Natural Polymorphisms in Mycobacterium tuberculosis Conferring Resistance to Delamanid in Drug-Naive Patients.

Mutations in the genes of the F420 signaling pathway of Mycobacterium tuberculosis complex, including dnn, fgd1, fbiA, fbiB, fbiC, and fbiD, can lead to delamanid resistance. We searched for such mutations among 129 M. tuberculosis strains from Asia, South America, and Africa using whole-genome sequencing; 70 (54%) strains had at least one mutation in one of the genes. For 10 strains with mutations, we determined the MIC of delamanid. We found one strain from a delamanid-naive patient carrying the natural polymorphism Tyr29del (ddn) that was associated with a critical delamanid MIC.

Whole-Genome Sequencing for Drug Resistance Profile Prediction in Mycobacterium tuberculosis.

Whole-genome sequencing allows rapid detection of drug-resistant Mycobacterium tuberculosis isolates. However, the availability of high-quality data linking quantitative phenotypic drug susceptibility testing (DST) and genomic data have thus far been limited. We determined drug resistance profiles of 176 genetically diverse clinical M. tuberculosis isolates from the Democratic Republic of the Congo, Ivory Coast, Peru, Thailand, and Switzerland by quantitative phenotypic DST for 11 antituberculous drugs using the BD Bactec MGIT 960 system and 7H10 agar dilution to generate a cross-validated phenotypic DST readout. We compared DST results with predicted drug resistance profiles inferred by whole-genome sequencing. Classification of strains by the two phenotypic DST methods into resistotype/wild-type populations was concordant in 73 to 99% of cases, depending on the drug. Our data suggest that the established critical concentration (5 mg/liter) for ethambutol resistance (MGIT 960 system) is too high and misclassifies strains as susceptible, unlike 7H10 agar dilution. Increased minimal inhibitory concentrations were explained by mutations identified by whole-genome sequencing. Using whole-genome sequences, we were able to predict quantitative drug resistance levels for the majority of drug resistance mutations. Predicting quantitative levels of drug resistance by whole-genome sequencing was partially limited due to incompletely understood drug resistance mechanisms. The overall sensitivity and specificity of whole-genome-based DST were 86.8% and 94.5%, respectively. Despite some limitations, whole-genome sequencing has the potential to infer resistance profiles without the need for time-consuming phenotypic methods.

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae

Leprosy is a chronic human disease caused by the yet-uncultured pathogen Mycobacterium leprae. Although readily curable with multidrug therapy (MDT), over 200,000 new cases are still reported annually. Here, we obtain M. leprae genome sequences from DNA extracted directly from patients' skin biopsies using a customized protocol. Comparative and phylogenetic analysis of 154 genomes from 25 countries provides insight into evolution and antimicrobial resistance, uncovering lineages and phylogeographic trends, with the most ancestral strains linked to the Far East. In addition to known MDT-resistance mutations, we detect other mutations associated with antibiotic resistance, and retrace a potential stepwise emergence of extensive drug resistance in the pre-MDT era. Some of the previously undescribed mutations occur in genes that are apparently subject to positive selection, and two of these (ribD, fadD9) are restricted to drug-resistant strains. Finally, nonsense mutations in the nth excision repair gene are associated with greater sequence diversity and drug resistance.

Whole genome sequencing distinguishes between relapse and reinfection in recurrent leprosy cases.

BACKGROUND: Since leprosy is both treated and controlled by multidrug therapy (MDT) it is important to monitor recurrent cases for drug resistance and to distinguish between relapse and reinfection as a means of assessing therapeutic efficacy. All three objectives can be reached with single nucleotide resolution using next generation sequencing and bioinformatics analysis of Mycobacterium leprae DNA present in human skin. METHODOLOGY: DNA was isolated by means of optimized extraction and enrichment methods from samples from three recurrent cases in leprosy patients participating in an open-label, randomized, controlled clinical trial of uniform MDT in Brazil (U-MDT/CT-BR). Genome-wide sequencing of M. leprae was performed and the resultant sequence assemblies analyzed in silico. PRINCIPAL FINDINGS: In all three cases, no mutations responsible for resistance to rifampicin, dapsone and ofloxacin were found, thus eliminating drug resistance as a possible cause of disease recurrence. However, sequence differences were detected between the strains from the first and second disease episodes in all three patients. In one case, clear evidence was obtained for reinfection with an unrelated strain whereas in the other two cases, relapse appeared more probable. CONCLUSIONS/SIGNIFICANCE: This is the first report of using M. leprae whole genome sequencing to reveal that treated and cured leprosy patients who remain in endemic areas can be reinfected by another strain. Next generation sequencing can be applied reliably to M. leprae DNA extracted from biopsies to discriminate between cases of relapse and reinfection, thereby providing a powerful tool for evaluating different outcomes of therapeutic regimens and for following disease transmission.

Red squirrels in the British Isles are infected with leprosy bacilli.

Leprosy, caused by infection with Mycobacterium leprae or the recently discovered Mycobacterium lepromatosis, was once endemic in humans in the British Isles. Red squirrels in Great Britain (Sciurus vulgaris) have increasingly been observed with leprosy-like lesions on the head and limbs. Using genomics, histopathology, and serology, we found M. lepromatosis in squirrels from England, Ireland, and Scotland, and M. leprae in squirrels from Brownsea Island, England. Infection was detected in overtly diseased and seemingly healthy animals. Phylogenetic comparisons of British and Irish M. lepromatosis with two Mexican strains from humans show that they diverged from a common ancestor around 27,000 years ago, whereas the M. leprae strain is closest to one that circulated in Medieval England. Red squirrels are thus a reservoir for leprosy in the British Isles.