Isolation of Mycobacterium lepromatosis and Development of Molecular Diagnostic Assays to Distinguish Mycobacterium leprae and M. lepromatosis

Abstract

Background: Mycobacterium leprae was thought to be the exclusive causative agent of leprosy until Mycobacterium lepromatosis was identified in a rare form of leprosy known as diffuse lepromatous leprosy (DLL).

Methods: We isolated M. lepromatosis from a patient with DLL and propagated it in athymic nude mouse footpads. Genomic analysis of this strain (NHDP-385) identified a unique repetitive element, RLPM, on which a specific real-time quantitative polymerase chain reaction assay was developed. The RLPM assay, and a previously developed RLEP quantitative polymerase chain reaction assay for M. leprae, were validated as clinical diagnostic assays according to Clinical Laboratory Improvement Amendments guidelines. We tested DNA from archived histological sections, patient specimens from the United States, Philippines, and Mexico, and US wild armadillos.

Results: The limit of detection for the RLEP and RLPM assays is 30 M. leprae per specimen (0.76 bacilli per reaction; coefficient of variation, 0.65%-2.44%) and 122 M. lepromatosis per specimen (3.05 bacilli per reaction; 0.84%-2.9%), respectively. In histological sections (n = 10), 1 lepromatous leprosy (LL), 1 DLL, and 3 Lucio reactions contained M. lepromatosis; 2 LL and 2 Lucio reactions contained M. leprae; and 1 LL reaction contained both species. M. lepromatosis was detected in 3 of 218 US biopsy specimens (1.38%). All Philippines specimens (n = 180) were M. lepromatosis negative and M. leprae positive. Conversely, 15 of 47 Mexican specimens (31.91%) were positive for M. lepromatosis, 19 of 47 (40.43%) were positive for M. leprae, and 2 of 47 (4.26%) contained both organisms. All armadillos were M. lepromatosis negative.

Conclusions: The RLPM and RLEP assays will aid healthcare providers in the clinical diagnosis and surveillance of leprosy.

Keywords: Mycobacterium leprae; Mycobacterium lepromatosis; leprosy diagnostic assay; real-time PCR.

Figure 1.

Genome-wide comparison of Mycobacterium lepromatosis NHDP-385 isolated in mouse footpads (MFPs) to the earlier sequenced strain Mx 1-22A (GenBank no. JRPY00000000.1). Colored blocks represent locally collinear blocks; red lines, contig boundaries; proportion of block filled with color, the percentage of the block filled with color equals the percent identity with the Mx 1-22A assembly; white spaces indicate insertions or SNPs. A total 196 SNPs and 41 indels are identified. Abbreviations: NCBI, National Center for Biotechnology Information; NHDP, National Hansen’s Disease Program; SNPs, single-nucleotide polymorphisms.

Figure 2.

Standard curves and limit of detection. A, B, Standard curves were plotted between cycle threshold (Ct) values from 4-fold serial dilutions of 3 samples and microscopic counts for Mycobacterium lepromatosis (A) and Mycobacterium leprae (B) . C, For the limit of detection (LOD), a series of 8 samples was prepared by diluting a high-concentration standard. Each sample was tested 8 times in duplicate. The solid vertical line indicates the RLEP LOD of 30 bacilli per specimen (0.76 bacilli per reaction), and the dashed vertical line, the RLPM LOD of 122 bacilli per specimen (3.05 bacilli per reaction). Abbreviations: RLEP, Repetitive element in M. leprae; RLPM, repetitive element in M. lepromatosis.

Figure 3.

Tolerance and acceptability limits of the RLEP and RLPM assays. The low-positive controls were monitored over 12 separate runs using a Levey-Jennings plot. The plot shows the target value (RLEP cycle threshold [Ct], 34.63; RLPM Ct, 34.55 solid line) as well as expected limits of 1 and 2 standard deviations (SDs). Abbreviations: NHDP, National Hansen’s Disease Program; PCR, polymerase chain reaction; RLEP, repetitive element in M. leprae; RLPM, repetitive element in M. lepromatosis; SD, standard deviation.

Figure 4.

Prevalence of Mycobacterium leprae and Mycobacterium lepromatosis. Clinical specimens from Mexico (n = 47), Philippines (n = 180), and the United States (n = 218; tested by the National Hansen’s Disease Program [NHDP] in 2017), and samples from US wild armadillos (n = 106) were tested using RLEP and RLPM quantitative polymerase chain reaction (qPCR) assays for M. leprae and M. lepromatosis, respectively. Abbreviations: RLEP, repetitive element in M. leprae; RLPM, repetitive element in M. lepromatosis.