The tuberculosis diagnostics pipeline

14 July 2016. Related: Pipeline report, TB coinfection.

Erica Lessem, TAG

Introduction

Over the past decade, several new options for improving TB diagnosis have become available. The past year saw considerably more progress than the previous one. However, the reality of how most TB is diagnosed – or not – remains largely unchanged. The world failed to detect 3.6 million of the estimated 9.6 million new cases of TB in 2014. [1]

Sputum smear microscopy – which misses over half of TB cases [2] and gives no indication of drug susceptibility to guide appropriate treatment – is still the diagnostic standard in most of the world, despite the availability of the far more sensitive GeneXpert MTB/RIF for six years. [3] Late in 2015, the World Health Organization (WHO) approved Alere’s TB lipoarabinomannan (LAM) test – a very affordable, simple, rapid, noninvasive, point-of-care (POC) rule-in test for people with HIV with very low CD4 counts – but no country has begun to implement it yet. New versions of line probe assays – Hain’s MTBDRplus and MTBDRsl and a product from Nipro – received WHO recommendation, facilitating rapid drug susceptibility testing (DST), but the world is still a long way from universal DST, with an estimated 59 percent of cases of multidrug-resistant TB (MDR-TB) undetected. [4]

Research and development (R&D) for TB diagnostics features some promising developments since last year (see table 1). Improvements on nucleic acid amplification tests (NAATs) such as GeneXpert Omni and Ultra and Molbio’s TrueNAT are being validated, positioning them for possible WHO recommendation. Further upstream, encouraging research into gene sets that can predict active TB disease and reliably distinguish it from latent TB and other infections may eventually underpin new blood tests (currently, there is no effective serological test for active TB). Incremental advances are being made to improve detection of pediatric TB (see Extending quality).

Yet, overall, with a mere US$65 million in 2014 funding out of an estimated annual need of $340 million, [5] the pipeline for evidence-based new diagnostics has largely remained stagnant (see table 2). Dismayingly, some companies continue to move forward with marketing for their products when the data are unavailable or scream that they should not, as is the case with Epistem, which is marketing GeneDrive in India despite the test’s having flopped in studies.

With use of poor tests predominating, poor uptake of good extant options, poor evidence bases to support the introduction of new tests, and poor funding to support the development of better tests, it’s no wonder we’ve made little headway in diagnosing TB.

CI: confidence interval
FLD: first-line drugs (isoniazid, rifampin, ethambutol, pyrazinamide)
FQ: fluoroquinolone
ICMR: Indian Council of Medical Research
INH: isoniazid
LAM: lipoarabinomannan
MDR-TB: multidrug-resistant tuberculosis
MTB: Mycobacterium tuberculosis
NAAT: nucleic-acid amplification test
qPCR: quantitative polymerase chain reaction
RIF: rifampin
RT-PCR: real-time polymerase chain reaction SLID: second-line injectable drug (e.g., amikacin, capreomycin, or kanamycin) WHO: World Health Organization

CXR: chest x-ray
DR: drug resistance
EMB: ethambutol
FLD: first-line drugs (isoniazid, rifampin, ethambutol, pyrazinamide)
FQ: fluoroquinolone
INH: isoniazid
MDR-TB: multidrug-resistant TB
MIC: minimum inhibitory concentration
MTB: Mycobacterium tuberculosis
NAAT: nucleic-acid amplification test
PCR: polymerase chain reaction POC: point of care
PZA: pyrazinamide
RIF: rifampin
RT-PCR: real-time polymerase chain reaction
SLD: second-line drug
SLID: second-line injectable drug (e.g., amikacin, capreomycin, or kanamycin)
TB: tuberculosis
WHO: World Health Organization

Trot, Trot to Market

Perhaps the most exciting advance in TB diagnostics came in late 2015, when the WHO recommended Alere’s Determine LAM Ag simple urine dipstick test for ruling in TB in people with HIV with CD4 counts below 100/mm3 or who are seriously ill. [17] The test’s imperfect sensitivity (56% pooled sensitivity based on five studies of people with CD4 counts below 100/mm3) means that a negative test must still be followed up with other testing to rule out TB. [18] However, given the extremely high mortality of people with TB and HIV (TB is thought to be the cause of death in nearly 40% of HIV-positive patients, half of which is undiagnosed) [19] and challenges in diagnosing TB in people with low CD4 counts, having an inexpensive ($2.26 per test), simple, and noninvasive test to use in this very high-risk population is a major advance. In fact, the LAM test is the first TB test to ever demonstrate a mortality benefit in a randomized controlled clinical trial: among 578 people with HIV in hospitals in South Africa, Tanzania, Zambia, and Zimbabwe, using LAM was associated with an absolute reduction of all-cause mortality at eight weeks of 4% (95% confidence interval [CI]: 1%–7%) from 25% to 21%, and a relative risk reduction of 17% (95% CI: 4%–28%). [20] This difference appeared to be attributable to the test’s allowing earlier initiation (by one day on average) of anti-TB therapy. [21] Countries with large burdens of TB/HIV, including many countries in sub-Saharan Africa, should roll out LAM testing immediately, along with proper accompanying training to ensure the test is used only in the recommended population and that follow-up tests are done as necessary. [22] Whoever ends up with the rights to the test – Abbott is trying to pull out of a putative acquisition of Alere – should ensure its continued manufacture as well as marketing. [23] Further upstream, funding from the Global Health Innovative Technology Fund (GHIT Fund, which is itself funded by the Japanese government, pharmaceutical companies, the Bill & Melinda Gates Foundation, and the Wellcome Trust) to FIND and Fujifilm will support the development of what is hoped to be a more sensitive LAM test. [24, 25, 26]

Other policy advances can help improve timely detection of drug-resistant TB. In late 2015, the WHO extended its 2008 guidance, which recommended the use of the Hain version 1 line probe assay (LPA), to recommend the use of two alternative LPAs with the capability to detect TB and rifampin resistance: the Hain version 2 LPA (also called the GenoType MTBDRplus assay) and the Nipro Assay. [27] The WHO still does not recommend using LPAs on smear-negative samples. In 2014 and 2015, FIND conducted a cross-sectional noninferiority study to compare the accuracy of these two tests to that of Hain Version 1 assay, evaluating their performance both on clinical isolates and on sputum specimens from people with pulmonary TB; both tests showed comparable performance in detecting Mycobacterium tuberculosis (the bacterium that causes TB infection and disease) and rifampin resistance in smear-positive samples: on clinical isolates, sensitivity and specificity compared with the phenotypic reference standard for Hain V1, HainV2, and Nipro were 90.3%/98.5%, 90.3%/98.5% and 92.0%/98.5%, respectively, for detection of rifampin resistance and 89.1%/99.4%, 89.1%/99.4%, and 89.6%/100.0%, respectively, for detecting isoniazid resistance. In sputum testing, sensitivity and specificity were 97.1%/97.1%, 98.2%/97.8%, and 96.5%/97.5% for rifampin resistance and 94.4%/96.4%, 95.4%/98.8%, and 94.9%/97.6% for isoniazid resistance. [28] The WHO will update its guidance on LPAs later in 2016. While this certainly reflects progress, Hain and Nipro launched these assays in 2011; it’s taken five years to optimize and fully evaluate them.

In May 2016, the WHO also recommended and issued guidance on Hain’s MTBDRsl, an LPA capable of detecting resistance to fluoroquinolones and second-line injectables. [29] LPAs can produce results in 24–48 hours, much quicker than the two weeks that liquid culture or two to three months that solid culture take. As such, the MTBDRsl LPA can guide appropriate treatment selection. The announcement of the WHO’s MTBDRsl recommendation accompanied its recommendation of the shortened or “modified Bangladesh” regimen, whose introduction the test can help facilitate, as the shortened (9- to 12-month) regimen is not suitable for fluoroquinolone- or injectable-resistant TB (pre-XDR-TB; see Tuberculosis Treatment). [30] Countries and donors must scale up the introduction of this test and work with Hain to further reduce the price. FIND negotiated a public sector price of €7.50 (approximately $10) per test strip in 138 countries; however, the total cost of running a test (which requires other laboratory supplies) can result in costs of $20–$30. The test equipment itself can cost $8,000–$40,000, depending on its size and whether it automatically reads results or not. [31]

Newer iterations of GeneXpert are moving closer to market. Recent investments may make it more suitable for use in a variety of settings and more sensitive. GeneXpert Omni, a smaller and more rugged single-cartridge version of the test that is dustproof and runs on batteries could be a point-of-care test for TB. The test device’s anticipated cost is $2,895. [32] Cepheid claims that another new product, the GeneXpert Ultra cartridge, is more sensitive than the MTB/RIF, approximating the sensitivity of culture, and has a shorter processing time. [33] FIND is currently validating both the Ultra cartridge and Omni platform: Ultra results are expected at the end of 2016; Omni results have been further delayed. If studies show them to indeed be as promising as the company claims, the WHO will issue recommendations and formulate guidance accordingly. Data on the use of Ultra in smear-negative specimens are expected in 2017, which could inform recommendations on whether Ultra can be used to replace culture. Cartridge prices are expected to remain consistently high at $9.99, as even though MTB/RIF sales volumes have increased, [34] those profits are said (by Cepheid) to have been reinvested in R&D. In 2017, Cepheid plans to release the XDR assay, designed to genotype resistance to isoniazid, fluoroquinolones, and second-line injectables when MTB/RIF (or Ultra) indicates rifampin-resistant TB, though no peer-reviewed data yet exist on this product. [35] A FIND evaluation is expected in 2018.

Molbio’s TrueNAT, an Indian GeneXpert competitor that has been on the market since 2013, is finally being validated by outside parties (FIND and the Indian Council on Medical Research). [36] A recent study compared TrueNAT to Xpert MTB/RIF on 274 patient specimens, using culture as the reference standard. The assays had similar sensitivity on sputum-smear-positive samples: TrueNAT had 99% sensitivity (95% CI: 94.2%–99.95%) versus MTB/RIF’s 100% (95% CI: 96.5–100.0%, respectively). With sputum-smear-negative, culture-positive samples, the sensitivity of the TrueNAT was 86.2% (95% CI: 74.1%–93.4%) as compared with 90.1% (95% CI: 88.7%–94.35%) for Xpert MTB/RIF. [37] The cartridge-based sample preparation extraction tool, TruePrep – which is rugged and portable – costs $7,000, and each assay costs $14; the public sector will receive a further discount. [38]

Other products without the data to back them up continue to be marketed by unscrupulous manufacturers. Epistem’s Genedrive performed dismally in a recent clinical study of 336 participants: sensitivity was 45.4% (95%CI: 35.2%–55.8%) versus 91.8% (95% CI: 84.4%–96.4%) for Xpert MTB/RIF and 77.3% (95% CI: 67.7%–85.2%) for smear microscopy. In smear-negative cases, sensitivity of GeneDrive was 0% (95% CI: 0, 15.4) versus 68.2% (95% CI: 45.1%–86.1%) for Xpert. [39] Yet just after those data were published, Epistem announced full commercial launch of Genedrive TB tests in India, claiming it “enables early detection of TB and antibiotic resistance without need for central laboratory facilities.” [40] The regulatory authority in India should ban the marketing of this test, and private providers should be extremely wary and not waste patients’ time, money, and effort by subjecting them to it (see box).

In Development

Back in the lab, a promising development came from a team at Stanford University that identified a three-gene set indicative of active TB (GBP5, DUSP3, and KLF2) in whole blood across eight data sets containing over 1,000 samples from both adult and pediatric patients in ten countries. This gene signature could accurately separate people with active TB from healthy controls, from people with latent TB, and from people with other diseases. HIV status, bacillus Calmette-Guérin (BCG) vaccination, and drug resistance did not confound expression of the three-gene set. The gene set may be of use in monitoring treatment, as its expression increases with disease severity and decreases with time of treatment, though this must be validated prospectively. Further validation and development are required. [47]

Researchers from the University of Washington and the University of Cape Town have received funding to further study a simple oral swab to test for TB DNA, and it detected TB well in 18 of 20 patients in a proof-of-concept study (90.0% sensitivity compared with GeneXpert MTB/RIF; 95% CI: 66.9%–98.2%). [48, 49]

Several researchers are exploring the value of immune activation markers such as C-reactive protein (CRP) to indicate active TB disease or to identify good responses to TB therapy. One small study pre- and post-treatment in Gambia showed that CRP levels showed the most significant decrease by two months of treatment (P < .0001), whereas two other markers, beta2 microglobulin and neopterin, showed little change by two months but a significant decrease by six months of treatment (P = .0002 and P < .0001, respectively). [50] A larger prospective study identified a seven-marker biosignature including CRP as well as transthyretin, interferon-γ, complement factor H, apolipoprotein-A1, inducible protein 10, and serum amyloid A that identified TB disease in the test set (N = 210) with a sensitivity of 93.8% (95% CI: 84.0%–98.0%) and a specificity of 73.3% (95% CI: 65.2%–80.1%), regardless of HIV infection status. [51] CRP may be of particular interest for pediatric development (see Tuberculosis Diagnostics Research for Children below).

DST research saw some developments. Development of a rapid colorimetric method for detection of resistance to pyrazinamide – one of the most important drugs to treat TB, for which DST development remains challenging due to the enormous number of resistance-associated mutations in the M. tuberculosis pncA gene – using a dye called 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) progressed when initial testing in a small test of 50 isolates showed DST results could be available in four to six days with 97.1% sensitivity and 81.3% specificity, in comparison with liquid culture. [52] Unfortunately, research that could underpin DST development for bedaquiline has not yet been as successful – examining the 12 cases who had developed over fourfold increases in bedaquiline minimum inhibitory concentrations (MICs) in study C209 revealed that all had M. tuberculosis with mutations in the Rv0678 gene, but there was no correlation between MIC change and treatment response, making it unclear what might be a clinically meaningful breakpoint. [53] Developing DST for bedaquiline will be important, as resistance to it has already started to develop. [54]

Recommendations

With new products moving forward and interesting new leads to pursue, we are pleased to report progress with the TB diagnostics pipeline. But the world is still far from ensuring all those with TB get appropriate diagnostics. Both R&D and access need dramatic infusions of funding and political will. In particular:

• National governments and donors must substantially increase funding for TB programs to allow for best diagnostic practices. This includes the widespread scale-up of NAAT to supplant microscopy, universal DST using liquid culture or LPAs, digital X-ray, and the rapid adoption of LAM testing in areas with high HIV burdens.
• National governments, donors, and the private sector must invest far more in TB R&D to advance better tests, including those for children. This should include a commitment to rapidly and rigorously evaluating new technologies and to publishing peer-reviewed results. Greater resources are necessary to achieve the requirements set out in Target Product Profiles (TPPs). [55]
• National governments and donors should work closely with the nonprofit and private sectors to ensure only quality and affordable tests are used. In countries with large proportions of care-seeking in the private sector, access to appropriate diagnostics is extremely limited and can be catastrophically expensive. Good programs such as IPAQT to address this exist and should be expanded and replicated.
• Developers must commit to timely and rigorous validations of their tests prior to marketing, and health and regulatory authorities and private practitioners should hold them accountable for doing so. Epistem and other companies who market ineffective or as-yet-unproven tests must cease doing so immediately. National governments should ban the import and use of inappropriate tests and enforce those bans. Those working in TB globally should call to task companies such as Epistem that inappropriately market them.

Acknowledgments

Erica Lessem thanks Mark Harrington for his editing, Rachel Schiff for her support for references, and the contributions of many researchers, developers, and technical experts that made this chapter possible. Lindsay McKenna sends special thanks to Dr. Anne Detjen, Dr. Jennifer Cohn, and Dr. Devasena Gnanashanmugam for their thoughtful reviews and comments on the pediatric section.

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