TB or not TB? How NGS will win the battle against drug-resistance

The stats on TB are deeply worrying: one–third of the world's current population has been infected with Mycobacterium tuberculosis; new infections occur at a rate of one per second – and drug-resistance is an increasing problem. Here we find out why metrology is a vital part of the development of new detection methods… Following major declines in TB over most of the 20th century, its incidence increased steadily in England from the late 1980s to 2005, and has remained at relatively high levels ever since – higher than most other Western European countries¹. By 2012, there were 8,130 cases reported. Drug-resistant TB is an increasing problem in England with numbers of cases increasing from 48 in 2003 to 78 in 2012². The World Health Organisation (WHO) estimates that there are nine million people globally with TB. About 3.6% – or 325,000 – have multi-drug resistant (MDR) strains, where the bacteria are resistant to the two core antibiotics (Rifampicin and Isoniazid) of the four drug treatments used to treat the infection. Drug resistant TB can be caused by transmission of already resistant strains (primary resistance) in the same way that drug susceptible (regular) TB is transmitted, or it can be caused by the development of resistance mutations during inadequate therapy (secondary resistance). This can lead to the development of further resistances such as extensively drug-resistant (XDR) TB, a less common type of multi-drug resistant TB. XDR-TB is resistant to almost all drugs used to treat TB, together with the best second-line medications and at least one of three injectable drugs. If the current pandemic is to be brought under control, accurate and rapid diagnosis – alongside methods for monitoring resistance to therapeutic agents, and transmission and spread in the community – is needed. The sputum smear microscopy currently recommended by NICE³ for diagnosis in those patients with chest X-ray appearances suggestive of TB was developed more than 100 years ago. This method is inexpensive and diagnosis can be made in a day; however it is prone to false negatives and is unable to identify if the bacteria are resistant to antibiotics. To distinguish between drug-susceptible TB and drug-resistant TB the bacteria need to be grown and tested in a specialised laboratory and final diagnosis for TB, especially for XDR-TB, routinely takes one to three months. The application of next generation sequencing (NGS) methods for the sequencing of whole genomes in this area of pathogen identification has recently been boosted by the introduction of new technologies, offering a range of sequencing strategies and supported by a number of reference databases. Clinically, the capacity of NGS to generate large data sets offers new strategies for typing pathogens and identifying drug resistance, thus enabling the rapid identification of emerging infectious diseases and tracking of outbreaks. The data could inform the identification and description of drug resistant TB which would help tackle the global incidence of the disease.

The microscopy currently recommended by NICE for diagnosis of TB was developed more than 100 years ago

LGC scientists are exploiting this approach as part of a multi-partner European Metrology Research Programme (INFECTMET), which focuses on metrology for monitoring infectious diseases, antimicrobial resistance and harmful micro-organisms. Dr Denise O’Sullivan, LGC Researcher, Molecular Biology, said: “Through the INFECTMET project, we are attempting to investigate sources of error in methods for the identification of drug resistant M. tuberculosis. There are new strains of M. tuberculosis which are very difficult to treat as they are resistant to most of the current drug treatments. We hope next generation sequencing can be used to gain a better understanding of mutations and drug-resistance, which can then help with containment, prevention, and more effective treatment.” In collaboration with University College London’s Centre for Clinical Microbiology, LGC scientists are sequencing the DNA extracted from clinical samples of drug resistant TB and looking at the ability of current methods to reliably identify unique mutations which are conferring the drug resistance. The samples include XDR-TB. The team is assessing the performance characteristics of the sequencing methods used to identify the mutations. Each XDR-TB sample is being sequenced multiple times (all stages, from the first step of library preparation right through to sequencing and the analysis of the data, are performed separately) on the Illumina HiSeq – an ultra-high-throughput sequencing system. The findings will inform the identification of a single nucleotide polymorphism (SNP) – a DNA sequence variation occurring commonly within a population (e.g. 1%) in which a single nucleotide in the genome (or other shared sequence) differs between members of a biological species or paired chromosomes. This makes it possible to determine a particular strain type which can be associated with an increased risk of spreading amongst communities and also may have an association with multiple drug resistance. Dr O’Sullivan said: “This INFECTMET study will assess screening methods for the surveillance and monitoring of disease load and antibiotic resistance. In partnership with our European National Measurement Institute and clinical collaborators, we hope to develop novel measurement procedures and validation frameworks to support current and emerging molecular approaches for efficient, harmonised and rapid diagnosis, surveillance and monitoring of infectious diseases, with a particular focus on respiratory tract infections like TB.” The team is also investigating the ability of the molecular (PCR) methods which are currently used, in clinical laboratories to accurately diagnose TB. This is being achieved by sending samples containing safe versions of M. tuberculosis to different laboratories around the world for analysis. This study aims to improve the molecular diagnosis of TB in the future by enabling laboratories to compare results and assess laboratory performance. Dr O’Sullivan added: “Overall this should improve methods currently used for the monitoring of M. tuberculosis and drug resistance. The ultimate aim is to establish routes for improving the accuracy, robustness, comparability and traceability of measurements within the metrology and diagnostics/epidemiological communities across Europe and link these into international standardisation initiatives through the Centres for Disease Control and Prevention, and WHO.” References

1. http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317140970182
2. http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317140970182
3. http://www.nice.org.uk/nicemedia/live/13422/53642/53642.pdf

Author Ellie Gadd is a science writer at LGC. She writes on behalf of the Science and Innovation Division, which houses the unique function of the UK Government Chemist and the UK’s designated National Measurement Institute for chemical and bio-measurement. Contact www.lgcgroup.com