Genome Sequencing of Multidrug Resistant Tuberculosis (MDR TB) in Sputum

Drug resistant M. tuberculosis is an increasing problem in the United Kingdom and abroad. In the United Kingdom (UK) as a whole the number of isolates that were shown to be resistant to at least one of the first line drugs was nearly 400, with the total number of isolates approaching 5000. A total of 9040 cases were reported in the UK in 2009 and of these 6.9% demonstrated resistance to at least one first line drug(1). In certain populations the incidence of drug resistance is higher: in London, homeless patients, those who have been in prison and those from certain countries abroad, particularly Eastern Europe.

The difficulty of drug resistant tuberculosis is that the treatment duration, cost and complexity is increased. Typically the patient will be on treatment for 18 months or more and the Health Protection Agency (HPA) has estimated the cost of this as being upto £50000.

Current diagnosis rests on the culture of the M. tuberculosis and drug sensitivity testing. This can take six or eight weeks, meaning that patients may be on ineffective therapy for some time, leading to further transmission and deterioration of the patient's clinical condition. In this study the investigators would hope to develop a new test to improve and expedite the diagnosis of multi- drug resistant or MDR TB.

The predominant mechanism by which resistance occurs in M. tuberculosis is by the development and selection of mutants containing single nucleotide polymorphisms (SNP's)(2). Present commercial assays enable only a common subset (5-10) of the (900+) documented resistance mutations to be detected (www.tbdreamdb.com). It is highly likely that many more exist particularly in regions of the genome that may modulate sensitivity or resistance. This complexity is compounded by the requirement to treat TB with cocktails of antibiotics even for fully drug sensitive M.tuberculosis for which treatment consists of isoniazid (H), rifampicin (R), pyrazinamide (Z) and ethambutol (E). Multidrug resistant (MDR) and extensively drug resistant (XDR) TB cases are treated with second line drugs such as moxifloxacin, amikacin, capreomycin, para-aminosalicylic acid (PAS), thiocetazone and others for which genotypic markers of resistance are not tested. Such complex treatment combinations increase the need to screen multiple gene targets with the imperative to treat immediately with correct combinations of antibiotics.

The large number of mutations makes exhaustive detection of all known SNP's impossible with existing diagnostic procedures. Whole genome sequencing offers the potential to interrogate the genome of clinical isolates of M. tuberculosis for all known mutations and from this to infer an antimicrobial sensitivity pattern.

The extensive cost of treating and managing MDR cases(3) could potentially be reduced by obtaining a rapid genomic resistance profile early within the patients treatment. Cost benefit analysis of immediate whole genome sequencing (WGS) on all TB cases would be highly beneficial financially as well as clinically. The investigators propose to conduct a limited pilot study to assess the potential to acquire whole genome sequence directly from sputum specimens, early in a patient's treatment and to retrospectively define the potential impact of the availability of this data on patient care. This proposal will thus provide an evidence base for WGS to be developed into a routine diagnostic test/process with medical potential both at local National Health Service (NHS) level and globally so as to improve care pathways for MDR and XDR TB.