Molecular Drug Susceptibility Testing and Genotyping of Mycobacterium leprae Strains from South America

Abstract

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).

TEXT

The success of leprosy control programs relies heavily upon multidrug therapy (MDT) (rifampin, dapsone, and clofazimine). Therefore, it is important that drug resistance trends be monitored periodically and Mycobacterium leprae strains genotyped in order to understand transmission patterns and genetic diversity. In the present study, strains of M. leprae from Bolivia, Brazil, Uruguay, and Venezuela were genotyped using single nucleotide polymorphism (SNP) analysis (17, 18), and their drug resistance-determining regions (DRDRs) in the rpoB, folP1, and gyrA genes were sequenced. In parallel, a real-time PCR assay that targets the most frequent sites of drug resistance in rpoB and folP1 was established.

A total of 233 M. leprae strains were obtained from patient biopsy specimens or from slit skin smears; the majority of the patients were from Venezuela (n = 197), and the remainder were from Bolivia (n = 10), Brazil (n = 24), and Uruguay (n = 2). Reference DNA from drug-susceptible strains TN, Br4923, NHDP63, and Thai53 was used as a control for the sequencing and the real-time PCR-based TaqMan assay. A known rifampin-resistant strain, 85054 (French West Indies), with an rpoB mutation (Ser425Leu) (8), was included as a positive control. This strain also has a dapsone resistance mutation (Pro55Leu) in folP1.

DNA extraction from skin biopsy specimens/slit skin samples was carried out by the “freeze-boiling” method as described previously (24), followed by PCR amplification and DNA sequencing (18). Primers used for analyzing the drug resistance-associated loci are shown in Table 1. The sequences, obtained from an ABI 3130xl genetic analyzer (Applied Biosystems, Life Technologies Corporation, CA), were aligned against the sequences of the TN reference strain using CodonCode Aligner software (Dedham, MA) to identify polymorphisms. SNP genotyping was done as described previously (17, 18). The frequency of SNP7614 (presence of “T” at this position in subtype 3I strains) (18) was also determined.

Table 1.

Primers used for molecular drug susceptibility testing

Primer name	Primer sequence (5′–3′)
rpoB47	TGG TCC GGG AGC GGA TGA C
rpoB48	AAC CGA TCA GAC CTA TGT TCG
folP1-F2	AAC TGA TGC TGC TTC TCG TG
folP1-R2	CCC TGT GCT GCA AGT TCT TT
gyrA-F3	GGT ACA TCG TCG GGT CTT GT
gyrA-R3	GCC CAT CAA CCA AGG GAT AC

TaqMan probe assays using real-time PCR were performed in triplicate. Each 20-μl reaction mixture contained 100 nM probe, 200 nM primers (Table 2) in 1× TaqMan PCR master mix (Applied Biosystems, CA), and 2 μl of DNA. PCR mixtures were subjected to thermal cycling for 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C with a 7900HT Fast real-time PCR system (Applied Biosystems, CA). An increase in fluorescence indicates perfect complementarity between template and probe. The cutoff is decided based upon the known controls (horizontal red line in Fig. 1). A rise in fluorescence above the “cutoff” only in the presence of the wild-type probe indicates wild-type template sequences, while strains harboring target mutations show an “above-cutoff” rise in fluorescence when used with the “mutation-specific probe” and not with the wild-type probe. No increase in fluorescence or an increase below cutoff while using either set of probes indicates an uninterpretable result.

Table 2.

Primers and probes used for real-time PCR-based hybridization assay

Primer/probe name (target)	Sequence (5′–3′)a
qPCR-Rif-forward (rpoB)	AGC CAG CTG TCG CAG TTC
qPCR-Rif-reverse (rpoB)	GCT CAC GCG ACA AAC CAC
qPCR-Dap-forward (folP1)	ACC TTG ATC CTG ACG ATG CT
qPCR-Dap-reverse (folP1)	TAA TCCCCT GTG CTG CAA GT
Probe-Rif-Resistant
(Ser425Leu mutation)	FAM-CGG CTG TTG GCG CT-MGB
Probe-Rif-Sensitive	FAM-CGG CTG TCG GCG CT-MGB
Probe-Dap-Sensitive	FAM-CGA ATC GAC CCG GCC CGG T-TAMRA

^aUnderlining denotes the site of the target mutation in the probe. FAM, 6-carboxyfluorescein; MGB, minor groove binder; TAMRA, 6-carboxytetramethylrhodamine.

Fig. 1.

PCRs performed with a wild-type probe versus rifampin (Rif)-sensitive M. leprae (perfectly complementary sequences) show an exponential increase in fluorescence; the same is true for reactions with the strains harboring the target mutation and the corresponding detection probe (matching colors). However, reactions performed with a mutation probe versus a wild-type template or with a wild-type probe versus a mutated template show no rise in fluorescence or a rise that is below the cutoff (horizontal red line). The control strain TN is a rifampin-susceptible strain with wild-type sequences, while strains 85054 and B23 harbor the Ser425Leu (TCG→TTG) mutation targeted by the mutation probe (red labeling). Strain V4S exhibits wild-type sequences at the target region in rpoB. ΔRn is the normalized reporter signal minus the baseline signal.

The aim of the present study was to use molecular drug susceptibility testing (MDST) to survey drug resistance and to determine genotypes of M. leprae strains from South America. Except for three relapse cases (defined according to WHO criteria [26]), all of the remaining strains were from newly diagnosed leprosy cases of either sex ranging in age from 13 to 74 years. Patients with various clinical presentations of leprosy were biopsied in the field or in private or government leprosy clinics in rural as well as urban areas of the 20 different states of Venezuela as well as 3 other South American countries.

The results of all 233 cases are shown in Table 3. Among the 230 strains from new cases of leprosy, we found no drug resistance-associated mutations in the rpoB, folP1, and gyrA genes. However, among the three relapsed cases (V4B and V4S from Venezuela and B23 from Brazil), two harbored drug resistance-associated mutations: strain V4B exhibited the folP1 mutation Pro55Leu, while the Brazilian strain (B23) had two mutations, Pro55Leu in folP1 and Ser425Leu in rpoB. These mutations have previously been shown to confer resistance to rifampin and dapsone, respectively. These two patients exhibited recurring lesions; indeed, the Brazilian case also presented with grade 2 disability at the time this biopsy specimen was taken. Strain V4S was obtained from a patient who displayed clinical signs of leprosy three times in his life and became bacteriologically negative following each treatment. This strain exhibited no mutation in the DRDRs of the rpoB, folP1, and gyrA loci, consistent with the treatment outcome.

Table 3.

Results of drug resistance surveillance

Country of origin	Total no. of strains	No. of strains containing:
		rpoB		folP1		gyrA
		Wild type	Mutation	Wild type	Mutation	Wild type	Mutation
Bolivia	10	10	0	10	0	10	0
Brazil	24c	23	1a	23	1b	24	0
Venezuela	197d	197	0	196	1b	197	0
Uruguay	2	2	0	2	0	2	0
Total	233	232	1	231	2	233	0

^aSer425Leu (TCG→TTG).

^bPro55Leu (CCC→CTC).

^cOne from a relapse case.

^dTwo from relapse cases.

For settings lacking a sequencing facility, a real-time PCR-based TaqMan assay for analyzing the DRDRs in rpoB and folP1 was also developed, and its performance was evaluated by using PCR sequencing results as the gold standard. One hundred sixty-eight of these samples were tested by the TaqMan assay. The results were in complete agreement with the sequencing results. However, this method requires more M. leprae DNA than is used for sequencing, as each mutant allele is targeted separately in real-time PCR.

Subtype 3I was found to be the most prevalent, accounting for nearly three-quarters of the strains (172/233, or 73.8%), followed by subtypes 4P (27/233, or 11.6%), 1D (16/233, or 6.9%), 4N (14/233, or 6%), and 4O (4/233, or 1.7%). Strains from relapsed cases belonged to SNP subtypes 4N (strains V4B and V4S) and 3I (strain B23). The presence of subtype 1D strains in Venezuela is most likely a reflection of Asian migrants, as this subtype is prevalent in Southeast Asia (18). SNP7614 in the gyrA locus (18) was found in all 172 strains belonging to subtype 3I, exhibiting “T” at position 7614, while the strains belonging to the other subtypes exhibited “C” at the corresponding position. It should be noted that this synonymous SNP is not associated with drug resistance but that interrogating the DRDR in gyrA provides information about drug resistance and the strain genotype simultaneously.

Though drug resistance among new cases is still rare (14), several reports from around the world have confirmed the emergence of resistance to one or more antileprosy drugs, mostly among relapsed cases (1–16, 19–23, 25, 27, 28). Despite the common use of fluoroquinolones for treating a wide range of bacterial infections, we found no gyrA mutations associated with resistance, which is reassuring for fluoroquinolone use in leprosy control programs.

In conclusion, the present MDST study of 230 new cases of leprosy found no indication of resistance to rifampin, dapsone, or ofloxacin. The absence of drug resistance among the new cases indicates the success and effectiveness of leprosy control programs. However, the gradual selection of resistance-associated mutations among the treatment failure/relapsed cases (mainly as a result of noncompliance, as seen in the drug-resistant cases in the present study) could be a concern. Presently, while the proportion of such strains is very low, the risk of their transmission to healthy contacts cannot be ignored. Such cases may then present with primary resistance (14, 22). In order to check the spread of such strains by instituting effective therapy, the relapsed cases or the treatment failure cases, where the suspicion of resistance is higher, should routinely be analyzed by MDST.