TB-DRUG OLIGOCOLOR - Development of a molecular platform for the simultaneous detection of Mycobacterium tuberculosis resistance to rifampicin and fluoroquinolones

Background:

Treatment success and containment of drug-resistant tuberculosis (TB) rely on a timely laboratory diagnosis. In view of this, a versatile and user-friendly molecular platform was proposed for the identification of Mycobacterium tuberculosis in clinical specimens and the simultaneous detection of resistance to two key anti-TB agents: rifampicin and fluoroquinolones. The platform will be initially developed for the detection of resistance to rifampicin because the associated mutations are well defined and their prevalence is sufficiently known worldwide. A small, single-stranded primer covalently-linked to an activated solid surface will be used to amplify a specific DNA target in a microplate well strip format, and an enzymatic chromogen system will be applied for detection. The technique will be validated for reproducibility and proof-of-principle.

Subsequently, mutated sequences encoding resistance to quinolones will be investigated. To this end, the gyrA gene will be sequenced in a collection of M. tuberculosis clinical isolates with known phenotypical resistance to fluoroquinolones. Relevant segments will be added as probes to the platform. The gyrA gene will serve also as a source for M. tuberculosis-specific target segments. A pre-clinical trial will be performed to evaluate the combined platform directly in clinical specimens and early liquid cultures. In this trial, the performance of the system will be verified by measuring: i) sensitivity, ii) specificity, iii) predictive values in settings with different prevalence rates of drug resistance, and iv) turnaround time to diagnosis. This project will add value to the leadership of the European initiative in biotechnology research and confront the emergency of MDRTB through the proposal of a promising deliverable useful to support targeted interventions.

Problem:

The management and control of multidrug resistant tuberculosis (MDRTB) relies upon a solid laboratory support. Even in countries with excellent TB control programmes, 8 out of 10 cases of TB with treatment failure harbour MDR bacilli and the prolongation of first-line drug regimens in these cases merely amplifies the epidemiological problem. The spread of MDRTB can be prevented only if patients with drug resistant disease are detected and treated with a combination of effective drugs. The treatment outcome of MDRTB improves consistently when it is recognized and treated early. To maximize the efficacy of a combined drug regimen, drug resistance to first- and second-line drugs should be investigated, if not in every new TB case, at least in those who remain smear-positive at the end of the intensive treatment phase. In practice, however, this goal is difficult to achieve in clinical settings with high burden of drug resistance. It would be very useful to count on a tool able to simultaneously anticipate failure of first-line chemotherapy and susceptibility to a major second-line drug.

Phenotypic drug susceptibility assays entail sub-cultivation in the presence of a set of anti-tuberculosis agents. Thus, the possibilities for obtaining rapid results, even using liquid culture, are limited by the slow growth of the bacilli. By requiring only small amounts of bacterial nucleic acids, genotypic approaches may circumvent this hindrance, thus shortening the turnaround time. Moreover, DNA-based technologies can in principle be applied directly to the clinical specimen, provided it contains enough bacilli. PCR-sequencing is the undisputed standard for molecular resistance detection. New designs involving real-time PCR promise to be highly efficient. However, these methods, including sequencing, are not readily available and require sophisticated, expensive equipment and/or highly skilled personnel. These disadvantages preclude their implementation on a routine basis in most diagnostic laboratories.

Electrophoresis-based approaches and other PCR-based techniques are relatively easy to implement but require expertise in polyacrylamide gel preparation and fail to detect some mutations. Methods based on immobilized probes rely on the capture of amplified product of a target DNA sequence by a set of single chain oligonucleotides representing wild and mutated segments linked to a solid support. The capture oligos are bound either to microtiter plate wells or membranes. This kind of format has proved to be accurate and technically unsophisticated.

In the development of molecular tools, special attention has been paid to the detection of rifampicin resistance because the associated mutations are well defined and their prevalence is sufficiently known worldwide. Fluoroquinolones are second-line agents with considerable activity against M. tuberculosis and are preferred in the treatment of all MDRTB cases unless resistance to this class is also demonstrated. Up to now, M. tuberculosis resistance to fluoroquinolones has been relatively rare and knowledge on its genetic basis is still limited. High levels of resistance to quinolones have so far found to be encoded by specific mutations clustered in a very small region of the gyrA gene. Together with other second-line drugs, quinolones will become widely available to many countries as a result of the Green Light committee of the WHO and, therefore, an emergence of resistance to these agents is expected to occur. It has already been reported that under a sub-optimal therapeutic scheme M. tuberculosis develops resistance to fluoroquinolones at an alarmingly rapid rate. Therefore, there is the urgent need to develop a rapid system for the detection of resistance to these agents.

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