1. 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

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

3. 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

4. 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.

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

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

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

8. MB/PB Ratio (TLMTI Hospitals)

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

10. 2017
WHO calls for early detection, intensified efforts to eliminate leprosy
2015
time for action, accountability and inclusion
RIF Resistance arises

11. 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.

12. 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).

13. 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

14. 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.

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

16. 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

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

18. 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.

19. 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.

20. 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

21. 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…)

22. 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

23. 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

24. Increase in resistance of Rifampicin

25. Percentage of drug-resistance among M.leprae relapse cases

26. Multidrug Patient with mutation in rpoB gene (Ser456Leu), folP gene (Pro55Ser) and gyrA gene (Ala91Val)
 Query TCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGGCTGTTGGCGCTGGGCCCGGG 120
Sbjct TCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGGCTGTCGGCGCTGGGCCCGGG
Query KFFGTSQLSQFMDQNNPLSGLTHKRRLLALGPGGLSRERAGLEVRDVHPSHYGRMCPIE 66
Sbjct KEFFGTSQLSQFMDQNNPLSGLTHKRRLSALGPGGLSRERAGLEVRDVHPSHYGRMCPIE 487 
Query TCGCGGGAAGGCGCGGCGATTGTCGACGTCGGTGGCGAATCGACCCGGTCCGGTGCCATT 60
Sbjct TCGC-GGAAGGCGCGGCGATTGTCGACGTCGGTGGCGAATCGACCCGGCCCGGTGCCATT   
Query EGAAIVDVGGESTRSGAIRTDPRVELSRIVPVVKELAAQGITVSIDTTRADVARAALQSG 62
Sbjct EGAAIVDVGGESTRPGAIRTDPRVELSRIVPVVKELAAQGITVSIDTTRADVARAALQSG 100 
rpoB (Ser456Leu)
folP (Pro55Ser)
Query TGGGCAATTACCATCCGCACGGCGACGTATCGATTTATGACACGTTAGTGCGCATGGCGC 121
Sbjct TGGGCAATTACCATCCGCACGGCGACGCATCGATTTATGACACGTTAGTGCGCATGGCGC 7621
Query SGFRPDRSHAKSARSVAETMGNYHPHGDVSIYDTLVRMAQPWSLRYPLVDGQGNFGSPG 59
Sbjct SGFRPDRSHAKSARSVAETMGNYHPHGDASIYDTLVRMAQPWSLRYPLVDGQGNFGSPG 121
gyrA (Ala91Val)

27. 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)

28. Regions of samples with Resistance23 13 4 10 2 7 22 1

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

30. 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

31. 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.

33. 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