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

Abstract

BackgroundSince 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.MethodologyDNA 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 findingsIn 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/SignificanceThis 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.

Human Genetic Susceptibility of Leprosy Recurrence

“…It is interesting to note that recurrent patient #1126 presented allelic and genotypic global counts similar to the controls and in contrast with the two other recurrent individuals. Patient #1126 has been described previously as the only case of recurrence caused by distinct M. leprae strains isolated in the first and the second diagnosis 8 . Also, patient #1126 presented the shortest time to relapse and the latest age of diagnosis -32 years -similar to the controls (Table 3).…”

“…Based on comparative analysis between M. leprae genomes obtained in the first and second infection of each patient, the authors concluded for two cases of relapses caused by same strain and one case of reinfection caused by a different M. leprae strain 8 .…”

“…However, to date, no attempt has been made to translate this genetic knowledge into the definition of an individual susceptibility profile that could help explain complex leprosy phenotypes such as recurrence/reinfection. Here we genotyped 19 Single Nucleotide Polymorphism (SNP) distributed across these 11 loci associated with leprosy per se on six leprosy cases from the U-MDT/CT-BR cohort: two cases previously reported as relapses and one as reinfection 8 , and three matching, successfully treated leprosy patients that did not relapse. Allele frequencies were compared across the two groups and with publicly available databases.…”

Phylogenomics and antimicrobial resistance of the leprosy bacillus Mycobacterium leprae

“…For decades, the sole source of M. leprae DNA suitable for genomics was from bacteria isolated 12 months after infection of armadillos or mice. Recently, we have developed and optimized methods that enable M. leprae DNA to be extracted directly from fresh or formalin-fixed skin biopsies from leprosy patients 28 . These methods include enrichment of M. leprae DNA by array capture 17 but this is less practical for large population-based investigations.…”

“…RpoB numbering is based on M. leprae , E. coli numbering in brackets: 51 (126), 52 (127), 171 (246), 200 (275), 432 (507), 433 (508), 441 (516), 448 (523), 451 (526), 456 (531), 681 (756), 791 (866). 1 Coverage below the threshold, both “2188” isolates come from the same patient but after an interval of 7 years 28 …”