, as shown by pragmatic trials in high burden settings [6,7]. In many countries, the private sector plays a major role in TB care, yet this sector has limited access to subsidized pricing [8]. Lastly, decentralized deployment of Xpert in low-income countries is challenging because of technical and infrastructure constraints at the primary care level.

In the short term (the next 5 years), we need to expand the range of molecular technologies that can replace sputum smear microscopy at the primary care level, where most patients with suspected TB seek medical care [9]. However, since molecular assays are not suitable for treatment monitoring, smear microscopy may still have a role, until a better test for cure is identified.

In 2016, WHO endorsed the use of the TB-LAMP assay (Eiken Chemical Co., Tokyo, Japan), which utilizes an isothermal amplification process that produces a result that can be visualized by the naked eye. WHO recommends that TB-LAMP be used as a replacement for microscopy for the diagnosis of pulmonary TB in adults with signs and symptoms of TB [10]. It can also be considered an add-on test to microscopy in adults with signs and symptoms of pulmonary TB, especially when further testing of sputum smear-negative specimens is necessary. TB-LAMP does have several manual steps, making it unsuitable for implementation outside of reasonably well-resourced laboratories. Furthermore, lack of DST makes it challenging to implement this assay in areas with high prevalence of multidrug-resistant TB (MDR-TB). It is unclear whether LMICs can successfully implement this manual technology at the level of peripheral microscopy centers which have limited infrastructure and resources [11,12].

For expanded DST at the reference laboratory level, WHO recently endorsed the use of second-line LPAs [13]. This assay, MTBDRsl version 2.0, detects the mutations associated with fluoroquinolone and second-line injectable drugs. This is particularly relevant for the use of new TB drugs such as bedaquiline and delamanid, as well as the roll-out of the shorter MDR regimen endorsed recently by WHO [14]. Once a diagnosis of rifampicin-resistant TB (RR-TB) or MDR-TB has been established, second-line LPA can be used to detect additional resistance to second-line drugs. For patients with confirmed rifampicin-resistant TB or MDR-TB, WHO recommends that MTBDRsl may be used as the initial test, instead of phenotypic culture-based DST, to detect resistance to fluoroquinolones, as well as to the second-line injectable drugs [13].

3. Options for the future

Existing and emerging molecular diagnostics span the health-care delivery value chain, from primary, district to reference settings. It is clear that no single product will provide all required solutions. Since most LMICs have only microscopy to offer at the primary care level [15], we must find ways to move high-sensitivity molecular technologies down the value chain to the primary care level, in order to diagnose TB early in the patient pathway.

In this context, the development of more rugged, automated systems such as the GeneXpert OMNI system (a portable, battery-operated platform intended for peripheral microscopy centers) is a welcome development [16]. Aligned with this device, a next-generation test cartridge is in development, the Xpert® MTB/RIF Ultra. Ultra is expected to have higher sensitivity than the existing MTB/RIF assay and will soon be commercialized; its utility will be reviewed by WHO in 2017. In addition, there are NAATs developed indigenously in India (i.e. TrueNAT MTB, Molbio Ltd, India) and China (EasyNAT TB, UStar, China) that are now being validated for use in decentralized settings [17]. Although their impact is yet to be demonstrated, these domestic products might be more affordable and scalable in LMICs.

Besides their diagnostic application, new molecular tools can identify drug resistance mutations and help reach the post-2015 target of universal DST for all individuals with active TB disease at the time of diagnosis. New forthcoming TB drug regimens will require adequate companion diagnostics to ensure timely completion of the ‘test and treat’ approach [18]. To this end, Cepheid Inc. is developing another cartridge, the Xpert XDR cartridge that will provide drug resistance information for additional key drugs (isoniazid, fluoroquinolones, and aminoglycosides). In the longer term, next-generation sequencing tools hold great promise but considerable translational work is required to make them affordable and deployable in low-income, high-burden countries [19].

While it would be ideal for DST to be available as close as possible to where patients first seek care, it must at least be available in reference centers. Since expanded DST options are likely to be deployable (or necessary) only at the reference laboratory level, it is clear that we need to invest in systems for rapid, reliable transport of samples up the referral chain, enabled by digital tracking technologies; ideally, these could also deliver results to patients and care providers [20]. In addition to expanding DST, we need to develop shorter and less toxic regimens for MDR-TB, and make sure that patients with DST results are appropriately linked to care.

Molecular diagnostics are starting to make a difference in the field of TB. However, most patients in LMICs have yet to benefit from their potential. Investment in R&D must be continued to develop more affordable and point-of-care diagnostics at lower tiers of health care, while planning for specialized solutions at the referral level, with efficient systems for sample referral. Target product profiles have been established that describe the most urgently needed molecular diagnostics and to guide new diagnostic development [21]. In the longer term, we need simpler, non-sputum-based, biomarker tests that can rapidly detect TB at the point-of-care, especially in children.

Declaration of interest

M. Pai has no financial or industry conflicts. He serves as a consultant to the Bill & Melinda Gates Foundation, and on the Scientific Advisory Committee of FIND, Geneva. Neither BMGF nor FIND had any involvement in this manuscript. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

Additional information

Funding

This article was not funded