MOLECULAR PATTERNS OF MULTI DRUG RESISTANT MYCOBACTERIUM LEPRAE FROM INDIAN LEPROSY PATIENTS Mallika Lavania, Itu Singh, Ravindra Turankar, Madhvi Ahuja, Utpal Sengupta and Annamma S John

PRESENCE OF TLMTI IN INDIA 9 states with 14 hospitals, 6 VTCs, 7 Community development projects, 5 Snehalayas, 1 advocacy centre 1 Research Laboratory

WHAT IS LEPROSY? World’s oldest recorded disease Stigmatized disease Caused by Mycobacterium leprae Rod Shaped First bacterial disease inflicting humans It is a chronic infectious disease, multiplies slowly and the incubation period of the disease, varies from 5 years to 20 years Characterized by lesions of the peripheral nerve, skin and mucus membrane of the URT Leprosy is curable with multidrug therapy (MDT). Every year January 27th is World Leprosy day

NATURAL HOST? Armadillo, USA, Brazil Humans Red Squirrel, UK Although not highly infectious, leprosy is transmitted via droplets, from the nose and mouth, during close and frequent contacts with untreated cases. Untreated, leprosy can cause progressive and permanent damage to the skin, nerves, limbs, and eyes.

TREND IN NEW CASE DETECTION OF LEPROSY BY WHO 2006-2015

Patients were reported in 96% 211 973 newly diagnosed Patients were reported in end of 2015  from 138 countries Global statistics show that 203 600 (96%) of new leprosy cases were reported from 22 priority countries.

NUMBER OF NEWLY DIAGNOSED LEPROSY CASES REGISTERED FOR MDT TREATMENT AT TLM HOSPITALS

MB/PB Ratio (TLMTI Hospitals)

TLM approx. 70% Geographical distribution of new cases in 2015 by Country WHO WER SEP 2016

2017 WHO calls for early detection, intensified efforts to eliminate leprosy 2015 time for action, accountability and inclusion RIF Resistance arises

BACKGROUND Currently, leprosy treatment and control is based on World Health Organization (WHO)-recommended multidrug therapy (MDT) and is in use in India for treatment of leprosy for the last 33 years. Using MDT the prevalence of leprosy has come down drastically all over the world. It has been noted from earlier experience that any therapeutic control measure for prevention of disease with antibiotics ultimately leads to emergence of drug resistance. Therefore, a surveillance mechanism should function as a ‘watch dog’ for identification of drug resistance. Recent publications have indicated evidences for prevalence of drug resistance in leprosy in several endemic areas.

DEFINITION OF LEPROSY RELAPSE Relapse defined as the multiplication of M. leprae, suspected by the marked increase (at least 2+ over the previous value) in the BI at any single site, usually with evidence of clinical deterioration (new skin patches or nodules and/or new nerve damage).

CRITERIA OF LEPROSY RELAPSE Relapse was confirmed by reappearance of new symptoms and signs of the disease after successful completion of appropriate treatment and with any one of following findings A previously negative skin smear becoming positive with at least 2 units of Bacteriological Index (BI) at any site. Biopsy showing specific histopathological changes with a significant number of acid-fast bacillus (AFB) positivity

THREAT TO LEPROSY INTERVENTION PROGRAM: RELAPSED CASES NUMBER OF CLINICALLY DIAGNOSED RELAPSE CASES No. of cases No. of cases GLOBAL 1312 relapses were reported from 46 countries Year INDIA Year TLM The states with the highest numbers of relapse cases were Chhattisgarh, West Bengal and Maharashtra.

NUMBER OF RELAPSE CASES ACCORDING TO BI POSITIVITY FROM TLM HOSPITALS No. of cases BI

Total Number of Relapse Cases Processed at SB Lab Sentinel Site (reporting period) (The Leprosy Mission India) No. of cases relapsed and tested for drug resistance Clinical manifestations of MB cases who relapsed and tested for drug resistance New skin lesions BI>2 2009 27 23 2010 25 19 2011 13 2012 34 31 2013 53 41 50 2014 17 2015 59 45 52 2016 39 35 Total 250 224

OBJECTIVE To detect drug resistance in M. leprae strains in clinically relapsed leprosy patients from the hospitals of The Leprosy Mission Trust, India.

DRUG RESISTANCE: WHY IS THIS IMPORTANT? The most important problem associated with infectious diseases today is the rapid development of resistance by the pathogen to antibiotics. It compels clinicians to change their views about disease and the mode of treatment of patients with drug resistance.

DRUG RESISTANCE Condition in which there is insensitivity to drugs that usually cause growth inhibition or bacterial cell death at a given concentration of the drug. People cannot be effectively treated by the drug. People remain ill for longer time. People are at a greater risk of succumbing to infection. Epidemics are prolonged. Others are at a greater risk of getting infection.

FUNDAMENTAL MECHANISM OF ANTIBIOTIC RESISTANCE Microorganisms have developed various ways to resist the toxic effects of antibiotics and other drugs (A) Drug inactivation: enzymes, phosphorylate, adenylate, acetylate (B) Target alteration: mutation or enzymatic modification, decrease affinity (C) Prevention of drug influx: decrease permeability, losing porins (D) Activation of drug efflux: pumps, ATP / protons and ions

WHY DO PATIENTS HAVE RESISTANCE? Patients previously treated for leprosy? Patients previously treated by rifampicin? For tuberculosis For any other infection For prophylaxis (meningitis, TB, Leprosy…)

Sequencing or other methods DETECTION OF RESISTANCE CASES: WHO SENTINEL SURVEILLANCE OF DRUG RESISTANCE IN LEPROSY Leprosy relapse case With BI > 2+ Results to clinics Treatment DR* If RmpR => oflo + mino+ clo Mutation => R No mutation => S Standard MTD Rmp+Dds+clo skin samples clinics laboratory PCR rpoB folP gyrA PCR 0 Check RLEP PCR Search for inhibitors Ask for new sample PCR + Sequencing or other methods Data base shared with other labs WHO report WHO guidelines

Methods for detecting resistance in M. leprae 1. Phenotypic methods: Inoculation in MFP 2. Molecular Methods PCR SSCP, PCR heteroduplex analysis Allele specific PCR, reverse hybridization, Arrays, High resolution melting (HRM) analysis 3. DNA sequencing Perfect agreement with MFP

Increase in resistance of Rifampicin

Percentage of drug-resistance among M.leprae relapse cases

Multidrug Patient with mutation in rpoB gene (Ser456Leu), folP gene (Pro55Ser) and gyrA gene (Ala91Val)  Query 61 TCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGGCTGTTGGCGCTGGGCCCGGG 120 Sbjct 2275483 TCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGGCTGTCGGCGCTGGGCCCGGG 2275424 Query 7 KFFGTSQLSQFMDQNNPLSGLTHKRRLLALGPGGLSRERAGLEVRDVHPSHYGRMCPIE 66 Sbjct 428 KEFFGTSQLSQFMDQNNPLSGLTHKRRLSALGPGGLSRERAGLEVRDVHPSHYGRMCPIE 487  Query 1 TCGCGGGAAGGCGCGGCGATTGTCGACGTCGGTGGCGAATCGACCCGGTCCGGTGCCATT 60 Sbjct 296811 TCGC-GGAAGGCGCGGCGATTGTCGACGTCGGTGGCGAATCGACCCGGCCCGGTGCCATT 296869   Query 3 EGAAIVDVGGESTRSGAIRTDPRVELSRIVPVVKELAAQGITVSIDTTRADVARAALQSG 62 Sbjct 41 EGAAIVDVGGESTRPGAIRTDPRVELSRIVPVVKELAAQGITVSIDTTRADVARAALQSG 100  rpoB (Ser456Leu) folP (Pro55Ser) Query 62 TGGGCAATTACCATCCGCACGGCGACGTATCGATTTATGACACGTTAGTGCGCATGGCGC 121 Sbjct 7562 TGGGCAATTACCATCCGCACGGCGACGCATCGATTTATGACACGTTAGTGCGCATGGCGC 7621   Query 1 SGFRPDRSHAKSARSVAETMGNYHPHGDVSIYDTLVRMAQPWSLRYPLVDGQGNFGSPG 59 Sbjct 63 SGFRPDRSHAKSARSVAETMGNYHPHGDASIYDTLVRMAQPWSLRYPLVDGQGNFGSPG 121 gyrA (Ala91Val)

Mutation: Codon position Mouse Foot pad (MFP) growth at 10 months in mice of three patient isolates having mutation at codon position 442 Gln-His Patient code SJB   AAK SKS Mutation: Codon position 424 Val-Gly; 442 Gln-His 438 Gln-Val; 442 Gln-His Initial Incoulum 2.0x103 Control 1.4x105 2.9x104 3.0x104 Rifampicin 2.1x104 Dapsone 1.17x104 Clofazimine 2.0x104 No growth in MFP Growth in MFP (Confirmed Resistance)

Regions of samples with Resistance 23 13 4 10 2 7 22 1

Regions of samples with Resistance Kharsia Rifampicin resistant Dapsone resistant Ofloxacin resistant Primary Rifampicin resistance

TREATMENT FOR RIFAMPICIN-RESISTANT CASES (WHO) 2 year regimen 6 months of daily clofazimine (50mg) daily Ofloxacin (400mg) daily Minocycline (100 mg) or Clarithromycin (500 mg) Followed by 18 months of daily clofazimine Daily Ofloxacin (400 mg) or Minocycline (100 mg) Should know about ofloxacin susceptibility, minocycline susceptibility? Clarithromycin susceptibility? 3 drugs daily 2 drugs daily

CONCLUSION Results from this study revealed presence of M. leprae resistant strains to rifampicin in relapsed cases of leprosy. Rise in number of cases with resistance to rifampicin is likely that resistant strains are actively circulating in some endemic regions of India. Therefore an urgent need for development of drug-resistant monitoring policy and a careful post-treatment follow-up of cured patients in order to detect relapse earlier and rapidly identify resistant strains. This further indicates an urgent need for identification and inclusion of new drugs in the regimen for treating such cases.

With thanks to...... TLM Hospitals Field staff Mr Atul Roy Dr Loretta Das Dr Joydeepa Darlong Dr Archana Kumar Dr Helen Roberts Dr Asha Massey Dr Saurabh Misra Dr Neeta Maximus Mr Pankaj Gupta Mr R Senthil Kumar Mr Ravichandran The Leprosy Mission Trust India Indian Council of Medical Research Field staff Mr Atul Roy