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.

Mycobacterium lepromatosis was isolated and propagated in mouse footpads. We developed a real-time polymerase chain reaction (PCR) assay using a unique repetitive element, RLPM. We validated this and the RLEP PCR Mycobacterium leprae assay for the clinical diagnosis of leprosy.

Leprosy is a curable disease. A delay in diagnosis and treatment, however, may lead to permanent nerve damage that cannot be reversed by antibiotic treatment. Multidrug therapy has reduced the global prevalence of leprosy by 90% in the last 3 decades, but the incidence of disease remains high, with >200 000 new cases annually [1].

The leprosy spectrum ranges from tuberculoid leprosy, with high cell-mediated immunity and a low bacterial index, to lepromatous leprosy (LL), with a high bacterial index and low cell-mediated immunity. Across the spectrum, infection occurs primarily in the skin, mucosal membranes, and the peripheral nervous system; however, the diagnosis and treatment of leprosy is often complicated by immune-mediated reactional episodes. In addition, a form of leprosy with diffuse cutaneous infiltration of the skin by histiocytes and no visible skin lesions, known as diffuse lepromatous leprosy (DLL), is not uncommon in western Mexico and the Caribbean region. Patients with DLL who have a very high bacterial load can experience severe, life-threatening reactions called Lucio reactions (or erythema necroticans) [2, 3]. The Lucio reaction is a vasculopathy resulting in cutaneous infarcts, which produce severe necrosis and desquamation of the skin [4].

Mycobacterium leprae was the only etiological agent known for leprosy until another closely related species, Mycobacterium lepromatosis, was identified by means of a nested polymerase chain reaction (PCR) assay in 2 Mexican patients with DLL [5, 6]. Subsequent studies using the same nested PCR assay reported a high prevalence of M. lepromatosis from Mexico (63.21%) and south Brazil (21.7%) [7, 8]. These findings suggested that M. lepromatosis, rather than M. leprae, may be the cause of DLL and Lucio reactions.

In the current study, we isolated the first strain of M. lepromatosis, propagated it in mouse footpads (MFPs), and sequenced its genome. Comparative genomics identified a multicopy genomic element specific to M. lepromatosis, RLPM, for which a real-time quantitative PCR (qPCR) assay was developed. This RLPM qPCR assay, along with a previously developed RLEP qPCR for M. leprae [9], were validated as laboratory-developed molecular diagnostic assays for infectious diseases [10]. We used these assays for the detection of each causative agent of leprosy among cohorts of clinical specimens from Mexico, the Philippines, and the United States. We also examined samples from wild 9-banded armadillos from areas in the United States where they are known to carry leprosy.

METHODS

Animals

Experiments using mice and armadillos were performed in accordance with US Public Health Service Policy on Humane Care and Use of Laboratory Animals and the US Department of Agriculture Animal and Plant Health Inspection Service. The Institutional Animal Care and Use Committee reviewed and approved all protocols.

Isolation of M. lepromatosis in Athymic Nude MFPs

A skin biopsy specimen was obtained (with informed consent) from a patient of Costa Rican origin who presented at the National Hansen’s Disease Program (NHDP) with DLL. The biopsy was homogenized in Roswell Park Memorial Institute medium (Sigma), and a total of 50 µL of suspension (2.3 × 10⁵ acid-fast bacilli [AFB]/mL) was inoculated into the hind footpads of athymic nude mice, as described elsewhere [11]. The MFPs were harvested 6 months after inoculation, and M. lepromatosis bacilli were passaged into 4 additional athymic nude mice. Freshly harvested organisms were inoculated onto culture medium (detailed in Results) and incubated at 33°C and 37°C, each for 60 days [12].

Whole-genome Sequencing of M. lepromatosis

The M. lepromatosis–infected MFPs were homogenized in Roswell Park Memorial Institute medium, and mouse DNA was reduced by treating the suspension with 0.1N sodium hydroxide [13]. DNA was extracted using a DNeasy kit (Qiagen) with overnight proteinase K lysis at 56°C to ensure lysis of the bacteria, and 2 ng of DNA was converted into a genomic library using the Nextera XT kit (Illumina) after amplification of genomic DNA using the GenomiPhi kit (GE Healthcare). Libraries were sequenced by means of paired-end sequencing using the V3 kit (two 300–base pair [bp] reads) on a MiSeq instrument (Illumina).

Development of a qPCR Assay for M. lepromatosis

Comparative genomic analysis of the available M. lepromatosis genomes identified a multicopy element, designated RLPM. A TaqMan probe (5’-AA GTG ACG CG GGC GTG GATT- 3’) and primers (forward, 5’-TTG GTG ATC GGG GTC GGC TGG A-3’; reverse, 5’-CCC CAC CGG ACA CCA CCA ACC-3’) were designed from the areas specific to M. lepromatosis, using Primer3 software [14]. PCR was performed on DNA using an initial denaturation at 95°C for 3 minutes, followed by 40 cycles of denaturation for 30 seconds at 95°C and annealing/extension at 60°C for 30 seconds, using the 7500 RealTime PCR System (Applied Biosystems, ThermoFisher). Assay specificity was determined by testing purified genomic DNA from 17 mycobacterial species, 11 of which are associated with human diseases, 3 gram-positive microorganisms associated with skin infections, and Escherichia coli [9].

Standard Curves

DNA extracted from 2 × 10⁶M. leprae or M. lepromatosis into a 100-µL volume was serially diluted (4-fold; 12 times), and 2.5 µL from each dilution was added to the PCR reaction. Standard curves were plotted from 3 different samples, counted independently in duplicate, and repeated 8 times. Each PCR assay was performed in duplicate using TaqMan technology. This standard-curve DNA also served as an exogenous internal amplification control for assessing the quality of the sample DNA extraction and the efficiency of amplification.

Limit of Detection

A standard curve from a lower 4-fold serial dilution (7812.50 to 0.48 bacilli) of the internal amplification control was generated, and linear regression analysis with >98% confidence was used to define the limit of detection (LOD) and the corresponding cycle threshold (Ct) value for qualitative diagnostic assays [10].

Tolerance and Acceptability Limits

Tolerance and acceptability limits were determined by calculating the mean and standard deviation (SD) of LOD Ct values over 12 repeats of the lower end of the standard curve. A temporary target allowable range was set to 2 SDs.

Interference and Accuracy Analysis

The effects of interference and accuracy were evaluated by spiking DNA from approximately 12.20 bacilli (M. leprae or M. lepromatosis) into each patient sample DNA (n = 180) tested at the NHDP Clinical Laboratory in 2017 (12 runs). A known quantity of MFP-derived tissue was used as a positive control alongside the clinical samples. The Ct values and an AFB count of each species were recorded from the negative and positive samples. Accuracy of qualitative assays and iterance values were determined by comparing the spiked value (12.20 genomic DNA templates per reaction) to the actual values determined by the assays.

Precision Analysis

The interassay precision (reproducibility) was assessed by calculating the coefficient of variation for all runs combined, using the standard-curve data.

Clinical Specimens

All patient specimens used in this study were archived excess diagnostic material and were exempted by the institutional review boards. Analyses were done only on bacterial nucleic acids and not on host nucleic acids. The following types of specimens evaluated are outlined below.

Formalin-fixed, Paraffin-embedded Biopsy Specimens

Ten 10-µm serial sections were cut from formalin-fixed, paraffin-embedded (FFPE) blocks of 4-mm skin lesion biopsy specimens. The sections were deparaffinized by xylene treatment followed by gradual rehydration. After centrifugation at 12 000g, tissue was resuspended in DNeasy kit lysis buffer (Qiagen). DNA was extracted as described above and eluted in a 100-µL volume, and 2.5 µL was added to the PCR reaction.

Slit-skin Smears

Slit-skin smear (SSS) samples were fixed in 70% ethanol. The samples were centrifuged, and the pelleted tissue material was resuspended in lysis buffer (Qiagen). DNA was extracted as described above.

Archived Fite-stained Histological Sections

Fite-stained histological sections of skin biopsy specimens obtained from 1968 to 1994 were examined. The blocks for these specimens were lost in the 1994 Northridge earthquake [3]. The clinical diagnosis was blinded before laboratory testing. Total DNA was extracted with 3 additional freeze-boil cycles after the enzymatic lysis of host tissue to maximize DNA yield. Total DNA was eluted in 30 µL, and 1–2 µL was used for PCR.

Wild Armadillo Samples

Blood and reticuloendothelial tissue were collected from 645 wild 9-banded armadillos from 8 locations in the United States. DNA was extracted from lymph nodes or spleens of animals seropositive for PGL-1 or LID-1 antigens (n = 106), as described elsewhere [15], and tested in duplicate for M. lepromatosis and M. leprae infection.

RESULTS

Isolation of M. lepromatosis in Athymic Nude MFPs

M. lepromatosis was isolated from a biopsy specimen from a patient with DLL and confirmed by amplification and sequencing of a genomic region (hemN) specific to M. lepromatosis but absent in M. leprae [16] and by sequencing the 16S ribosomal RNA gene. This isolate, designated NHDP-385, was negative when tested with the M. leprae–specific RLEP qPCR [9]. M. lepromatosis grew in athymic nude MFPs but did not grow when viable bacteria were inoculated on Löwenstein-Jensen medium and Middlebrook 7H11 agar medium, which are routinely used for growing cultivable species of mycobacteria. M. lepromatosis also did not multiply on blood agar, thioglycollate broth, and tryptic soy broth (soybean-casein digest medium).

Genome Sequencing of M. lepromatosis NHDP-385

A total of 14.67 million paired-end reads were obtained on sequencing of M. lepromatosis NHDP-385 (National Center for Biotechnology Information BioSample accession no. SAMN12872980). High-quality (quality score [Q] > 30) reads were aligned against the mouse genome; approximately 76% of reads were 100% homologous to the mouse genome so were excluded from further analysis. The remaining approximately 24% were assembled into 77 large contigs, along with an additional 26 contigs of shorter length (<500 bp), with an assembly of L50 = 16 (50% of assembly comprised in 16 largest contigs) and % guanine/cytosine content (GC%) = 57.87 (Figure 1 and Table 1). We used a previously published M. lepromatosis genome assembly of the Mx1-22A strain for genome-wide comparison [16]. A 136× comparative genome coverage was obtained, and 239 variants between the 2 genomes were identified.

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.

Table 1.

Genome Sequence Summary

		Mycobacterium lepromatosis
Feature	Mycobacterium leprae (TN)	EPFL Mx1-22A Strain	NHDP-385 Strain
Source	MFPs	Clinical sample	MFPs
Size	3 268 212	3 206 741	3 258 667
Contigs	Closed genome	126	77 (+26 small contigs)
GC%	57.79	57.89	57.87

Abbreviations: EPFL, Ecole Polytechnique Fédérale de Lausanne; GC%, % guanine/cytosine content; MFPs, mouse footpad; NHDP, National Hansen's Disease Program; TN, Tamil Nadu strain of M. leprae.

Development of a M. lepromatosis Specific qPCR Assay

Comparative genomic analysis identified a genomic sequence that had much higher coverage (>2000×) than the average genome coverage (approximately 136×). We hypothesized that these areas might represent multicopy regions in the genome and thus a potential target for a highly sensitive qPCR assay. Basic Local Alignment Search Tool (BLAST) analysis of these high-coverage regions revealed a unique, approximately 200-bp region, designated RLPM, which was present in 6 contigs of the reference Mx1-22 genome assembly and in 5 contigs of the NHDP-385 assembly. Therefore, we designed a M. lepromatosis specific qPCR assay targeting this sequence. Sequencing of the qPCR products confirmed that the appropriate targets were amplified.

The RLPM assay was positive for M. lepromatosis but negative when tested against the purified genomic DNA from 17 mycobacterial species, including M. leprae and 10 other mycobacteria associated with human diseases, 3 gram-positive microorganisms associated with skin infections, and E. coli (Table 2). Amplification of genomic DNA from each species was verified in a separate PCR assay that detected 16S recombinant DNA. The specificity of the M. leprae RLEP assay was previously determined using the same potential sources of false-positivity [9, 17]. Neither assay reacted with human, mouse, or armadillo DNA.

Table 2.

Specificity of RLPM Quantitative Polymerase Chain Reaction for Mycobacterium lepromatosis Detection

	Assay Result
Organism	16S rDNA	RLPM
Mycobacterium species
M. lepromatosis	Positive	Positive
M. leprae	Positive	Negative
M. avium	Positive	Negative
M. bovis	Positive	Negative
M. bovis BCG	Positive	Negative
M. chelonei	Positive	Negative
M. flavescens	Positive	Negative
M. gordonae	Positive	Negative
M. intracellulare	Positive	Negative
M. kansasii	Positive	Negative
M. lepraemurium	Positive	Negative
M. lufu	Positive	Negative
M. marinum	Positive	Negative
M. phlei	Positive	Negative
M. simiae	Positive	Negative
M. smegmatis	Positive	Negative
M. tuberculosis	Positive	Negative
M. ulcerans	Positive	Negative
Clostridium perfringens	Positive	Negative
Staphylococcus epidermidis	Positive	Negative
Streptococcus pyogenes	Positive	Negative
Escherichia coli	Positive	Negative

Abbreviation: BCG, Bacillus Calmette–Guérin; rDNA, recombinant DNA; RLPM, repetitive element in M. lepromatosis.

Validation of the RLPM and RLEP assays for the diagnosis of leprosy was performed according to Clinical Laboratory Improvement Amendments (CLIA) guidelines for molecular diagnosis of infectious diseases [10, 18]. Standard curves were prepared from M. leprae and M. lepromatosis DNA and tested using the RLEP and RLPM assays. As shown in Figure 2A and 2B, Ct values were plotted against the log of the bacterial count. Nonlinear regression analyses (performed with GraphPad Prism software; version 8.2.0) had excellent correlation (RLPM, r² < 0.9961; RLEP, r² < 0.9991). The mean (SD) percent efficiency of the RLEP and RLPM assays was 94.829 (3.273) and 93.058 (6.782), respectively.

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.

The percentages of detectable Ct values in the lower dilutions were plotted against the number of bacilli in the samples (Figure 2C) to determine the LOD. The RLPM qPCR assay could consistently (100% of the time) detect 122 M. lepromatosis in the specimen, corresponding to 3.05 bacilli per reaction. The RLEP qPCR assay could consistently detect 30 M. leprae in the specimen, corresponding to 0.76 bacilli per reaction. For qualitative assays, mean (SD) cutoff Ct values of 34.18 (0.49) and 34.84 (0.85) are used to diagnose M. lepromatosis and M. leprae, respectively, at the LOD concentration (Table 3). Interassay precision (reproducibility) was obtained by calculating the coefficient of variation (CV%) for all runs combined, and ranged from 0.65% to 2.44%.

Table 3.

Precision of Assays Across the Range of Detection and Inter-run Variation

		RLEP Assay (n = 12 Runs)		RLPM Assay (n = 12 Runs)
No. of Bacilli in Sample (10 sections)	No. of Bacilli in Reaction (2.5 μL)	Ct Mean (SD)	CV%	Ct Mean (SD)	CV%
7.81 × 103	195.31	26.65 (0.28)	1.05	28.71 (0.26)	0.91
1.95 × 103	48.83	28.71 (0.32)	1.11	30.83 (0.26)	0.84
4.88 × 102	12.21	30.71 (0.20)	0.65	32.83 (0.79)	2.41
1.22 × 102	3.05	32.73 (0.56)	1.71	34.18 (0.49)	1.43
3.05 × 101	0.76	34.84 (0.85)	2.44	35.53 (1.03)	2.9
7.63	0.19	36.35 (0.71)	1.95	…	…
1.91	0.05	…	…	…	…
0.48	0.01	…	…	…	…

Abbreviations: Ct, cycle threshold; CV, coefficient of variation; RLPM, repetitive element in M. lepromatosis; SD, standard deviation.

Tolerance and acceptability limits were determined by calculating the mean and SD of LOD Ct values over 12 repeats. The final target and SDs were graphed on Levey-Jennings plots. The means of the data were used as the expected values. As shown in Figure 3A and 3B, all control results were within 2 SDs of the mean target value and are therefore in range using Westgard multirule analysis [19].

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.

To determine possible interference and quantitative precision, samples (n = 180) were spiked with DNA from 12.20 bacilli (M. leprae or M. lepromatosis). Both assays precisely counted M. lepromatosis (mean count [SD], 12.47 [5.43]) and M. leprae (11.96 [4.09]) during the 12 assays performed over a year, and molecular counts did not differ significantly from the spiked values.

Molecular Diagnosis of Clinical and Epidemiological Specimens

We used the RLPM and RLEP assays for the detection and differentiation of M. lepromatosis and M. leprae in clinical and armadillo samples. As shown in Figure 4, 15 of 47 FFPE samples from Mexican patients (31.91%) were positive for M. lepromatosis, compared with 19 (40.43%) that were infected with M. leprae. Interestingly, 2 samples (4.26%) were infected with both organisms. Both assays were negative for the remaining 11 (23.40%) samples. In addition, we tested 180 SSS samples of clinically confirmed leprosy cases from the Philippines. All were positive for M. leprae; however, none were positive for M. lepromatosis. Of the 218 US samples submitted to the NHDP during 2017 for qPCR testing, 69 (31.65%) were positive for M. leprae, and, 3 (1.38%) were infected with M. lepromatosis. The remaining 146 US samples were negative for both organisms.

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.

Nine-banded armadillos are highly susceptible to leprosy and approximately 16% of wild armadillos in the southern US harbor M. leprae infection [15, 20]. Considering the similarities between M. leprae and M. lepromatosis, it is likely that armadillos can acquire M. lepromatosis infection. However, we found no M. lepromatosis–infected animals in this cohort (Figure 4).

Association With Lucio Reactions

We examined 15 archived Fite-stained histological sections of skin biopsy specimens, including 9 with Lucio reactions, 2 with DLL, and 4 with LL without DLL or Lucio reactions (Table 4). Owing to the age of the specimens and very limited material, sufficient DNA was obtained from only 10 patients’ sections. Of these, M. leprae was detected in 2 LL and 2 Lucio reaction sections; M. lepromatosis was found in 1 LL, 1 DLL, and 3 Lucio reaction sections. Both species were present in 1 LL specimen.

Table 4.

Association of Mycobacterium leprae and Mycobacterium lepromatosis With Lucio Reactions

Patient No./Sex/Age at Diagnosis, y	Place of Birth	Clinical-pathological Diagnosis	Species of Mycobacterium
1/M/45	Mexico	LL (control)	Both
2/M/30	Mexico	LL (control)	M. leprae
3/F/25	Mexico	LL (control)	M. lepromatosis
4/M/29	Mexico	LL (control)	M. leprae
5/M/31	Guanajuato, Mexico	DLL	M. lepromatosis
6/F/35	Jalisco, Mexico	DLL	No DNA
7/M/30	Jalisco, Mexico	Lucio reaction	M. lepromatosis
8/M/29	Guerrero, Mexico	Lucio reaction	M. lepromatosis
9/M/58	Mexico	Lucio reaction	M. leprae
10/M/36	Sinaloa, Mexico	Lucio reaction	M. leprae
11/M/45	Sinaloa, Mexico	Lucio reaction	No DNA
12/F/41	Jalisco, Mexico	Lucio reaction	No DNA
13/M/25	Jalisco, Mexico	Lucio reaction	No DNA
14/M/31	Pecos, Texas	Lucio reaction	No DNA
15/F/30	Sinaloa, Mexico	Lucio reaction	M. lepromatosis

Abbreviations: DLL, diffuse lepromatous leprosy; F, female; LL, lepromatous leprosy; M, male.

DISCUSSION

Leprosy is no longer an incurable affliction; however, this ancient disease still presents challenges. For over a century, M. leprae was believed to be the exclusive etiological agent of leprosy until M. lepromatosis was identified in 2008 [5]. M. lepromatosis has now been reported in Canada, Brazil, Singapore, and Myanmar [21–24]. We are the first laboratory to isolate this new species and propagate this isolate, NHDP-385, in athymic nude MFPs. Establishment of M. lepromatosis in MFPs will be instrumental for comprehensive comparative studies elucidating the pathogenicity of this leprosy-causing species.

Because leprosy bacilli do not grow in axenic media, the diagnosis of leprosy relies mainly on clinical symptoms and histopathological interpretation of skin biopsy specimens. Unfortunately, the expertise and experience needed for clinical diagnosis of leprosy is becoming rare. Moreover, although microscopic detection of AFB in a peripheral nerve is pathognomonic for leprosy, it does not differentiate between M. leprae and M. lepromatosis. New technologies to complement clinical and histopathological diagnosis and monitor transmission are needed. Advanced genomic techniques, especially PCR-assisted diagnoses [25–30], can help bridge these gaps and are now deployed in leprosy control programs with great benefit. The addition of the RLPM qPCR assay to this arsenal will enable detection of M. lepromatosis in otherwise M. leprae PCR-negative leprosy cases.

The RLPM qPCR assay detects a unique multicopy element in the M. lepromatosis genome and has an LOD of approximately 3.0 M. lepromatosis bacteria. The RLEP qPCR assay, which also detects a multicopy element, has an LOD of approximately 0.80 M. leprae bacteria. This difference in detection rate is likely due to the copy number of the target templates. There are 5–6 copies of RLPM in the M. lepromatosis genome, compared with 29–36 copies of RLEP in the M. leprae genome. Regardless, both repetitive elements render the assays more sensitive than detection of a single copy gene and enable an increased likelihood of diagnosing paucibacillary cases.

The RLPM and RLEP assays successfully detected M. lepromatosis and M. leprae in a variety of specimen preparations, including FFPE tissues, SSS samples, Fite-stained sections, and ethanol-fixed tissues. They are specific, reproducible, and easily adaptable to large-scale batch processing of samples. At the LOD, both assays have a 100% detection rate. Furthermore, we validated both assays according to CLIA guidelines, which regulate testing of laboratory-developed assays. Consequently, the Joint Commission accredited the NHDP Clinical Laboratory for molecular diagnosis of leprosy in the United States.

Because M. lepromatosis was first discovered in patients with DLL, there was speculation that this species may be the cause of DLL and Lucio reactions. However, on examination of archived histological sections, we found that Lucio reactions occurred in patients infected with either M. leprae or M. lepromatosis. Although the total number of cases examined was small, these results have particular merit because the patients’ clinical status had been carefully assessed and documented [3]. Our findings suggest a host component, rather than the species of Mycobacterium, as the risk factor for this reaction.

Molecular methods, such as our RLPM and RLEP qPCR assays, will help to advance clinical diagnosis and surveillance of leprosy infections [28, 29]. By CLIA-validating both assays, we have ensured that the tests’ LOD, tolerance, acceptability, accuracy, and precision meet the stringent standards of clinical laboratory practice. Using these resources, we also showed that the Lucio reaction can be caused by either M. leprae or M. lepromatosis. Finally, our laboratory successfully established M. lepromatosis in MFPs, providing a continuous source of this species for future studies.

Notes

Acknowledgments. The authors thank Vilma Marks, Heidi Zhang, Kyle Andrews, and Roena Stevenson, as well as the GeneLab staff at Louisiana State University School of Veterinary Medicine, Baton Rouge.

Disclaimer. The views expressed in this article are solely the opinions of the authors and do not necessarily reflect the official policies of the U. S. Department of Health and Human Services or the Health Resources and Services Administration, nor does mention of the department or agency names imply endorsement by the U.S. Government.

Financial support. The National Institutes of Health, National Institute of Allergy and Infectious Diseases funded the research for this article through the Interagency Agreement No. AAI15006 with the Health Resources and Services Administration, Healthcare Systems Bureau, National Hansen’s Disease Program.

Potential conflicts of interest. The authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest.