Some strains of the tuberculosis (TB) bacterium have mutated to become resistant to some of the drugs commonly used to treat TB. Accordingly, one of the first questions to ask when someone has TB is which drugs will be effective in fighting that person's TB. Unfortunately, quick answers to this question are not always available. As a consequence, people are at times treated with drugs that won't work for their particular strain of TB. What's worse, these drugs often have significant side effects.
As a result, the race is on to develop quick and affordable resistance tests for all commonly used TB drugs.
But first, some alphabet soup to set the scene.
Drug-sensitive TB (DS-TB) is TB that is not resistant to any of the four standard drugs used to treat TB. RR-TB is TB that is resistant to rifampicin (one of those standard four drugs). MDR-TB (multi-drug resistant) is resistant to both rifampicin and isoniazid (isoniazid is also one of the standard four). People with RR or MDR-TB are treated with anything from three to seven drugs, mostly different from those first four. Pre-XDR-TB (extensively drug-resistant) is MDR-TB that is also resistant to a class of drugs called fluoroquinolones (most common examples are moxifloxacin and levofloxacin). XDR-TB is pre-XDR-TB that is also resistant to one of an additional list of drugs (the most notable on this list being bedaquiline).
Why better testing for drug-resistant TB is needed
Many people undergo a long diagnostic process and might not be properly diagnosed for TB, let alone DR-TB, explains David Branigan, TB Project Officer at the New York-based Treatment Action Group (TAG). As a result, he says, some TB patients may be undergoing treatment failure due to being on the wrong treatment regimen.
"That's not appropriate. It's not okay to treat people like that. There needs to be a better standard of care that's implemented," he says.
Part of the solution is to make better use of existing resistance tests, but since quick tests for resistance to all the key drugs are not yet available, there is also general agreement that better tests are needed. The World Health Organization (WHO) last year published a Target Product Profile setting out what they are looking for in new drug-susceptibility tests for TB.
Ideally, Branigan says the implementation of new technologies for drug-susceptibility testing needs to go hand-in-hand with the strengthening of laboratory systems and improving systems of implementation. The technologies need to be brought to, or as close as possible to, the point of care "so that people are given the most effective treatment regimen as soon as possible".
The current tests
There are four types of tests that look for drug susceptibility in TB samples, according to Branigan. The first, (and most widely used in South Africa), is rapid molecular tests. These typically test both for the presence of TB and for resistance to rifampicin.
Many people undergo a long diagnostic process and might not be properly diagnosed for TB, let alone DR-TB and as a result, some TB patients may be undergoing treatment failure due to being on the wrong treatment regimen. - David Branigan, TAG
In South Africa, diagnostic company Cepheid's GeneXpert platform is widely used - Molbio's Truenat is an alternative. GeneXpert tests come in the form of cartridges that are inserted into GeneXpert machines, of which we have many across South Africa. Cepheid also produces an XDR-TB cartridge that tests for resistance to isoniazid, the fluoroquinolones, and second-line injectables.
Then there are high throughput molecular tests, Branigan explains, which are large instruments generally located in centralised labs, manufactured by companies such as BD, Roche, and Abbott that test for TB and resistance to rifampicin and isoniazid. These tests are typically less decentralised than the GeneXpert tests.
The third type, he says, is line probe assays, which are complex laboratory tests that are performed primarily in central labs, and test for resistance to several other second-line DR-TB drugs.
And finally, there is mycobacterial culture. This involves attempting to grow TB bacteria from a sample in the lab in the presence of a drug. If the TB culture grows in the presence of the drug, it is resistant to that drug. If it doesn't grow, it is susceptible to that drug. For new and repurposed DR-TB drugs bedaquiline, delamanid, pretomanid, linezolid, and clofazimine, mycobacterial culture is currently the only available drug-susceptibility test. The process takes two to six weeks per drug being tested. This is also known as phenotypic drug-susceptibility testing. (TAG's excellent Activist's Guide to TB diagnostics provides more detail on these different types of tests.)
Drug-susceptibility tests used in South Africa
The initial diagnostic test for TB in South Africa is the GeneXpert molecular test. It replaced the old, much slower method of diagnosing TB using microscopy around a decade ago. Apart from giving quick results, the standard GeneXpert test has the additional benefit that it also indicates resistance to rifampicin.
If a sample is identified as rifampicin-resistant, additional testing will take place to determine the sample's susceptibility to other drugs, explains Dr Shaheed Vally Omar, a Medical Scientist, and the Scientific Lead at the Centre for Tuberculosis, (incorporating the National TB Reference Laboratory and WHO TB Supranational Reference Laboratory), who spoke to Spotlight on behalf of the National Health Laboratory Service (NHLS). "Additional testing includes molecular methods known as the line-probe assays, which provide information on isoniazid, fluoroquinolones, and second-line injectables," he says.
"The GeneXpert assay is effective at detecting rifampicin resistance. Based on systematic reviews it is estimated that approximately 95% of resistance to rifampicin can be detected. The line probe assays for isoniazid resistance detection are estimated at 90% and for the fluoroquinolones and second-line injectables in the region of 85%," he adds.
For many of the new and repurposed TB drugs, however, no commercial molecular assays have been approved. In these cases, explains Omar, the public sector has to rely on phenotypic testing, which has a protracted turnaround time of between three and six weeks.
The good news is that this is set to change with the introduction in South Africa of the new GeneXpert XDR-TB tests, which can detect resistance to isoniazid, the fluoroquinolones, ethionamide, and some injectable medicines.
"The NHLS has announced the implementation of this technology by its CEO. An implementation plan is in place using a phased approach," says Omar. "The first phase is expected to be complete by the end [of] December 2022."
DR-TB tests in the pipeline
Branigan says that the ideal drug-susceptibility test would be an initial TB test that includes upfront resistance testing to the core TB drugs rifampicin, isoniazid, the fluoroquinolones, and bedaquiline that can be implemented at the primary care level. The next best test would be a rapid follow-on test for resistance to these drugs following positive TB test results.
It is best to have resistance information on rifampicin, isoniazid, the fluoroquinolones, and bedaquiline as early as possible, according to Branigan. "Based on this, a person can be immediately given the best available treatment regimen, which can be further adapted as needed," he says.
Whatever new tests make it to market, they will need to be affordable in order to be implemented in high TB burden countries like South Africa.
So far, there is no initial test for TB and DR-TB that tests for all four of the core drugs, but two companies have been developing rapid molecular tests that will introduce some competition for the GeneXpert XDR-TB test. But so far, there isn't a timeline for when these will be available, says Branigan.
SD Biosensor's rapid molecular test will test for TB as well as resistance to rifampicin and isoniazid and could be implemented at the same level as Gene Xpert. And Bioneer's rapid molecular test is the closest to the four-drug ideal with a test that tests for resistance to rifampicin, isoniazid, and fluoroquinolones. (More information on these tests can be found in TAG's 2022 pipeline report)
According to Branigan, there is not yet a rapid molecular test for bedaquiline because the mutation associated with resistance to this drug has not yet been defined clearly.
The promise of whole genome sequencing
One of the most exciting areas in TB testing these days is targeted Next-Generation Sequencing (tNGS). Branigan describes it as a form of molecular test that is capable of mapping out the targeted portion of the TB genome, where mutations associated with TB drug resistance occur. Resistance to each drug will have a different set of gene mutations, and tNGS is capable of detecting all of them.
Branigan explains that this is made possible by whole genome sequencing (WGS). WGS is when the whole genome of any molecule is sequenced. In this case, the TB genome. Resistance of the TB bacteria to a particular TB drug, for example, rifampicin, will have mutations that are only present in a particular part of the genome, and this will be unique to bacteria resistant to rifampicin. Thus, it is only necessary to sequence the portion of the TB genome where TB resistance mutations occur.
Because it uses software to interpret the results, there technically isn't any limit for the kind of mutations (and thus drug resistance) that it can test for. The software can simply be updated when new mutations associated with resistance to TB drugs are defined.
"What makes tNGS different from rapid molecular tests and high throughput molecular tests is that it's not limited by the number of resistance targets it can test for," Branigan explains. "So, you get resistance information to basically all of the TB drugs from a single test. You get a comprehensive look at TB drug resistance and you can make an informed clinical decision based on that information."
Resistance to each drug will have a different set of gene mutations, and tNGS is capable of detecting all of them. - Branigan
The turnaround time for targeted next-generation sequencing is about 24 to 48 hours.
According to Branigan, evidence around tNGS is currently being reviewed by the WHO as a new class of TB diagnostics. And the WHO will likely be formulating policy recommendations next year. Based on these recommendations, country programmes will be able to start scaling up and implementing targeted next-generation sequencing, most likely only at central labs to start with.
tNGS also hasn't yet been approved for widespread use in South Africa, but it is probably only a matter of time.
"There has been much activity evaluating, assessing, and improving this technology for diagnostic use," says Omar. "Once this technology has been approved for diagnostic use, the NHLS would be in a position to consider its use. The technology has [been] implemented at the National TB Reference Laboratory for surveillance purposes."
For Omar, what is needed to ensure that future testing methods for drug-resistant TB become easier to conduct and less expensive to use is to simplify or introduce automation for the complex processes of tools like next-generation sequencing or whole genome sequencing. This will allow for the adoption of these technologies in "high-volume routine laboratory environments such as ours".
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The Resistance Sniffer
While tNGS has obvious potential in a high TB burden country like South Africa, one challenge to its use in clinical settings is that the data is quite complex and requires certain skills to use the technology and interpret the results. This is according to a South African study that was presented at this year's PHASA conference in Durban.
What is needed to ensure that future testing methods for drug-resistant TB become easier to conduct and less expensive to use is to simplify or introduce automation for the complex processes of tools like next-generation sequencing or whole genome sequencing. - Shaeed Vally Omar
To make NGS datasets from TB samples easier to interpret, the researchers developed an online tool called the Resistance Sniffer that uses complete or partial NGS datasets to predict resistance to thirteen TB drugs. While this is not a test per se, it is a tool that makes interpreting data from a TB sample that has undergone NGS much easier.
"Any DNA extracted either from a cultured bacterium or from a clinical specimen can be sequenced by NGS (through Illumina-a type of sequencing method) and the output files from the sequencer can be used directly for processing by this programme," says Professor Oleg Reva, a lecturer in Bioinformatics at the University of Pretoria.
According to Reva, if you already have a TB sample that has been sequenced by NGS and is in the right format, it's only a matter of uploading that file onto the site, which is free. Within a few seconds, it will produce a graph showing which antibiotics the bacterium in the sample is resistant to - shown in red columns - or may develop resistance to - shown in yellow columns. The results can also be shown in text format.
"Sequencing is becoming affordable even for small labs at clinics. Sequence data can be stored in files of many different formats. [But] non-experts generally cannot understand what to do with this data and how to convert one format to another. This tool provides users with an easy-to-understand interface to upload files in different formats and receive a graphical output," Reva explains. "It looks for multiple informative genomic regions and estimates the likelihoods of drug resistance, even if half of the lookup regions are missed in the provided sequence file."
However, according to Reva, there are no plans to commercialise the programme at this point. He is also not fully satisfied with the sensitivity or specificity of the programme and says it can be improved. It can also be re-designed to predict the antibiotic resistance of other pathogens.
