Development of drug resistance

The development of drug resistance is a feature of most infectious agents and mycobacteria make no exception. Indeed, drug resistance appeared quickly during the first clinical trials assessing tuberculosis treatment. In the British Medical Research Council trial testing monotherapy with streptomycin, half of the patients had M. tuberculosis strains with drug resistance less than two months after treatment start.¹⁰ In a second trial, the addition of a second anti-tuberculosis agent like PAS allowed to prevent the emergence of drug resistance (Figure 5).¹²

Mycobacteria have no mechanism to transmit drug resistance (i.e. plasmids), which conversely are common in other bacteria. Resistance in M. tuberculosis is conferred by chromosomal mutations. Chromosomal mutations appear spontaneously at a rate that is different for each drug, ranging for example between 10^-8 for rifampicin and 10^-5 for streptomycin: this rate represents the resistance threshold of the drug and is a key element in the selection of drug resistance. The second main factor driving the in vivo selection of resistance is the bacterial burden present in the body of individual patients: it is generally estimated that patients with moderate lung involvement with infiltrates may harbour around 10⁶ mycobacteria, while lung cavities may contain from 10⁹ up to 10¹² mycobacteria. In the classic model of drug resistance genesis, spontaneously-appeared resistant mutants are selected by the exposure of the bacterial population to the drug, which kills susceptible isolates, and select for the resistant ones; progressive exposure to different drugs could eventually lead to the development of multidrug-resistance (Figure 6).³⁹

Figure 6. Selection of drug resistance in Mycobacterium tuberculosis.

Schematic representing drug resistance selection during isoniazid monotherapy, followed by treatment with isoniazid plus rifampicin. Source: Dheda et al.⁴⁰

11 While this model is universally recognised and is adequate to explain the development of drug resistance in cases where suboptimal treatments are used (i.e. patients exposed to a single drug), a much more debated topic is the cause of the emergence of drug-resistance in patients receiving an adequate combination therapy. A common explanation is the scarce treatment adherence by the patient. This theory has led the tuberculosis national programs to include different tools to monitor treatment compliance, like directly-observed therapy.

However, in vitro hollow-fibre model studies have repeatedly failed to show a strong association between either missing treatment doses or taking them at different times, rather than all together (the so-called PK mismatch), and the selection of drug resistance.^41,42 Conversely, the same model was able to predict that interindividual variability in pharmacokinetics of the administered drugs, due to alterations in absorption and metabolism, would lead to the development of drug resistance in 0.68% of patients even in case of perfect treatment adherence. This finding was confirmed in vivo in a prospective cohort study in South Africa, where among 142 tuberculosis patients, 0.7% developed drug resistance despite good adherence after two months of treatment. All cases of acquired drug resistance and more than 90% of the cases of treatment failure were associated with low blood levels of the drugs.⁴³ Another element predisposing the acquisition of drug resistance is the penetration properties of drugs into lung cavities, which has been shown to be highly variable between different compounds.⁴⁴ The relevance of the variability of drug concentrations is confirmed by the finding that M. tuberculosis strains in different areas of lung cavities harbour different resistance-conferring mutations and different MICs for drugs being administered.⁴⁵ The lineage of M. tuberculosis strains also appears to play a role in the acquisition of drug resistance. For instance, it has been demonstrated that strains belonging to lineage two (East Asian, including the Beijing strain that is clonally expanding in association with the spread of MDR-TB) have a higher spontaneous mutation rate than strains belonging to lineage four (Euro-America), and are therefore more likely to acquire resistance.⁴⁶ Finally, the role of efflux pumps in the selection of drug resistance remains to be elucidated: it has been postulated that efflux pumps genes may be induced after a few hours of drug pressure, leading to a status of low-level resistance that in turn might favour the selection of the resistant mutants.⁴⁰

Resistance-conferring mutations were historically thought to come with a fitness cost, leading for example to a reduced multiplication rate and to the progressive disappearance of the resistant sub-population in absence of selective pressure by chemotherapy. This theory has

12 received multiple experimental confirmations.⁴⁷ However, it has been recently shown that compensatory mutations may appear in drug-resistant strains, leading to a high competitive fitness both in vitro and in vivo.⁴⁸

Similarly, drug-resistant tuberculosis has been traditionally labelled as a man-made disease and the direct consequence of suboptimal regimens or insufficient treatment adherence. The emphasis was therefore placed on the prevention of cases of secondary resistance and the presence of risk factors, such as previous treatment with first- or second-line anti-tuberculosis drugs, was regarded as the main predictor of drug-resistant tuberculosis.

However, this paradigm has been challenged by the increasing circulation of M/XDR-TB strains in the population. A recent modelling study has estimated that primary drug resistance transmission would account for a median of 95.9% (95% uncertainty range 68.0 – 99.6) of all incident MDR-TB cases and 61.3% (16.5 – 95.2) of incident MDR-TB cases in previously treated individuals.⁴⁹

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