Molecular Detection of Multidrug-Resistant Mycobacterium Leprae From

Journal of Global Antimicrobial Resistance 12 (2018) 214–219

Contents lists available at ScienceDirect

Journal of Global Antimicrobial Resistance

journal homepage: www.elsevier.com/locate/jgar

Molecular detection of multidrug-resistant Mycobacterium leprae from

Indian leprosy patients

a, a a a a

Mallika Lavania *, Itu Singh , Ravindra P. Turankar , Madhvi Ahuja , Vinay Pathak ,

a b c d e

Utpal Sengupta , Loretta Das , Archana Kumar , Joydeepa Darlong , Rajeev Nathan , f

Asha Maseey

Stanley Browne Research Laboratory, The Leprosy Mission (TLM) Community Hospital, Nand Nagri, Shahdara, New Delhi 110093, India

The Leprosy Mission Hospital, Naini, Uttar Pradesh, India

The Leprosy Mission Bethesda Hospital, Champa, Chhattisgarh 495 67, India

The Leprosy Mission Home & Hospital, Purulia, West Bengal 723 101, India

TLM Community Hospital Nand Nagri, Shahdara, Delhi 110093, India

TLM Kothara Community Hospital, Kothara, Maharashtra 444 805, India

A R T I C L E I N F O A B S T R A C T

Article history: Objectives: The emergence of multidrug-resistant (MDR) organisms for any infectious disease is a public

Received 18 May 2017

health concern. Global efforts to control leprosy by intensive chemotherapy have led to a signiﬁcant

Received in revised form 13 September 2017

decrease in the number of registered patients. Currently recommended control measures for treating

Accepted 20 October 2017

leprosy with multidrug therapy (MDT) were designed to prevent the spread of dapsone-resistant

Available online 31 October 2017

Mycobacterium leprae strains. Here we report the identiﬁcation of MDR M. leprae from relapse leprosy

patients from endemic regions in India.

Keywords:

Methods: Resistance profiles to rifampicin, dapsone and ofloxacin of the isolated strains were confirmed

Mycobacterium leprae

by identiﬁcation of mutations in genes previously shown to be associated with resistance to each drug.

Multidrug-resistant

Between 2009–2016, slit-skin smear samples were collected from 239 relapse and 11 new leprosy cases

Multidrug therapy

Rifampicin from hospitals of The Leprosy Mission across India. DNA was extracted from the samples and was

Dapsone analysed by PCR targeting the rpoB, folP and gyrA genes associated with resistance to rifampicin, dapsone

Quinolones and oﬂoxacin, respectively, in M. leprae. M. leprae Thai-53 (wild-type) and Zensho-4 (MDR) were used as

reference strains.

Results: Fifteen strains showed representative mutations in at least two resistance genes. Two strains

showed mutations in all three genes responsible for drug resistance. Seven, seven and one strain,

respectively, showed mutations in genes responsible for rifampicin and dapsone resistance, for dapsone

and oﬂoxacin resistance and for rifampicin and oﬂoxacin resistance.

Conclusion: This study showed the emergence of MDR M. leprae in MDT-treated leprosy patients from

endemic regions of India.

rights reserved.

1. Introduction population compared with 125 785 cases in 2014–2015 according

to the National Leprosy Eradication Programme (NLEP) [1]. As a

Following the introduction of multidrug therapy (MDT) in 1983, result of this signiﬁcant decline in prevalence there has been also a

a total of 86 028 leprosy cases are on record as of 1 April 2016, signiﬁcant fall in the ANCDR.

giving a prevalence rate of 0.66 per 10 000 population, compared Consequent to this decline in leprosy prevalence, in 2005 the

with 88 833 cases on 1 April 2015 in India [1]. A total of 127 334 vertical programme of the NLEP was gradually merged with the

new leprosy cases were detected during the year 2015–2016, giving general health system [1]. At this juncture of elimination, in recent

an annual new case detection rate (ANCDR) of 9.71 per 100 000 times there have been reports of relapses from many endemic

countries, indicating that these relapses might be due to a mutated

resistant strain of Mycobacterium leprae under drug pressure or due

to re-infection. According to the World Health Organization (WHO),

* Corresponding author.

E-mail address: [email protected] (M. Lavania). 3039 relapse cases were reported globally from 105 countries in

https://doi.org/10.1016/j.jgar.2017.10.010

M. Lavania et al. / Journal of Global Antimicrobial Resistance 12 (2018) 214–219 215

2015 [2] and a total of 276 relapse cases were conﬁrmed from India Slit-skin smear scrapings were collected in 70% ethanol and

in 2015 [1]. The Leprosy Mission Hospitals in India reported 275 were transported to the laboratory. Tubes were centrifuged and

relapse cases in the last 5 years. the pellet was air-dried to remove ethanol (Merck). The sample

India is known to constitute more than 50% of new leprosy cases was kept for lysis in lysis buffer for 12–16 h at 60 C and was

worldwide [2] and therefore the emergence of drug resistance in inactivated at 95 C for 10 min. DNA was kept at À20 C until PCR

M. leprae is of great concern at this stage. Drug resistance to anti- analysis.

leprosy drugs has been reported previously for dapsone in 1964

[3], rifampicin in 1976 [4] and oﬂoxacin in 1996 [5]. 2.5. Detection of mutations by PCR targeting rpoB, folP and gyrA

To prevent the development of multidrug-resistant (MDR) M. genes

leprae strains, current leprosy control strategies are based on early

detection of cases and treatment with MDT as recommended by PCR-based gene ampliﬁcation was done using primers/proto-

the WHO. The Global Leprosy Programme initiated the establish- cols according to the WHO ‘Guidelines for global surveillance of drug

ment of a sentinel surveillance network for monitoring drug resistance in leprosy’ [6] for detection of mutations in rpoB, gyrA and

resistance in leprosy in 2009. Data were systematically collected in folP in the M. leprae genome. The primer sequences used in this

six endemic countries (Brazil, China, Colombia, India, Myanmar study were as per WHO guidelines [6]. Following detection of the

and Vietnam) after consultations and deliberations at two work- PCR product on a 2% agarose gel, amplicons were excised from the

shops held in India (2006) and Vietnam (2008) [6]. The network is gel and were puriﬁed using a QIAGEN Gel Extraction Kit (QIAGEN,

currently operating in 12 leprosy-endemic countries (Brazil, China, Franklin Lakes, NJ). PCR products were sent for commercial

Colombia, India, Myanmar, Pakistan, Philippines, Vietnam, Burkina sequencing (Euroﬁns Lab, Hyderabad, India). Sequence data were

Faso, Indonesia, Mali and Nigeria). analysed using blast and MEGA 5.1 (http://megasoftware.net/).

In this study, molecular analysis of M. leprae strains was

employed to determine the spread of drug-resistant leprosy. The 3. Results

identiﬁcation of MDR M. leprae strains from relapse leprosy

patients is reported. The drug resistance proﬁles of the isolated 3.1. Demographic characteristics of the patients

strains were conﬁrmed by identiﬁcation of mutations in genes

previously shown to be associated with resistance to each drug. A total of 239 relapse leprosy patients (as per the clinical criteria

of relapse) and 11 new cases were enrolled in this study. Their

2. Materials and methods demographic characteristics, including age, sex and type of leprosy,

are given in Table 1 and are summarised below.

2.1. Ethical approval A total of 203 relapse cases (81.2%) were categorised as

multibacillary (MB) and the remaining 47 (18.8%) were pauciba-

Written informed consent was obtained from all patients who cillary (PB) at the time of relapse. Among the MB relapse cases 12

were recruited into this study, and the study was approved by the were previously PB at the ﬁrst time of diagnosis, and among the PB

Institutional Ethical Committee of The Leprosy Mission Trust India. relapsed cases only 1 case was previously MB. The BI of the MB

cases ranged between 1+ and 6+. The time to relapse ranged

2.2. Clinical specimens and bacterial strains between 1 year and 36 years (mean duration 8.09 years). Among

reported relapse cases, 183 (73.2%) were male and 67 (26.8%) were

Between 2009–2016, slit-skin smears from 250 samples were female, and the age of all cases at the time of relapse ranged from

collected from relapse leprosy cases (n = 239) and from new 17 years to 79 years. All of the patients were declared cured after

leprosy cases (n = 11) at different hospitals of The Leprosy Mission completion of the treatment regimen with MDT and were

across India. Each of the patients had shown a persistent or conﬁrmed as relapse cases based on the abovementioned clinical

increased bacteriological index (BI) in skin smears with the criteria of relapse and by BI examination.

appearance of new lesions during or after routine treatment with

MDT, including dapsone, rifampicin and clofazimine. For molecular 3.2. Clinical complaints at the time of relapse

determination of drug-resistant strains, a drug-susceptible strain

of M. leprae (Thai-53) and a MDR strain (Zensho-4) were used as A total of 77% of the relapsed patients visited hospitals with a

reference strains. Samples were stored at 4 C until transportation complaint of new skin lesions with either multiple hypopigmented to the laboratory.

Table 1

Demographic characteristics and treatment regimen of leprosy patients at the time

2.3. Deﬁnition of relapse

of relapse.

ﬁ

A relapse is de ned as the re-occurrence of the disease at any Characteristic No. (%) of patients

time after the completion of a full course of treatment with WHO- Sex

recommended MDT [6]. Relapse is diagnosed by the appearance of Male 183 (73.2)

Female 67 (26.8)

deﬁnite new skin lesions and/or an increase in the BI of two or

Type of leprosy at time of relapse

more units at any single site.

MB 203 (81.2)

PB 47 (18.8)

2.4. Preparation of M. leprae DNA Treatment regimen

No MDT (new case) 11

MDT for 12 months 134

Biopsies were minced with disposable scalpels on glass slides,

MDT for 24 months 62

followed by addition of 100 mL of lysis buffer containing proteinase

MDT for 36 months 18

K (10 mg/mL) (Sigma, USA), 1 M Tris (Merck, Kenilworth, NJ)

MDT for >36 months 6

(10 mM) and Tween-20 (Sigma, USA) (0.5%) at pH 8.0. The minced Irregular/defaulters 7

biopsy was transferred to a microcentrifuge tube and was MDT + DDS monotherapy 2

DDS monotherapy 10

incubated at 60 C for 18 h and was then inactivated at 95 C for

10 min. The lysate was used as template DNA for PCR. MB, multibacillary; PB, paucibacillary; MDT, multidrug therapy; DDS, dapsone.

216 M. Lavania et al. / Journal of Global Antimicrobial Resistance 12 (2018) 214–219

Fig. 1. Patient with Mycobacterium leprae isolate with mutations in the genes folP (Thr53Ile) and rpoB (Ser456Leu).

Fig. 2. (a) Percentage of sensitive strains versus drug-resistant strains. (b) Number of drug-resistant samples to an individual drug or to a combination of drugs.

M. Lavania et al. / Journal of Global Antimicrobial Resistance 12 (2018) 214–219 217

Fig. 3. Patient with multidrug-resistant Mycobacterium leprae isolate with mutations in the genes rpoB (Ser456Leu), folP (Pro55Ser) and gyrA (Ala91Val).

anaesthetic patches all over the body, swelling of ankle joints or fall in the BI following the introduction of moxiﬂoxacin. For

nodular inﬁltration all over the body with Grade II deformity of the example, in CHP 3, the BI fell from 4.33+ to 4.0+ after 6 months

hands. Type 2 reactions (erythema nodosum leprosum) were noted and to 3.33+ following administration of moxiﬂoxacin.

in 28.8% of patients after release from treatment and 6.8% had type 1 Contact tracing of patients (SHD 1, 2, CHP 3, 6, and PUR 1) was

reactions. Neuritis was observed in 3.8% of relapse patients. done up to 6 months and none of the contacts showed clinical signs

of leprosy. However, one contact (the father of SHD 2) was found to

3.3. Results of PCR and sequencing have suffered from leprosy that was responsive to MDT.

Following isolationof DNA from the PCR-ampliﬁed producton gel 4. Discussion

bandsforindividualgenes, mutationsineachgene were searchedfor.

All mutations were missense variants caused by a single nucleotide Currently, leprosy control is mainly based on WHO-recom-

substitution. Mutations in folP, rpoB and gyrA were determined by mended MDT. During the elimination stage, the emergence of drug

direct DNA sequencing. Most of the isolates showed a point resistance is a major concern for any infectious disease interven-

mutation of a single gene, but mutations conferring multidrug tion programme. Strains of M. leprae resistant to single and

resistance (to dapsone and rifampicin) were detected in seven cases multiple drugs were observed in the present study. Discontinua-

(Fig. 1). Among the 250 samples, 71 (28%) were resistant to any one tion of or inappropriate treatment as well as monotherapy play a

of the drugs and the remaining 179 (72%) were sensitive to all three major role in emergence of MDR bacilli. Recent publications have

drugs (Fig. 2a). Fifteen strains showed representative mutations in reported cases of rifampicin resistance from several endemic areas.

at least two resistance genes and two strains showed a mutation in Since rifampicin is the backbone of MDT, it is important to monitor

all three genes responsible for rifampicin, dapsone and oﬂoxacin the emergence of rifampicin-resistant mutants. Dapsone resis-

resistance (Fig. 3). Among these, seven strains showed resistance to tance has been reported since the late 1960s, but convincing data

dapsone and oﬂoxacin, seven to dapsone and rifampicin and one to supporting the existence of clofazimine-resistant strains of M.

rifampicin and oﬂoxacin. A total of 26 patients were rifampicin- leprae have not yet been reported.

monoresistant, 18 were dapsone-monoresistant and 10 were Developing countries started reporting drug-resistant M. leprae

oﬂoxacin-monoresistant. The results for individual genes from among new cases [15–18]. A recent publication from West Africa

clinical strains are summarised in Table 2 and Fig. 2. reported a cluster of Guinean patients with drug-resistant leprosy

Most of the resistance cases were observed from North East infections [19]. Some of the patients were identiﬁed to have

India, and mainly rifampicin resistance from Eastern India (Fig. 4). dapsone-resistant M. leprae, as well as a single case demonstrating

rifampicin resistance.

3.4. Treatment follow-up of cases MDR Mycobacterium tuberculosis has become a major problem

globally. At this stage, we should take proactive measures against

Patients with leprosy that was resistant to dapsone and MDR M. leprae by recommending alternative drug regimens to

oﬂoxacin but sensitive to rifampicin showed response to re- prevent the emergence of MDR M. leprae under the leprosy

administration of MDT. eradication programme. However, to understand the magnitude of

Moxiﬂoxacin (400 mg) was administered to patients with emerging resistance, the WHO has already initiated a Global

leprosy that was resistant to rifampicin along with dapsone/ Sentinel Surveillance of Drug Resistance programme for leprosy

oﬂoxacin. All patients with rifampicin-resistant leprosy showed a control in 2008.

218 M. Lavania et al. / Journal of Global Antimicrobial Resistance 12 (2018) 214–219

Table 2

Mutations in folP1, rpoB and gyrA genes related to resistance to dapsone, rifampicin and oﬂoxacin, respectively, in clinical strains of Mycobacterium leprae.

Strain Previous BI at Current BI Mutation (position) in: Origin

time of ﬁrst at the time

diagnosis of relapse

folP1 rpoB gyrA

Resistance to any two drugs

SHD 1 2.33+ 3.66+ Pro55Leu [7–9,16] Ser456Leu [10,11,16,18] No mutation Delhi

SHD 2 2.33+ 3.33+ Pro55Leu Ser456Leu No mutation Delhi

SH 3 3+ 3+ Pro55Leu No mutation Ala91Val [10,15,16,18] Delhi

SH 4 3+ 3.33+ Pro55Leu No mutation Ala91Val Delhi

CHP 1 NA 4+ Pro55Leu Ser456Leu No mutation Champa

CHP 2 NA 4+ Thr53Ile [8,12,16] Ser456Val [NC] No mutation Champa

CHP 3 3+ 4.33+ Pro55Ar [13] Ser456Val No mutation Champa

CHP 4 5+ 6+ Pro55Arg No mutation Ala91Val Champa

CHP 5 NA 4+ Pro55Leu Ser434Cys [NC] No mutation Champa

CHP 6 (new case) – 5+ Ala53Asp [NC] Val424Gly [14]; Gln442His [14] No mutation Champa

NAN 2 NA 3+ Pro55Leu No mutation Ala91Val Naini

NAN 3 4.33+ 5.33+ Pro55Leu No mutation Ala91Val Naini

PUR 1 NA 3+ Pro55Leu No mutation Ala91Val Purulia

MUZ 1 NA 2.33+ Pro55Leu No mutation Ala91Val Muzaffarpur

CV1 NA 3+ No mutation Asp441Tyr [10] Ser92Ala (NC) Vellore

Resistance to all three drugs

NAN 1 2+ 3+ Pro55Arg Ser456Leu Ala91Val Naini

KTH 1 1.66+ 1.6+ Pro55Leu Ser456Leu Ala91Val Kothara

BI, bacteriological index; NC, no conﬁrmation of resistance in mouse footpad assay.

Fig. 4. Regions in India for samples with drug resistance.

The most important ﬁnding of the present study is that patients different codon positions in the drug resistance-determining region

who relapsedafteradministration ofMDTmostly harbouredM. leprae were also noted in folP1, rpoB and gyrA (data not shown). Whether

strains having more than two resistance mutations, suggesting the these mutations are linked to actual drug resistance in M. leprae is

emergence of multidrug resistance. As shown in Table 2 and Fig. 2b, not known as these mutations have not been conﬁrmed by in vivo

15 isolateshad mutations in two genes (resistance totwo drugs) and studies in the mouse foot pad. We are now investigating the

2 strains (NAN 1 and KTH 1) showed mutations in three genes relationship between genotypic mutations and phenotypic resis-

(resistance to three drugs). MDR M. leprae (resistant to dapsone, tance using M. leprae isolates in a mouse foot pad assay.

rifampicin and oﬂoxacin) was ﬁrst reported in 1997 [15]. Some The current results strongly suggest the need for a survey of drug

reports of MDR leprosy were also reported from Japan and the resistance in leprosyas well as the developmentof rapid methodsfor

Philippines [16], However, this is the ﬁrst report from India detectionof drug-resistantleprosy bacilli.Molecular-based methods

regarding evidence of MDR cases. In this study, other mutations at help as a powerful tool for rapid detection of drug-resistant M. leprae

M. Lavania et al. / Journal of Global Antimicrobial Resistance 12 (2018) 214–219 219

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