Presentations made during the Informal Consultation on ...Molecular basis of rifampicin resistance and methods for detection Stewart Cole Nadine Honoré 3-D structure of RNA Polymerase

SEA/GLP/2007.1 Add.1 Distribution: Limited

Presentations made during the Informal Consultation on

Rifampicin Resistance in Leprosy

JALMA, Agra, India, 30 November – 1 December, 2006

Regional Office for South-East Asia New Delhi, March 2007

Page 1

Molecular basis of Molecular basis of rifampicinrifampicinresistance and methods for resistance and methods for

detectiondetection

Stewart ColeStewart ColeNadine Nadine HonoréHonoré

33--D structure of RNA D structure of RNA PolymerasePolymerasefromfrom ThermusThermus aquaticusaquaticus

β-subunit

β’-subunit

α-subunit

Rifampicin

Campbell et al. 2001Cell 104, 901-912

ResiduesResidues thatthat contact contact rifampicinrifampicin

Altered in Rif-R

M. leprae

Altered in Rif-R

M. leprae

Campbell et al. 2001Cell 104, 901-912

LandmarkLandmark studystudy

Honoré & Cole, 1993. AAC 37: 414-418

Same mechanismas in other bacteria.

Not due to drughydrolysis.

Page 2

DetectingDetecting RifampicinRifampicin ResistanceResistance in in MycobacteriumMycobacterium lepraeleprae

• Phenotypic methods:

Inoculation of mouse footpads, BACTEC

• Molecular methods:

PCR-SSCP, PCR-heteroduplex analysis,

allele-specific PCR, reverse hybridization, arrays

• DNA sequencing

Honoré et al. (1994) A simple and rapid techniquefor the detection of rifampin resistance in

Mycobacterium leprae. Int. J. Leprosy 61: 600-604.

Perfect agreement with MFP

RifRif--RR determiningdetermining locuslocus

T S Q L S Q F M D Q N N P L S G L T H K R R L S A L G P

ACCAGCCAGCTGTCGCAGTTCATGGATCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGGCTGTCGGCGCTGGGCCC

D L F M

420 425416

C3 aa insertion

36%9% 41%Mutationalfrequency of Rif-Rin M. tuberculosis

1999: IMMYC SteeringCommittee Initiative

To develop simple & rapid test for Rif-R

Telenti et al. (1993) Detection of rifampicin-resistance mutationsin Mycobacterium tuberculosis. Lancet 341, 647-650

Reverse Reverse hybridizationhybridization strategystrategy

Positive controls

DetectionDetection strategystrategy

Page 3

4

2 3 4 5123

1

27303233343536373839

TypicalTypical reverse reverse hybridizationhybridization resultsresults

Wildtype Rif-R+ controls

+++

Honoré et al.(2001) A method

for rapid detectionof rifampicin-

resistant isolates of Mycobacteriumleprae. Lepr Rev

72: 441-448.

Mutations Mutations affectingaffecting probes 4 & 35probes 4 & 35

Confirmed as His420Asp by DNA sequencing

Reverse Reverse hybridizationhybridization strategystrategy

Positive controls

Typical resultswith

reverse Hybridization

&NepaleseStrain

1 2 3 4 5

1234273032333536373839

+++

+ controlsWild type

Rif-R strain

_

_

Field Field resultsresults

M. Macdonald et al.

Page 4

Mutations Mutations affectingaffecting probes 3 & 36probes 3 & 36

• Confirmed by DNA sequencing as Ser425Phe

ConclusionsConclusions

• Method validated in Paris, Madagascar & Kathmandu

• Two detection systems available chemiluminescent andcolorimetric

• Robust and reproducible

• Backup in form of DNA sequencing provided

RecommendationsRecommendations

• Further field testing required

• Possibly in framework of prospective study of Rif-R

• At least two sites: Asia, Africa, …

• All results to be confirmed independently by DNA sequencing

• Include DDS & FQ

Or simply direct PCR sequencing (or equivalent)!

Page 5

Anandaban Hospital, Kathmandu, Nepal

Development of a novel Multiplex PCR method for detecting

mutations conferring rifampicin resistance in M. leprae

Bishwa Raj Sapkota Chaman Ranjit

Murdo Macdonald

Mycobacterial Research Laboratory, Anandaban HospitalGPO BOX 151, Kathmandu, Nepal

[email protected]


Introduction

• MDT involves treatment with Rifampicin, Dapsone and Clofazamine, which remains the mainstay of leprosy elimination strategies worldwide

• Drug resistance, does occur as a result of this regimen


Introduction• Detection remains problematic

– MFP remains gold standard• Alternative methods have not been

generally adopted in the field, – Many involve hybridization or other sophisticated

techniques– Unsuitable for use in field


• To develop a detection technique which can be reliably applied in laboratories in leprosy endemic countries. – We have adapted the Multiplex PCR based technique

described by Fan et al. for M. tuberculosis (J Clin. Microbiol. 2003), which uses an amplification refractory mutation system to detect mutations in the rifampin resistance-determining region of the rpoB gene.

Aim

Page 6


Materials and Methods• Evaluation of assay in plasmid and reference

controls• Clinical samples subjected to

– Multiplex-PCR in the presence of three primers• Mutant specific primer has deliberate mismatch near 3’-OH

end, to enhance differential amplification– MFP drug susceptibility test (Shepard 1960, Ji 1987,

Grosset 1989)


Primers designGene Bank accession no. AL583923, ML1891: 1 to 3537bp

• Control forward primer (CFP)*: – 5’ - CAG GAC GTC GAG GCG ATC AC - 3’ (1219 to 1238)

• Common reverse primer (CRP)*: – 5’ - TCC TCG TCA GCG GTC AAG TA - 3’ (1607 to 1588)

• M-531 forward primer: – 5’- ACC CAC AAG CGC CGG CAG TC - 3’ (1348 to 1367)

* Honore and Cole, 1993


3`1 3537

5`1219 1607

Amplicon

CFP CRP

rpoB gene

81bp-RRDR 1367 M-531 forward primer: 5’- ACC CAC AAG CGC CGG CAG TC – 3’ 1294 - GGC ACC AGC CAG CTG TCG CAG TTC ATG GAT CAG AAC AAC CCT CTG TCG GGC CTG ACC CAC AAG CGC CGG CTG TCG GCG CTG - 1374 507 - Gly Thr Ser Gln Leu Ser Gln Phe Met Asp Gln Asn Asn Pro Leu Ser Gly Leu Thr His Lys Arg Arg Leu Ser Ala Leu - 533

TTGLeu

ATGMet

TTCPhe

PCR scheme

Figure 1: The entire nucleotide sequence of rpoB gene spans 3537 bp of M. leprae as shown. Control forward primer (CFP) and common reverse primer (CRP) were designed from position of 1219 onward and 1607 backward of the rpoBgene. M- 531 forward primer was designed from 1348 position onward up to 1367 position and altering the nucleotide of Ainstead of T at position 1364 to detect the mutation in the rpoB gene (shown by line with the dot). Boldface and underline letter A indicate the nucleotide alteration introduced to enhance the 3’ mismatch effect. Codon numbers are assigned on the basis of alignment of the translated E. coli rpoB sequence. Alteration of the codon and amino acids products after mutation in the 531 positions are shown in the boldface codon and amino acids in the box.


Result Interpretation

531 Mutation-+Wild type ++

InterpretationM-531+CRP (260bp)

CFP+CRP (390bp)

Primers designrifampicin- resistance conferring mutation in the rpoB gene, a single band was observed on agarose gel electrophoresis, while wild type conferred two distinct bands

Page 7


Figure 2: Agarose gel electrophoresis of PCR products of rpoB gene.

1, 3, 4 & 5: Show two PCR bands produced from wild type strains

2 & 6: Show the single PCR band produced from mutant strains

M: 100 bp MW marker (NEB)


Evaluation of the assay in plasmid and reference controls

Wild typeCSUWild typeGenomic M. leprae12

Wild typeCSUWild typeArmadillo derived M. leprae11

Wild typeMatsuoka, 2006Wild typeJapan - 410

Wild typeMatsuoka, 2006Wild typeJapan - 39

MutantMatsuoka, 2006Ser531LeuJapan - 98



MutantMatsuoka, 2006Ser531LeuZensho-45

MutantNadine, 2001Ser531PhePlasmid 684

MutantNadine, 2001Ser531MetPlasmid 673

MutantNadine, 2001Ser531LeuPlasmid 662

Wild typeNadine, 2001Wild typePlasmid 651

PCR result

References (personal communication)

Genotype Test DNASN


Total analysis of the categorical samples

21404444Total1222Under MB MDT1111PB MDT relapse 1111DDS and MB MDT relapse1222DDS relapse 1333MB MDT defaulter 1ND - 41599MB MDT relapse

14262626New untreated case

Confirmed by seq.

MFP sen.PCR-WTTotal Sample type


Results• A number of M. leprae strains proven to be

sensitive and resistant to rifampicin by MFP and sequence were tested by this method

• In all clinical samples examined, the results obtained using the PCR method developed were consistent – with the MFP “gold standard” – some were confirmed by sequencing

Page 8


Conclusion• These results indicate that this technique

can reliably identify Rifampicin resistant strains of M. leprae at 531 position

• Its ease of use means that it can be adopted by many laboratories in the field


Recommendation• It is desirable to extend this study for all

reported rifampicin resistant strains• Development and use of multiplex PCR

systems is desirable to address all MDR Leprosy


AcknowledgementsWe would like to thank our colleagues who have shared samples from their labs:– Dr. N. Honoré, Institute Pasteur, France.– Dr. M. Matsuoka, Tokyo, Japan.– Prof. Dr. P.J. Brennan, Colorado State University, USA.– Dr. T. P. Gillis, Louisiana State University, USA.– Dr. D. L. Williams, Louisiana State University, USA.– Dr. V.P. Shetty, Foundation for Medical Research, India


Thank you

Page 9

Exploitation of dot blot hybridization method to detect mutation

Masanori Matsuoka Ph. D.National Institute of Infectious Diseases, Leprosy Research Center, Tokyo, Japan

Many drug resistant cases have been reported for single drug resistant and for the multidrug resistant cases. The emergence of drug resistant case threatens the usefulness of the MDT.

First case of dapsone resistance clinically suspectedWolcott, R. R. et al. Exacerbation of leprosy during present day treatment. Int. J. Lepr. 21 437-440 (1953)

First dapsone resistant isolates confirmed by mouse footpad testPeyit, J. H. S. et al. Sulphone resistance in leprosy. An experimental and clinical study. Lancet 2 673-674 (1964)

Increase of primary dapsone resistant isolates.Dela Cruz E. et al. Primary dapsone resistance in Cebu, The Philippines; cause for concern. Int. J. Lepr. 64 253-256 (1996)

Increasing of multidrug resistant cases.Jacques H. et al. Study of 39 documented relapses of multibacillaryleprosy after treatment with rifampin. Int. J. Lepr. 57: 607- 614 (1989)

Cambau, E. et al. Multidrug-resistance to dapsone, rifampicin and ofloxacin in Mycobacterium leprae. Lancet 349: 103-104 (1997)

Matsuoka, M. et al. A Mycobacterium leprae isolate resistant to Dapson, rifampin, ofloxacin and sparfloxacin. Int. J. Lepr. 68: 452-455 (2000)

Matsuoka, M. et al. A second case of multidrug resistant Mycobacterium leprae isolated from a Japanese patient with relapsed lepromatous leprosy. Int. J. Lepr. 71: 240-243 2003

Drug susceptibility of Zensho-4 isolate to anti-leprosy drugs

3

3.5

4

4.5

5

5.5

6

6.5

Antileprosy drugs

Bci

llary

num

ber i

n fo

otpa

ds (l

og.)

Control RMP OFLO SPAR DDS 0.01%

DDS0.001%

DDS 0.0001%

Inoculum

Page 10

Inoculation 1.0 X 106 bacilli/footpadprepared from clinical specimens

1 year

Harvest the bacilli from the footpadsPrepare the inoculum by Nakamura’s method

Inoculation 5.0 X 103 bacilli/footpadFeed the diets containing drugs

DDS: 0.01%, 0.001%, 0.0001%Rifampin: 0.01%Ofloxacin: 0.15%Sparfloxacin: 0.02%Clarithromycin: 0.03%Clofazimine: 0.001%Minocycline: 0.08%

Enumerate the bacillary growth in footpads by Shepard’s methods

BALB/c-nu/nu

BALB/c

30 weeks

Susceptibility test by mouse footpad methodFolate Biosynthesis Pathway

Dihydropteridine diphosphate7,8- dihydropteroate

Dihydroptorate synthase(DHPS)

folPCOOHH2N

PO

OHCH2O

OHOH

OO P

HN H

H

N

N

OH

H2N

N COOHCH2 NH

HN H

N

N

OH

H2N

NH

Paraaminobenzoic acid (PABA)pyrophosphate

Binding Model ofDHPS/DDS/PABA

Nature Str. Biol. Vol.4 490

Bacteriostatic activity of DDS

Dapsone is analogue of PABA

DNA sequence of the folP1 and mutationsrelated to resistant to dapsone

wild type M.leprae

ACC53-Thr CCC55-Pro

Bactericidal activity of Rifampin

Without Rifampin

DNA

mRNA

DNA dependent RNA polymerase

σ

β

α

α

Amino acids, Proteins

RNA polymerase

No bacterial growth

Bacterial growth

With Rifampin

No transcription

β

β-subunit RNA polymerase

Page 11

CAG513-Gln

DNA sequence of the rpoB and mutations related to resistant to rifampin

GAT516-Asp CAC526-His TCG531-Ser CTG533-Leu

Bactericidal activity of Quinolones

Nature Vol.379 225

Quinolone binding site

DNA gyraseTopoisomerase II

2 A and 2 B subunit

DNA supercoilingReplication

DNA sequencing of the gyrA and mutatinsrelated to resistant to Quinolones

wild type M.leprae

GCA91-AlaGGC89- Gly

Mutations and drug resistance to anti-leprosy drugs.

DDS: folP1 Threonine (ACC) at 53, Proline (CCC) at 55

Rifampin: rpoB Glycine (CAG)at 513, Asparagine(GAT) at 516,Histidine(CAC) at 526, Serine (TCG) at 531, Leucine (CTG) at 533

Quinolone: gyrA Glycine(GGC ) at 89, Alanine(GCA) at 91

Page 12

Exploitation of simple method to detect mutations

by Dot Blot Hybridization

Principle of dot blot hybridization as a drug susceptibility testing assay

Genomic DNA of M.lepraeGenomic DNA of M.leprae

Streptavidin-horseradish peroxidaseCaptures Biotin-labeled target

Streptavidin-horseradishperoxidase incubation

Color developing and mutation detectionOxidation of 3,3’,5,5’-tetramethylbenzidine

b b

Biotin labeled single Strand target

bbb

Hybridize with Oligo array absorbed on glass slide

bb

b

bbb b

b bb

bWild-type dot

Mutant dot

bbb

b

b

bb

b

b

PCR

bbb

folPrpoB gyrA

Drug susceptibility of isolates examined by simultaneously sequencing and mouse footpad method

Isolate Dapsone Rifampin Quinolone Origin folP MFP rpoB MFP gyrA MFP Hoshizuka-4 55:Pro(CCC)

Ser(TCC) Resistant Intermediate

531:Ser(TCG) →Leu(TTG)

Resistant

91:Ala(GCA)→Val(GTA)

Resistant

Japan relapse

Zensho-4 53:Thr(ACC)→Ile(ATC)

Resistant High


Resistant

91:Ala(GCA)→Val(GTA)

Resistant

Japan relapse

Airaku-2 55:Pro(CCC)→Leu(CTC)

Resistant High


Resistant

No mutation Susceptible Japan relapse

Zensho-5 55:Pro(CCC)→Leu(CTC)

Resistant High


Resistant


Kusatsu-6 55:Pro(CCC)→Leu(CTC)

Resistant High

516:Asp(GAT) →Tyr(TAT)

Resistant


Kusatsu-3 53:Thr(ACC)→Ile(ATC)

Resistant High

No mutation Susceptible No mutation Susceptible Japan relapse

Zensho-2 55:Pro(CCC)→Leu(CTC)

Resistant High

No mutation Susceptible No mutation Susceptible Japan relapse

Amami-1 55:Pro(CCC)→Leu(CTC)

Resistant High

No mutation Susceptible No mutation Susceptible Japan new

Zensho-9 No mutation Susceptible 526:His(CAC) →Try(TAC)

Resistant


MFP: mouse footpad method.

Drug susceptibility of isolates examined simultaneously by sequencing and mouse footpad method

Isolate Dapsone Rifampin Quinolone Origin folP MFP rpoB MFP gyrA MFP Kanazawa No mutation Susceptible No mutation Susceptible No mutation Susceptible Japan

new Izumi No mutation Susceptible No mutation Susceptible No mutation Susceptible Japan

new Keifu-4 No mutation Susceptible No mutation Susceptible No mutation Susceptible Japan

new Ryukyu-2 No mutation Susceptible No mutation Susceptible No mutation Susceptible Japan

new Kyoto-2 No mutation Susceptible No mutation Susceptible No mutation Susceptible Japan

new Kitazato No mutation Susceptible No mutation Susceptible No mutation Susceptible Japan

new Thai-53 No mutation Susceptible No mutation Susceptible No mutation Susceptible Thailand

new Korea 3-2

No mutation Susceptible No mutation Susceptible No mutation Susceptible Korea new

Indonesia-1 No mutation Susceptible No mutation Susceptible No mutation Susceptible IndonesiaNew

MFP: mouse footpad method.

Page 13

Drug susceptibility of isolates examined simultaneously by sequencing and mouse footpad method

Isolate Dapsone Rifampin Quinolone Origin folP MFP rpoB MFP gyrA MFP 01Mat02 53:Thr(ACC)

→Val(GTC) Resistant High

No mutation Susceptible No mutation Susceptible Phillipinesrelapse

N.C.R 53:Thr(ACC)→Ser(AGA)

Resistant Intermadiate


02Mat49 55:Pro(CCC)→Leu(CTC)

Resistant High


01Mat01 No mutation Resistant Low


01Mat03 No mutation Resistant Low


E.E.R No mutation Resistant Low


M.M.R. No mutation Resistant Low


41 isolates No mutation Susceptible No mutation Susceptible No mutation Susceptible Phillipines3 relapse

38 new MFP: mouse footpad method.

Mutations confirmed to confer resistance

67

64

GTA(3)

GCA91

gyrA

8350No mutation

Total

Mutation type

DMDR

TTC(1)TCC(1)AGG(1)

GTC(1)

AGA(1)

ATG(1)TAC(1)CGC(1)GCC(3)

25211669

GTG(1)TTG(23)GAC(2)TAT(1)GTG(1)CTC(7)ATC(4)CTG533TCG531CAC526GAT516CAG513CCC55ACC53

rpoBfolP1

1. Our data 2. Cambau E. et al. Clin. Infect. Dis. (2002)34:39-45.3. Honore N. et al. Antimicrob. Agents Chemother. (1993) 37: 414-418.4. Honore N. et al. Int. J. Lepr. (1993) 61:600-604.5. Williams D. L. Antimicrob. Agents Chemother. (1994) 38: 2380-2386.5.

References:

514 AGTTCA 515(1)

BiotinCCGrpoB533CTG

BiotingyrANegativecontrol

gyrAPositivecontrol

Biotin

TTCTTGATGrpoB531GTC

GTAgyrA91GCA

GACTACrpoB526CAC

TGCgyrA89GGC

TATAATrpoB516GAT

CTCCGCTCCFolP55CCC

BiotinGTGrpoB513CAG

BiotinAGAAGGATCGTCGCCfolP53 ACC

Biotin

Oligo nucleotides probes labeled with amino were covalently bound to the slide glass coated with polycarbodimide.

Position of dots for wild strains and mutants

Dot blot array

folP1: CGAATCGACCCGGCCCGGTGCCATTAGGACCGATCCTCGAGTTGAACTCTCfolP- CO1: CGAATCGACCCGGCCCGfolP- CO1m3: CGAATCGATCCGGCCCG

Zensho-4:ACC53ATC

Zensho-4:GCA91GTA

gyrA: ATGGGCAATTACCATCCGCACGGCGACGCATCGATTTgyrA –CO1: CCATCCGCACGGCGACGCATCGATTTgyrA –CO2m1: CCATCCGCACGGCGACGTATCGATTT

53

55

89

91

533

531

526

516

513

Zensho-4:TCG531TTG

rpoB1: AGCTGTCGCAGTTCATGGATCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGACTGTCGGCGCTGGTCC

rpoB- CO4 : GCCGACTGTCGGCGCTGGTrpoB- CO4m2: GCCGACTGTTGGCGCTGGT

Hybridization at 42℃, Washing at 47℃

Page 14

The procedure for mutation detection in the folp, rpoB and gyrA gene by dot blot hybridization

1.Amplification of target DNA fragments(1)Amplify three genes in one tube. Each reverse primer is labeled with biotin.(2)One cycle:, 40cycles 94℃for 45 sec, 58 ℃for 45 sec, 72℃for 30 sec.(3)Confirm the amplification by agarose gel electrophoresis.

2.Dot blot hybridization(1)Add 2 ul of PCR mixture to 38 ul of hybridization solution.(2)Denature fragments by heating at 98℃for 5 min and chilling on ice.(3)Apply 40 ul of denatured mixture to the slid glass with oligo array. Cover with film.(4)Incubate at 42℃ for 60 min to hybridize.(5)Wash with washing buffer at 47℃ for 20 min.(6)Treat with conjunction solution at room temperature for 30 min.(7)Wash with TBST.(8)Develop color at room temperature for 80 min.(9)Wash with distilled water.(10)Read the result by scanner.

Dot blot assay for drug susceptibility testing

Thai53 No mutation

Zensho-4folP:ACC53ATCrpoB:TCG531TTGgyrA:GCA91GTA

gyrA:141bp

rpoB:127bpfolP:119bp

Thai53

Zensho-4

531TTG

53ATC

91GTA

Page 15

Results of dot blot hybridization in Myanmar and sequencing Sample folP rpoB gyrA Dot blot

results Sequencing Dot blot


results Sequencing

S-7 new No mu. No mu. 516:GAT-TAT 516:GAT-TAT No mu. No mu. S-27 relapse 55:CCC-CGC 55:CCC-CGC 531:TCG-ATG 531:TCG-ATG. No mu. No mu. T-28 new No mu No Mu No mu. No mu. No mu. No mu. S-6 new No mu No mu. No mu. No mu. No mu. No mu. T-31 new 55:CCC-CTC No mu. No mu. No mu. No mu. No mu. T-35 new No mu No mu. No mu. No mu. No mu. No mu. T-37 new No mu No mu. No mu. No mu. No mu. No mu. T-44 new No mu No mu. No mu. No mu. No mu. No mu. T-51 new No mu No mu. No mu. No mu. No mu. No mu. T-53 new No mu No mu. No mu. No mu. No mu. No mu. T-54 new No mu No mu. No mu. No mu. No mu. No mu. T-57 new No mu No mu. No mu. No mu. No mu. No mu. T-62 new 55:CCC-CTC No mu. No mu. No mu. No mu. No mu. T-64 new No mu No mu. No mu. No mu. No mu. No mu. T-65 new No mu No mu. No mu. No mu. No mu. No mu. T-69 new No mu No mu. No mu. No mu. No mu. No mu. T-70 new No mu No mu. No mu. No mu. No mu. No mu. S-13 new No mu No mu. No mu. No mu. No mu. No mu. T-66 relapse No mu No mu. No mu. No mu. No mu. No mu.

Rate of concodance:52/54, 96%

N: New case, R: Relapse case

Results of dot blot hybridization and sequencing Sample folP rpoB gyrA Dot blot results Sequencing Dot blot results Sequencing Dot blot results Sequencing H001-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H002-05 R CCC-CTC CCC-CTC No Mutation No Mutation No Mutation No Mutation H003-05 R CCC-CTC CCC-CTC No Mutation No Mutation No Mutation No Mutation H004-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H005-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H006-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H007-05 N No Mutation ? CCC-CGT No Mutation No Mutation No Mutation No Mutation H009-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H010-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H011-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H012-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H013-05 N No Mutation ? CCC-CGT No Mutation No Mutation No Mutation No Mutation H014-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H015-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H016-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H018-05 N No Mutation No PCR No Mutation No Mutation No Mutation No Mutation H019-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H020-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H020-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H022-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.23-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.24 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.25-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.26-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.27-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.28 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.29 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.001-06 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.002-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.003-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.004-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.005-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.006-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation

Rate of concodance:97/99, 98%

DBH done in Myanmar

Susceptible to all

Resistant to dapsone and rifampin

folP rpoB

55:CCC→CGC 531:TCG→ATG

Page 16

Cambau E.et.al. Clin Infect. Dis. (2006)42:238


Results of dot blot hybridization and sequencing Sample folP rpoB gyrA Dot blot results Sequencing Dot blot results Sequencing Dot blot results Sequencing H001-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H002-05 R CCC-CTC CCC-CTC No Mutation No Mutation No Mutation No Mutation H003-05 R CCC-CTC CCC-CTC No Mutation No Mutation No Mutation No Mutation H004-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H005-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H006-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H007-05 N No Mutation ? CCC-CGT No Mutation No Mutation No Mutation No Mutation H009-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H010-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H011-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H012-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H013-05 N No Mutation ? CCC-CGT No Mutation No Mutation No Mutation No Mutation H014-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H015-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H016-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H018-05 N No Mutation No PCR No Mutation No Mutation No Mutation No Mutation H019-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H020-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H020-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H022-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.23-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.24 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.25-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.26-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.27-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.28 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.29 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.001-06 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.002-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.003-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.004-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.005-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.006-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation

Rate of concodance:97/99,98%

007-05

013-05

007-05

013-05

Dot blot hybridization method is useful and promising for the susceptibilitytest at places, where sequencing couldn’t be conducted.

A little improvements, such as increase proves, are need.

Page 17

Prevalence of drug resistance in Japan and some Asian countries

Masanori Matsuoka Ph. D.National Institute of Infectious Diseases, Leprosy Research Center, Tokyo, Japan

Mutations and drug resistance to anti-leprosy drugs.

DDS: folP1 Threonine (ACC) at 53, Proline (CCC) at 55

Rifampin: rpoB Glycine (CAG)at 513, Asparagine(GAT) at 516,Histidine(CAC) at 526, Serine (TCG) at 531, Leucine (CTG) at 533

Quinolone: gyrA Glycine(GGC ) at 89, Alanine(GCA) at 91

Prevalence of drug resistant M.leprae in Japanese patients

23(49%)

Susceptibleto all drugs

Resistant to

47

24 (51%)57228

TotalDDSRIFQuinolone

DDSRIF

QuinoloneRIFDDS

Relapsed or intractable cases

Two new cases were susceptible to all drugs.

Table 2. Prevalence of drug resistant M. leprae in Asian countries.

Indonesia (North Maluku)New case Relapse case

DDS RIF Quinolone DDS RIF Quinolone1/86 (1.12%) 4/106 (3.8%) N.D. 1/7 (14.2%) 2/9 (22.2%) N.D.

Myanmar ( Yangon)New case Relapse case

DDS RIF Quinolone DDS RIF Quinolone1/16 (6.2%) 1/15 (6.6%) 0/13 2/28 (7.1%) 0/36 0/21

Philippines ( Cebu)New case Relapse case

DDS RIF Quinolone DDS RIF Quinolone0/47 0/47 0/47 3/15 (20%) 0/15 0/15

Indonesia (Surabaya) New case Relapse case

DDS RIF Quinlone DDS RIF Quinlone 1/40

(2.5%) 0/40 0/40 N.D N.D N.D

Page 18

Results of dot blot hybridization in Myanmar and sequencing Sample folP rpoB gyrA Dot blot



results Sequencing

S-7 new No mu. No mu. 516:GAT-TAT 516:GAT-TAT No mu. No mu. S-27 relapse 55:CCC-CGC 55:CCC-CGC 531:TCG-ATG 531:TCG-ATG. No mu. No mu. T-28 new No mu No Mu No mu. No mu. No mu. No mu. S-6 new No mu No mu. No mu. No mu. No mu. No mu. T-31 new 55:CCC-CTC No mu. No mu. No mu. No mu. No mu. T-35 new No mu No mu. No mu. No mu. No mu. No mu. T-37 new No mu No mu. No mu. No mu. No mu. No mu. T-44 new No mu No mu. No mu. No mu. No mu. No mu. T-51 new No mu No mu. No mu. No mu. No mu. No mu. T-53 new No mu No mu. No mu. No mu. No mu. No mu. T-54 new No mu No mu. No mu. No mu. No mu. No mu. T-57 new No mu No mu. No mu. No mu. No mu. No mu. T-62 new 55:CCC-CTC No mu. No mu. No mu. No mu. No mu. T-64 new No mu No mu. No mu. No mu. No mu. No mu. T-65 new No mu No mu. No mu. No mu. No mu. No mu. T-69 new No mu No mu. No mu. No mu. No mu. No mu. T-70 new No mu No mu. No mu. No mu. No mu. No mu. S-13 new No mu No mu. No mu. No mu. No mu. No mu. T-66 relapse No mu No mu. No mu. No mu. No mu. No mu.

New case:0/17, rifampin 1/17, quinolone 0/17Relapse case: dapsone 1/2, rifampin 1/2, quinolone 0/2


Results of dot blot hybridization and sequencing Sample folP rpoB gyrA Dot blot results Sequencing Dot blot results Sequencing Dot blot results Sequencing H001-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H002-05 R CCC-CTC CCC-CTC No Mutation No Mutation No Mutation No Mutation H003-05 R CCC-CTC CCC-CTC No Mutation No Mutation No Mutation No Mutation H004-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H005-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H006-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H007-05 N No Mutation ? CCC-CGT No Mutation No Mutation No Mutation No Mutation H009-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H010-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H011-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H012-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H013-05 N No Mutation ? CCC-CGT No Mutation No Mutation No Mutation No Mutation H014-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H015-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H016-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H018-05 N No Mutation No PCR No Mutation No Mutation No Mutation No Mutation H019-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H020-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H020-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation H022-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.23-05 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.24 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.25-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.26-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.27-05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.28 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.29 -05 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.001-06 R No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.002-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.003-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.004-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.005-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation No.006-06 N No Mutation No Mutation No Mutation No Mutation No Mutation No Mutation New case: dapsone 2/27, rifampin 0/27, quinolone 0/27

Relapse case: dapsone 2/6, rifampin 0/6, quinolone 0/6

Prevalence of the drug resistance deduced in DBH study

Myanmar Philippines (Cebu)

New case Relapse case New case Relapse case

DDS Rif QNL DDS Rif QNL DDS Rif QNL DDS Rif QNL

0/17 1/17 0/17 1/2 1/2 0/2 2/27 0/27 0/27 2/6 0/6 0/6

The prevalence of drug resistance is generally low.

Comprehensive surveillance for the drug resistance is need to maintain effectiveness of MDT and for the prevention of spreading resistant cases.

Page 19

Detection of rpoB Mutations of M. tuberculosis and M. leprae

Hyeyoung Lee, Ph.D.Sang-Nae Cho, Ph.D.

Department of MicrobiologyYonsei U. Coll. MedSeoul, South Korea

CTG AGC CAA TTC ATG GAC CAG AAC AAC CCG CTG TCG GGG TTG ACC CAC AAG CGC CGA CTG TCG GCG CTG511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533

W-1

W-2

W-3

W-4

W-5

M-2 M-3 M-4 M-5

M. tuberculosis:rpoB probes for wild-type and mutants

M-1

H37Rv 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

Fortuitum-complex

Pan-mycobacteriaTB complex

AviumIntracellulareScrofulaceum

SzulgaiGastri

TerraeChelonaeAbscessusGordonae

KansasiiPeregrinum

UlceransGenavense/Simiae


WT1(509-514)WT2(515-520)WT3(521-524)WT4(525-529)WT5(530-534)

MT1(531TTG)

MT4(513CCA)MT5(511CCG)

Identification of Mycobacteria and Rifampin-susceptability test for clinical isolates of M. tuberculosis.

MT2(526AAC) MT3(516GTC)

SpeciesID

rpoBMutations

Fortuitum-complex


AviumIntracellulareScrofulaceum

SzulgaiGastriTerrae

ChelonaeAbscessusGordonae

KansasiiPeregrinum

UlceransGenavense/Simiae

Species identification of Mycobacterial species by reverse blot hybridization.

M. t

uber

culo

sisH

37R

v

M. b

ovis

M. b

ovis

BC

G

M.

afri

canu

m

M. m

icro

ti

M. a

vium

M. i

ntra

cellu

lare

M. s

crof

ulac

eum

M. g

ordo

nae

M. a

bsce

ssus

M. c

helo

nae

M. t

erra

e

M. g

astr

i

M. s

zulg

ai

M. k

asas

ii

M. f

ortu

itum

l

M. f

ortu

itum

ll

M. p

ereg

rinu

m

M. u

lcer

ans

M. m

arin

um

M. g

enav

ense

M. s

imia

e

M. n

onch

rom

ogen

icum

M. c

elat

uml

M. c

elat

umll

M. m

alm

oens

e

M. f

lave

scen

s

M. p

araf

ortu

itum

M. p

hlei

M. t

herm

ores

ista

ble

M. t

rivi

ale

M. v

acca

e

M. i

nter

med

ium

M. g

ilvum

Page 20

1 2 3 4

1:wild type, 2: 531TTG mutant, 3:516GTC mutant, 4:513CCA mutant

MycTB

S1(509- 514)

S4(524- 529)

S5(530- 534)

M1(531TTG)

M2(516GTC)

M3(513CCA)

S2(515- 520)

S3(521- 525)

Color RxnECL1 2 3 4

MycTB

S1(509- 514)

S4(524- 529)

S5(530- 534)

M1(531TTG)

M2(516GTC)

M3(513CCA)

S2(515- 520)

S3(521- 525)

1:wild type, 2: 531TTG mutant, 3:516GTC mutant, 4:513CCA mutant

rpoB Reverse Line-Blot Assay

CTG AGC CAA TTC ATG GAC CAG AAC AAC CCG CTG TCG GGG TTG ACC CAC AAG CGC CGA CTG TCG GCG CTG

CTG AGC CAG TTC ATG GAT CAG AAC AAC CCT CTG TCG GGC CTG ACC CAC AAG CGC CGG CTG TCG GCG CTG

511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

W-2

W-3

W-4

W-5

W-6

M-2 M-3 M-4 M-5 M-7M-6

M-1

W-1

507

W-8

M-8

M-10

M. tuberculosis

M. lepraeGGC

GGC

401

M-9

M. leprae:rpoB probes for wild-type and mutantion sites

Control DNAs for M. leprae rpoB with mutations in an E. coli plasmid

pUC19

Hot-spot region for mutationM. LepraerpoB

700 bpWild type

507 mutant

513 mutant

516 mutant

526 mutant

531TTG mutant

531ATG mutant

533ATG mutant

Lep-Rifa Reverse Blot Hybridization Assay

503-508

509-514

515-520

521-525

524-529530-534

507 AGC

513 GTG

516 TAT

531 ATG

531 TTG

533 GTG

∙W

ild t

ype

∙507 m

uta

nt

∙513 m

uta

nt

∙516 m

uta

nt

∙526 m

uta

nt

∙531T

TG

muta

nt

∙531A

TG

muta

nt

∙533 m

uta

nt

Wild probe

Mutant probe

∙W

ild ty

pe

∙50

7 m

utan

t ∙51

3 m

utan

t ∙51

6 m

utan

t ∙52

6 m

utan

t∙53

1TTG

mut

ant

∙53

1ATG

mut

ant

∙53

3 m

utan

t

503-508

509-514

515-520

521-525

524-529530-534

* 526GAC probe in progress

Page 21

0 : 100

0.1 : 99.9

0.5 : 99.5

1 : 99

2 : 98

5 : 95

10 : 90

15 : 85

20 : 80

50 : 50

80 : 20

85 : 15

90 : 10

95 : 598 : 299 : 199.5 : 0.5

99.9 : 0.1

100 : 0

Resista

nt DNA : S

usceptib

le DNA

C 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 C

C 1 2 3 4 5 6 7 8 9 10 11 12 NC C

SusceptibleDNAs

ResistantDNAs

PCR-SSCP:rpoB

Dot-blot

10099.9 99.5

99.0

98.095

.0 90.0

85.0 80.0

50.020.0

15.0 10.0 5.0 2.0 1.0 0.5 0.1 0

10050.

0

20.0 10.0 5.0 2.0 0

Mu:Wt (%)

Mu:Wt (%)

Innolipa

SusceptibleResistant

Radioisotope-labeled probe

Detection of rpoB mutations

Questions to be addressed:

• Which mutations at rpoB of M. leprae are associated with resistance to rifampicin?

• Criteria for resistance to rifampicin in M. leprae:1%, 10%, or what %? & How to verify, then?

• Which test can detect the lowest proportion of rpoBmutant population in M. leprae?

1%, 10%, 15%, or 20%?

• Comparison between PCR sequencing and dot-blot hybridization assay or other assay methods.

Page 22

3/30/20073/30/2007 8585

Rifampicin resistance in Rifampicin resistance in tuberculosis in the communitytuberculosis in the community--

Andhra Pradesh and Andhra Pradesh and OrissaOrissa

S. S. AparnaAparna, M.D, M.DLEPRALEPRA-- Blue Peter Research Center, Blue Peter Research Center,

HyderabadHyderabad

LEPRA LEPRA –– Operations in IndiaOperations in India

Anti TB drug susceptibility tests Anti TB drug susceptibility tests under RNTCP (Revised under RNTCP (Revised

National TB control National TB control programmeprogramme))

TB control TB control programmeprogramme areas studiedareas studied

ORISSA StateORISSA State

20032003--20052005KoraputKoraput

20032003--20062006AP Chest hospital, Hyderabad for TB AP Chest hospital, Hyderabad for TB lymphadenitislymphadenitis

Andhra Pradesh StateAndhra Pradesh State

20042004--20052005JunagarhJunagarh

20042004--20052005LEPRALEPRA-- Hyderabad, Vijayawada, Hyderabad, Vijayawada, AdilabadAdilabad

20022002--20042004MahaveerMahaveer hospital, Hyderabadhospital, Hyderabad20022002--20032003RangaRanga reddyreddy districtdistrict

Page 23

DefinitionsDefinitionsCategory I: Newly diagnosed smear positive pulmonary Category I: Newly diagnosed smear positive pulmonary TB patients (no history of previous anti TB treatment for TB patients (no history of previous anti TB treatment for more than a month)more than a month)

Category II: Smear positive pulmonary TB + previous Category II: Smear positive pulmonary TB + previous anti TB treatment (relapses, failures, treatment after anti TB treatment (relapses, failures, treatment after default etc)default etc)

Category III (extra pulmonary TB): lymph node swellings Category III (extra pulmonary TB): lymph node swellings refractory to treatment with three weeks broad spectrum refractory to treatment with three weeks broad spectrum antibiotics antibiotics

Standard terminology for drug resistanceStandard terminology for drug resistance

2 or 3 drugs excluding, MDR2 or 3 drugs excluding, MDROther resistanceOther resistance

Isoniazid+ rifampicin with or Isoniazid+ rifampicin with or

with out any other drugswith out any other drugs

MDRMDR

one specific drugone specific drugMono resistanceMono resistance

any one or more drugsany one or more drugsAny resistance Any resistance

in previously treated patientsin previously treated patientsAcquired DR*Acquired DR*

in newly treated patientsin newly treated patientsInitial (primary) DR*Initial (primary) DR*

* DR- drug resistance

Fine needle aspirate

ZN-stainingH & E staining Culture

LJ

AFB Growth

Drug Susceptibility testing(1% proportion method)

Sputum

Species identification

Presence of AFB

Spoligotyping

Epithelioid cell granulomas +/-multinucleate giant cells and caseationnecrosis;

MB BacT

M. tuberculosis

1% proportion method1% proportion method--

>1% growth >1% growth 4 4 StreptomycinStreptomycin

>1% growth>1% growth22EthambutolEthambutol

>1% growth>1% growth4040RifampicinRifampicin

>1% growth>1% growth0.20.2IsoniazidIsoniazid

Resistance Resistance Concentration Concentration (ug/mL) (ug/mL)

Drug Drug

Source: IUATLD. Minimum Requirements, Role And Operation In A Low Income Country. The Public Health Service National Tuberculosis Reference Laboratory And The National Laboratory Network. 1998: 62- 65

Page 24

Drug resistance in CategoryDrug resistance in Category I patientsI patients

222299331313113113Vijayawada (urban)Vijayawada (urban)

444477771212479479Hyderabad (urban)Hyderabad (urban)

Andhra PradeshAndhra Pradesh

RR= RR= RangaRanga reddyreddy, H=isoniazid, R=rifampicin, S=streptomycin, E=ethambutol, H=isoniazid, R=rifampicin, S=streptomycin, E=ethambutol

OrissaOrissa (tribal)(tribal)

Adilabad (tribal)Adilabad (tribal)

RR district (rural/semi urban)RR district (rural/semi urban)

Area (Number of isolates)Area (Number of isolates)

Drug resistanceDrug resistance

00335500883939

4444444410104848

2233335599175175

MDRMDREESSRRHHTotal Total

number of number of

isolatesisolates

% Drug resistance pattern in Category% Drug resistance pattern in Category--II

Andhra PradeshAndhra Pradesh

Drug resistanceDrug resistance

220033113113Vijayawada (urban)Vijayawada (urban)

442277479479Hyderabad (urban)Hyderabad (urban)

RR= RR= RangaRanga reddyreddy, H=isoniazid, R=rifampicin, S=streptomycin, E=ethambutol, H=isoniazid, R=rifampicin, S=streptomycin, E=ethambutol

OrissaOrissa (tribal)(tribal)

Adilabad (tribal)Adilabad (tribal)

RR district (rural/semi urban)RR district (rural/semi urban)

AreaArea

0000003939

4400444848

223355175175

MDRMDRMonoMono

resistanceresistance

Any Any

resistanceresistance

No. of No. of

isolatesisolates

% rifampicin resistance in Category% rifampicin resistance in Category--II ObservationObservation

Tribal isolates from Category I patients did Tribal isolates from Category I patients did not show not show rifampicinrifampicin resistance resistance

Page 25

Drug resistance in CategoryDrug resistance in Category II patientsII patients

0

5

10

15

20

25

30

35

Isoniazid

Rifampicin

Strpeotmycin

EthambutolMDR

Hyderabad (n=148)Orissa (n=25)

% Drug resistance pattern in % Drug resistance pattern in CategoryCategory--II patientsII patients

23.6 24

0.6

4

22.2

20

0

5

10

15

20

25

Any resistance Mono resistance MDR

Hyderabad ( n=148)Orissa (n=25)

% Rifampicin resistance in % Rifampicin resistance in CategoryCategory--II patientsII patients ObservationObservation

RifampicinRifampicin mono resistance is higher in mono resistance is higher in tribal isolates than that in Urban isolatestribal isolates than that in Urban isolates

Page 26

57827

56639

55451

504101

Total

R=resistant, S=sensitive, percentages are indicated in parentheses892 S2674R

<0.001Ethambutol892S1882R

<0.05Streptomycin

793S2278R

<0.001Rifampicin892S1387R

<0.1Isoniazid

P valueUn favourable outcome (51)

Favourable outcome (554)

Drug Drug resistanceresistance

Drug resistance vs. treatment outcomeDrug resistance vs. treatment outcome ObservationObservation

RifampicinRifampicin and and ethambutolethambutol resistance is resistance is significantly associated with significantly associated with unfavourableunfavourabletreatment outcometreatment outcome

TB lymphadenitisTB lymphadenitis160 patients presenting with 160 patients presenting with peripheral lymphadenopathy peripheral lymphadenopathy who were non responsive to who were non responsive to treatment with 3 weeks of treatment with 3 weeks of broad spectrum antibiotics broad spectrum antibiotics and clinically suspected of and clinically suspected of TB (TBLN)TB (TBLN)

Page 27

ProfileProfile

96% patients were from >12 years96% patients were from >12 years94% were cervical lymph nodes 94% were cervical lymph nodes 16% patients sero positive for HIV 16% patients sero positive for HIV 16% had H/O previous anti TB treatment. 16% had H/O previous anti TB treatment. Overall Overall -- more femalesmore femalesMore females in < 20 years More females in < 20 years Equal in 20Equal in 20--40 40 More males in 40More males in 40--5050

Drug resistance in lymph node TBDrug resistance in lymph node TB

11 (19)11 (19)Mono resistance Mono resistance (isoniazid)(isoniazid)

4 (7)4 (7)MDRMDR

4 (7)4 (7)RifampicinRifampicin15 (26)15 (26)IsoniazidIsoniazid15 (26)15 (26)Any ResistanceAny Resistance43 (74)43 (74)Susceptible to allSusceptible to all

N=58 (%)N=58 (%)Isolates Isolates

SpoligotypingSpoligotyping for Direct Repeat locus region for Direct Repeat locus region polymorphismpolymorphism

SpoligotypingSpoligotyping for Direct Repeat locus region for Direct Repeat locus region polymorphism polymorphism

Reference: Reference: GoguetGoguet YO et al,YO et al, J J ClinClin MicrobiolMicrobiol, 1997; 35: 2210, , 1997; 35: 2210, IsogenlifeIsogenlife sciences, Netherlandssciences, Netherlands

sequences matched with data base. .

DRaDRa ((5’5’--CCG AGA GGG GAC GGA AACCCG AGA GGG GAC GGA AAC--3’)3’)

DRbDRb ((5’5’-- GGT TTT GGGTCT GAC GACGGT TTT GGGTCT GAC GAC--33’)’)

Hybridisation with set of 43 oligonucleotides

Chromosomal Chromosomal DNA extractionDNA extraction

PCR

Spacers seen by chemiluminescence. .

Page 28

SpoligotypesSpoligotypes of MDR isolatesof MDR isolates

••Spacer 34 absent for allSpacer 34 absent for all

••Spacers 2,3, 29 Spacers 2,3, 29 --32, 34, 32, 34, 3737--39 were absent 39 were absent

••Two belonged to EAI3 Two belonged to EAI3 type (ST 11)type (ST 11)

••Also reported from Delhi*Also reported from Delhi*

••Two isolates lack spacer Two isolates lack spacer 34 alone34 alone

••Nearer to Manu subtype Nearer to Manu subtype (33&34 absent)(33&34 absent)

••Commonly seen cluster Commonly seen cluster from Mumbaifrom Mumbai****

**Mistry et al, JCM 2002;40 (7):2677–80, *Singh UB et al, EID 2004; 10(6): 1138-42

Overall observationsOverall observations

Rifampicin resistance in TB varies in different Rifampicin resistance in TB varies in different geographical areasgeographical areas

Significantly influences the treatment outcome Significantly influences the treatment outcome

M tuberculosisM tuberculosis isolates from tribal area did not isolates from tribal area did not show rifampicin resistanceshow rifampicin resistance

Different genotypes can be associated with the Different genotypes can be associated with the in vitro rifampicin resistance in vitro rifampicin resistance

However, studies with large number of isolates However, studies with large number of isolates are needed to confirm the findings.are needed to confirm the findings.

Need for drug resistance studies in Need for drug resistance studies in leprosyleprosy

Page 29

Mechanism of rifampicin resistanceMechanism of rifampicin resistance

Rifampin (RIF) resistance is due to mutation in Rifampin (RIF) resistance is due to mutation in rpoBrpoB gene encoding for the ßgene encoding for the ß--subunit of the subunit of the RNA polymerase RNA polymerase M. tuberculosisM. tuberculosis and and M. leprae share a similar M. leprae share a similar mechanismmechanismRifampicin resistance in leprosy may give rise to Rifampicin resistance in leprosy may give rise to either treatment failures or relapses as it does in either treatment failures or relapses as it does in TBTBHence all such cases need to be tested for Hence all such cases need to be tested for rifampicin resistancerifampicin resistance

3249753233103BPRCBPRC

9689607331828755754594OrissaOrissa

265197318260209159120APAPPBPBMBMBPBPBMBMBPBPBMBMBPBPBMBMB

Released Released from from treatmenttreatmentN=2417N=2417

Child Child CasesCasesN=159N=159

Treated Treated casescasesN=2089N=2089

New New casescasesN=1763N=1763

ProjectsProjects

LEPROSY cases during 2005-2006

Relapses at BPRC clinicRelapses at BPRC clinic

Total 40 cases (2000Total 40 cases (2000--2004)2004)5 cases (20055 cases (2005--2006)2006)

Case Case --1150 year old male patient 50 year old male patient already treated for leprosy, already treated for leprosy, reported with high fever, severe reported with high fever, severe joints pain, exacerbation of joints pain, exacerbation of existing lesion and appearance existing lesion and appearance of new lesion of new lesion

Diagnosed as BB and Diagnosed as BB and downgraded to BLdowngraded to BL

GranulomaGranuloma fractionfraction--90%90%

Even after giving 12 pulses of Even after giving 12 pulses of treatment both clinical as well treatment both clinical as well as bacteriological condition as bacteriological condition remained the same remained the same

4.664.6620/6/0620/6/063.333.3321/2/0621/2/063.03.07/7/057/7/05

BIBIDate Date

Page 30

CaseCase--22A female patient 24 years presented diffuse infiltration all oveA female patient 24 years presented diffuse infiltration all over the r the body with thickened ear lobes.body with thickened ear lobes.

Bacteriological examination:Bacteriological examination:

Clinical improvement (Nodules started resolving) and Clinical improvement (Nodules started resolving) and bacteriological clearance noticed bacteriological clearance noticed with in 2 months of replacing with in 2 months of replacing rifampicin with rifampicin with ofloxacinofloxacin

113.253.2512/07/0612/07/06

115.335.3325/05/0525/05/05

0.00.03.003.0011/10/0611/10/06

0.00.04.04.022/12/0422/12/04

0.00.05.665.6623/12/0323/12/03

MIMIBIBIDate Date

ConclusionConclusion

Observations on Observations on rifampicinrifampicin resistance in resistance in TB prompts to investigate for the same in TB prompts to investigate for the same in leprosyleprosyThis may lead to the knowledge on the This may lead to the knowledge on the probable causes of probable causes of rifampicinrifampicin resistance resistance Inadequate treatment or biological Inadequate treatment or biological properties of properties of M . M . LepraeLeprae ????????????

ACNOWLEDGEMENTSACNOWLEDGEMENTSProf. Prof. IndiranathIndiranath for the encouragementfor the encouragementDr. KV Krishna Murthy , Dr. Vijaya Dr. KV Krishna Murthy , Dr. Vijaya lakshmilakshmi and and Dr. Sonia for their contributionsDr. Sonia for their contributionsTravel is supported by BPRC,LEPRATravel is supported by BPRC,LEPRA--SocietySocietyMedical Officers and Project coordinators of Medical Officers and Project coordinators of LEPRA projects.LEPRA projects.Collaborators: Dr. NF Collaborators: Dr. NF MistryMistry, Dr KJR Murthy, Dr , Dr KJR Murthy, Dr AVV AVV SatyaSatya NarayanaNarayana, Dr CE Prasad. , Dr CE Prasad. Our technical teamOur technical team-- Kishore Kishore reddyreddy, Srinivas, , Srinivas, Triveni, Triveni, MeherMeher vanivani, , Mohd.IsmailMohd.Ismail, , PrameelaPrameelaChauhanChauhan, , SyedSyed MujafarullahMujafarullah and and AnuradhaAnuradha

Page 31

Overview of Drug Resistance Screening at Karigiri

Tom Gillis, Ph.D.Gift Norman, M.D.

Schieffelin Leprosy Research and Training Centre, Karigiri

National Hansen’s Disease ProgramsBaton Rouge, LA

Questions surrounding threats to leprosy control

• Operational Level– Can effective diagnosis/treatment capabilities be sustained with

integration into community health services?

• Technical Level– Will MDT alone reduce incidence?

• Other strategies (vaccines, new drug treatments)

– What are the long-term effects of immunomodulatory diseases and new treatments on leprosy?

– Is drug resistance a problem?

Patient History of relapse case with drug-resistant M. leprae (SLR&TC, Karigiri)

• 35 year old male• 1983 LL Diagnosis (dapsone, 3 months)• 1984-88 WHO MDT (rif/dap/clof, 40 pulses)

– (smear neg in 1987)• 1988 released from treatment• Clinically inactive next 14 years• 2002 suspected relapse (SSS = 4.25)• MFP report = dapsoneR*/ rifampinR**/clofazimineR***

• DNA sequence confirmed folP mutation for dapsoneR ONLY*Resistance at 0.01%**Resistance at 0.003%***Resistance at 0.0001%

RifampinRifampin--resistant strains of M. leprae:resistant strains of M. leprae:20002000--20062006

Patient No. RIF 0.003% RIF 0.03% Year1 6310/97 R 20002 538780 A R 20003 0.3661 R 20004 189204 R 20035 206036 R 20056 G 02 - 4232 R 20007 1569 / 91 R R 20068 251461 R R 20069 5686 / 05 R 200610 2157/ 99 R R 2006

Page 32

Drug resistance testing with mouse foot pad assay

• Infect foot pads with M. leprae• Treat mice with drugs (6-9 mos)• Harvest foot pads• Stain smears and count AFB• Advantage

– Test drug resistance without knowing mutation

• Disadvantages– available in very few labs– Labor intensive

40-50 mice/assay

Can we improve drug resistance testing for leprosy?

Drug Resistance DetectionPCR

• DNA amplification– low bacterial cell numbers

• crude biological specimens• highly specific • mutation detection analysis

PCR Mutation Detection Analyses

• PCR-DNA sequencing• PCR-HDA• PCR-SSCP• LiPA• Molecular beacon sequence

analysis• Microchip hybridization

Page 33

Specimen PreparationSkin biopsy– ethanol fix 30 min

• reduces risk of biohazards• preserves DNA for PCR for years

– remove ethanol– rehydrate-30 min– homogenize biopsy– prepare DNA extract

• Enzymatic extraction or F/T

PCR-DNA Sequencing

• PCR amplify extract– RRDR rpoB– SRDR folP– QRDR gyrA

• Obtain DNA sequence– Compare traces

• Assign phenotype– susceptible/resistant RMPS Strain (Ser425)RMPS Strain (Ser425)

RMPR Strain ( Leu425)

Rifampin PCR-Seq Assay

PCR-based MethodsAdvantages

Rapid and simple• assay crude biological specimens• 1-2 days

Sensitivity/Specificity• 103-104 bacteria• 100% (based on PCR specificity)

PCR-based MethodsDisadvantages

• Not routine test• Technically demanding• Contamination

– amplifies contamination• Expensive

– equipment and reagents• Trained technical personnel

Page 34

Recommendations

• Establish standard molecular test(s) for rifampin resistance/susceptibility

• Identify regional Reference Labs with drug susceptibility testing capabilities

• Acquire funding and evaluate rifampin resistance globally

History of drugs for leprosy and associated resistance

• Dapsone - 1950– decades of monotherapy…guess what?

• Rifampin, Clofazimine (1960-70’s)• WHO MDT-1980’s

– Karigiri, India - 5 % (primarily dapsone) – Cebu, PI - 37 % (dapsone) – Few MDR strains identified

• Current rates of drug resistance in most parts of the world…unknown

Anti-Leprosy Drugs

First-line drugs-MDT•Rifampin•Dapsone•ClofazimineSecond-line drugs• Minocycline• Ofloxacin• Clarithromycin

Page 35

Mechanism of ActionRifampin

D N A

R N AP o ly m e r a s e

m R N A

RMP

•$-subunit RNA polymerase•rpoB

O

O

CH3

OCH3

OH

CH3

OH

O

CH3 CH3

OCH3

CH3

NH

OHOH

CH3

O

OHOCH3

N N N CH3

mRNA elongation

rpoB

(Rifampin resistance determining region)

401 407 410 416 420 425 427

Gly Thr Ser Gln Leu Ser Gln Phe Met Asp Gln Asn Asn Pro Leu Ser Gly Leu Thr His Lys Arg Arg Leu Ser Ala Leu

Ser Val Asn Tyr Leu ProAsp Met Val

Rifampin

PheTrp

LysPhe

Cys

Mechanisms of Resistance

Mechanism of ActionClofazimine

CLFRCLF

N

N

Cl

N

CH3

CH3

NH

Cl

• Mechanism of action ?– Binds DNA

• guanine-rich regions– inhibits K+ transport

• Weakly bactericidal • Anti-inflammatory• Genomic arrays may help elucidate

Mechanism of Action Ofloxacin

• Derivative of nalidixic acid • Target

– DNA gyrase• gyrA and gyrB

– negative supercoiling activity• Bactericidal

NH

O

OH

O

F

N

NCH3

OCH3

CH3

H

Page 36

Mechanism of Resistance Ofloxacin

86 87 88 89 90 91 92 93 94 95 96 97CAT CCG CAC GGC GAC GCA TCG ATT TAT GAC ACG TTAHis Pro His Gly Asp Ala Ser Ile Tyr Asp Thr Leu

Cys Val

NH

O

OH

O

F

N

NCH3

OCH3

CH3

H

gyrAQuinolone resistance determining region

Mechanism of ActionMinocycline

MinMin

• Tetracycline (lipophilic)• Mechanism of action ?

– Binds 30S ribosomal subunit blocking protein synthesis

– Bactericidal

• Resistance ?– Efflux, ribosomal protection,

enzymatic inactivation

O H

O H

O HO H

H

NH2

OO O

HN

CH3 C H3NCH3 C H3

Thanks for your attention!

Thomas P. Gillis, Ph.DChief, Laboratory Research Branch

National Hansen’s Disease Programsat

School of Veterinary MedicineLouisiana State University

Skip Bertman Drive, Baton Rouge, [email protected]

Contact Information

Mechanism of ActionDapsone

Folate BiosynthesisGUANOSINE TRIPHOSPHATE

TETRAHYDROFOLATE

+ PABA

2-AMINO-4-HYDROXY-6-HYDROXYMETHYL-DIHYDROPTERIDINE

DIHYDROPTERIDINEDIPHOSPHATE

7,8-DIHYDROPTEROATE

DIHYDRONEOPTERIN TRIPHOSPHATE

DIHYDROPTEROATE SYNTHASE (folP)

H

N

N

OH

CH2O

N2 N

N P O P

OH

OH

OO

OHHH

N

N

OH

CH 2

NH2 N

N N COOH

H

H

H

H

H

H

NH2 COOH

NH2SO

OH2N

Dapsone

SNH2 NH2

O

O

Page 37

Mechanism of Resistance DapsoneSNH2 NH2

O

O

49 50 51 52 53 54 55 56

Ile Leu

Sulfone resistance determining region

folP1

GGT GGC GAA TCG ACC CGG CCC GGTGly Gly Glu Ser Thr Arg Pro Gly

Ala ArgArg

Page 38

Screening for drug resistant M.leprae using mouse foot pad in relapse cases of MB leprosy - A Multi-centric Study

OBJECTIVES:

• Detection of cases of MDR in relapsed leprosy cases and patients released from treatment after various regimens of FDT

• To correlate the results of mouse foot pad with detection in rpoB (target of rifampicin ) and folP(targetfor dapsone resistance).

MATERIALS AND METHODS :

Participating centres : • NJIL&OMD, Agra• GRECALTES, Kolkata• BLP, Mumbai• SLRTC, Karigiri

Identification of suspected cases :• Relapsed cases with clinical detail (TT, BT, I, BB, BL,LL ,smear state ,

histopathology if available) as well as cases with persistent BI related to the following treatment regimens will be included-(1). FDT -12month MB MDT treated cases. (2). FDT- 24 month MB MDT treated cases. (3). Cases treated with newer drugs

• RO (RFM+ OFLO) daily for 28 days • ROM (RFM+ OFLO+ MINO once a month) for 1, 3, 6 and /or12 months.

MATERIALS AND METHODS-Cont. :

• Cases treated with bizarre treatment regimens or regimens comprising of other drugs like Ofloxacin, Minocycline etc.

Definition of relapse :Relapse in PB Leprosy:

• Any one or more of the criteria observed after successful completion of the prescribed FDT is considered as relapse.

• increase in the size of the patch/s, • appearance of new lesions which do not respond to

steroid treatment and • patient becoming smear positive is taken as relapse.Relapse in MB Leprosy:

• Any one or more of the criteria observed after successful completion of the prescribed FDT is considered as relapse.

• The increase in BI by 1 log or more.


• Appearance of new lesions is taken as relapse.Non-responders:

• Cases where BI continues to persist despite completion of full course of treatment.

Released from treatment (RFT): • After successful completion of the prescribed treatment schedule

the patients are considered RFT in this study. Persisting BI at the same level as on stopping treatment for one year will be considered as non responders for this study.

• Biopsies collected from JALMA, BLP ,SLRTC and GRECALTES were included for mouse foot pad as well as molecular biological studies .

Page 39


BLP 13 FDT-12 m(5); FDT-24 m (2) new drugs (RO-28d-2);ROM 3m( 2); DDS mono(2).

SLRTC 10 FDT -12m MDT(4); FDT-24mMDT(1);DDS mono(2); MDT 3y(3).

GRECALTES 17 FDT-12m MDT(5 ) ; FDT-24 mMDT(2); DDS mono(2); MDT 4y (8).

NJIL&OMD 19 RFT 2y (2 ); DDS mono(1); MDT2y-(2);RIF+INH+TCH(1); MDT1y(1);MDT3y(12)

MATERIALS AND METHODS-Cont. :• Biopsies from the relapsed as well as non-responder refractory cases

were transported at 40C immediately by messenger for inoculation in the mouse foot pad. The biopsies with BI up to 2+ (Ridley Joplingscale) were inoculated in Tr 900r mice and normal mice at SLRTC, Karigiri for detection of resistance. Similarly the biopsies with BI of › 2 (Ridley Jopling scale) were tested for resistance at NJILOMD, Agra.

• The biopsy were minced with scissors, homogenised and suspended in Hanks balanced salt solution. After allowing the suspension to stand for 3 min. the supernatant fluid was collected and bacterial enumeration was carried out. A batch of random-bred BALB\ C Mice (8-10 mice depending upon the number of bacilli ) was inoculated in to each hind foot pad with a 0.03-0.04 ml suspension containing up to 104 bacilli.


• Drug sensitivity of M.leprae to DDS, Rifampicin, Clofazimine, Minocycline and Ofloxacin was carried out by giving the drug either through the feed or through the gavage at the conc. of 0.01g % DDS, 0.03g% Rifampicin 0.01g % Clofazimine, 0.01g% Minocycline and 50 mg /kg of body weight of Ofloxacin. The harvests were done after 8 and 10 months of innoculation.

• he

Molecular Biology

Different Biopsy samples were taken from Mumbai, Kolkata, Karigari, JALMA. DNAs from different biopsies samples were extracted by treatment with lysozyme /proteinase-K, deproteinization and precipitation (van Embeden et al (1993)The DNA sequencing reaction was performed with Big Dye Terminator cycle sequencing PCR.The amplicons were purified by precipitating with 3M sodium acetate and absolute alcohol.The sequence data was generated with ABI 310 automated sequencer. The sequences were analyzed with MEGALIGN program (DNASTAR) and compared with a susceptible rpoB and folP1 gene sequences.

Page 40

RESULTS:

Out of the 59 biopsies, results of 16 biopsies from MFP are available and none of the biopsy has shown any resistance.

Out of 48 samples, 35(73%) had no mutation in Rifampicin rpoB locus while in 13(27%) rpoB could not be amplified.

Out of 48 samples, 27(56%) samples were amplified for folP1gene. Out of 27 samples 2(7.4%) showed mutations ACC → ATC;Thr53Ile and CCC → CTC; Pro55Leu. Theses mutations were previously reported by Maeda et al (2001) in dapsone resistant samples of M.leprae.

Results

48

16

11

10

11

(Sample No)

Total

*NA(21)*NA(13) Amplified (27)Amplified (35)

No Mutation(9)

No Mutation (6)CCC→TC;Pro55Leu(1)

No Mutation (5)

No Mutation(5)ACC→ATC;Thr53Ile

(1)

folP1 GenerpoB Gene

73No Mutation(13)

JALMA

41No Mutation(10)

Karigari

54No Mutation(6)

Kolkata

55No Mutation(6)

Mumbai

Mutation DetectionCentres

*NA/WA=Not Amplified/Weak Amplified

CONCLUSIONS:

So far no resistance has been observed by MFP for dapsone and rifampicin resistance but 2 specimens showed mutations in folP1 gene . However, there is a need to analyze large number of Indian M.leprae samples so as to arrive at statistically valid conclusions.

Acknowledgment

Raj Kamal, M. Arora, B. Bansal, K. Katoch , J.K. Chakma , B.K. Girdhar, R. Ganapati , V. Pai , G. Shah, G. Norman and A. Joseph for providing biopsy specimens.Ram Das, Mallika Lavania, V.D. Sharma, D. S. Chauhan for Molecular Biology work.ICMR for financial assistance.

Page 41

Leprosy research at CSUNIH-NIAIDLeprosy Contract: PI: Patrick J. Brennan•Provide materials•Basic research objectives

PI: Varalakshmi VissaRO3: M. leprae strain typingRO1: Molecular epidemiology of leprosy

PI: Patrick J. BrennanRO1: Leprosy bacillus: Genotype to Phenotype

The Heiser Program for Research in Leprosy and TB of the New York Community Trust

Initiative for Diagnostic and Epidemiological Assays for Leprosy (IDEAL)PI: Hazel Dockrell

•Molecular Epidemiology Field Study•T cell assays for early diagnosis

Molecular epidemiology of leprosy

Continued incidence

Approach: Can molecular methods answer Transmission issues?

Reservoirs: patients, environmentDetection, Description, and TrackingViability of M. lepraeHost SusceptibilityEvaluation of Drug resistance

Molecular epidemiology of leprosyCollaborators

Beijing Tropical Medicine Research institute, China

Thai-NIH, Thailand

Leonard Wood Memorial, Philippines

Yonsei University, Korea

Blue Peter Research Center, India

Stanley Browne Laboratories, India

Instituto Colombiano de Medicina Tropical, Colombia

Leprosy bacillus: Genotype to Phenotype

Collaborators

Institut Pasteur, France

Oswaldo Cruz Foundation, FioCruz, BrazilUFG, Goias

Leonard Wood Memorial, Philippines

Yonsei University, Korea

Page 42

Resources and MethodologySelected Study sites

Local and National Ethical committee approvals

Retrospective and Prospective sampling

Patients Contacts

Biological SamplesBiopsy, SSS, blood (serum), nasal swabs

Sample banks

Epidemiological information

Transmission Causes/Patterns

Interruption

AssaysStrain typing

Drug resistance

Infection and immunitySerology: PGL-1 and new antigensT cell based assays

Viability testsmRNAs, 16SrRNA

Drug resistance

Target population?

CurrentAll new untreated patients

PastOld patient samples

Under treatment

Post MDT surveillance

High BIs ???Monotherapy?DDSRifHigh BIs

RelapseDrug resistance?

rpoB or folP1 mutations3/30/2007 168

CAGGACGTCGAGGCGATCACGCCGCAGACGCTGATCAATATCCGTCCGGTGGTCGCCGCTATCAAGGAATTCTTCGGCACCAGCCAGCTGTCGCAGTTCATGGATCAGAACAACCCTCTGTCGGGCCTGACCCACAAGCGCCGGCTGTCGGCGCTGGGCCCGGGTGGTTTGTCGCGTGAGCGTGCCGGGCTAGAGGTCCGTGACGTGCACCCTTCGCACTACGGCCGGATGTGCCCGATCGAGACTCCGGAGGGCCCGAACATAGGTCTGATCGGTTCATTGTCGGTGTACGCGCGGGTCAACCCCTTCGGGTTCATCGAAACACCGTACCGCAAAGTGGTTGACGGTGTGGTCAGCGACGAGATCGAATACTTGACCGCTGACGA

CTG CCG

GAT AATTCG TTG

TGGCAC TAC

rpoB

CTGTCGCACGAT003 rpoB

CAG (CTG)CGA (TCG)GTG (CAC)ATC (GAT)008 rpoB

CAG (CTG)CAA (TTG)GTG (CAC)ATC (GAT)006 rpoB

CTGTCGCACGAT001 rpoB

CTG → CCG

(458)

TCG → TTG(456) TGG

CAC → TAC(451)

GAT → AAT(441)

Upper primer

Lower primer

•The pink colors highlight the mutations in codons commonly reported in the literature for M. leprae and other organisms

As of 2-9-06rpoB and folP Colombia data page2

Page 43

>M. leprae |ML0224|folP1: 855 bp

GTGAGTTTGGCGCCAGTGCAGGTTATTGGGGTTTTGAACGTCACTGACAATTCGTTCTCA GATGGCGGACGTTACCTTGATCCTGACGATGCTGTCCAGCACGGCCTGGCAATGGTCGCG GAAGGCGCGGCGATTGTCGACGTCGGTGGCGAATCGACCCGGCCCGGTGCCATTAGGACC GATCCTCGAGTTGAACTCTCTCGTATCGTTCCTGTCGTAAAAGAACTTGCAGCACAGGGGATTACAGTAAGTATCGATACTACGCGCGCTGATGTTGCACGGGCGGCGCTGCAAAGCGGC GCACGGATCGTCAACGATGTGTCTGGTGGGCGAGCAGATCCCGCGATGGCTCCTCTGGTG GCTGAAGCCGGTGTTGCGTGGGTGTTGATGCACTGGCGACTGATGTCGGCTGAACGGCCG TATGAGGCTCCGAATTACCGCGACGTGGTGGCTGAAGTGCGTGCCGACCTACTGGCTGGT GTCGATCAGGCTGTGGCCGCAGGTGTTGATCCTGGGAGTCTAGTGATCGATCCCGGGCTT GGATTCGCCAAGACGGGACAGCACAATTGGGCGCTGCTGAATGCGTTACCGGAGTTGGTG GCTACTGGGGTCCCGATTCTACTTGGCGCCTCGCGTAAACGGTTCCTGGGTAGGTTATTA GCTGGGGCTGATGGCGCGGTACGACCGCCGGACGGACGTGAGACGGCGACCGCGGTGATT TCCGCACTTGCTGCCCTACACGGGGCTTGGGGTGTTCGGGTGCACGATGTGCGTGCCTCG GTCGACGCACTCAAGGTCGTCGGGGCTTGGCTGCATGCTGGGCCGCAGATTGAAAAGGTTAGATGTGATGGCTGA

Forward primer

Reverse primer

CCCACC006 FolP

CCCACC008 FolP

CCCACC003 FolP

CCCACC001 FolP

CCC→RCGACC→ATC

As of 12.6.05rpoB and folP Colombia data page4

177 bp 5’TTACTGTAATCCCCTGTGCTG 3’ & R5’-TTGATCCTGACGATGCTGTC

Evaluation of presence of mutations in genes associated with drug resistance of Mycobacterium leprae in patients from Brazil(submitted and approved for financing by CNPq)

Maria Leide Van-Del-Rey de Oliveira: EpidemiologyClaudio Salgado, Local Co-ordinator, State of ParáLigia Kerr Pontes, Local Co-ordinator, State of CearáNorma Lucena Silva, Local Co-ordinator, State of Pernambuco

FIOCRUZ – RJPhilip Noel Suffys, Main Investigator of the molecular area

Magnitude and characterization of leprosy relapses in patients

(submitted to the WHO/multi-drug therapy schemes)

•Evaluation of accuracy of the definition of relapse in Brazil

•Improvement of diagnosis and samples collection in different states

•Genetics analysis of the M. leprae isolates.

ObjectivesTotal of leprosy new cases and relapses in different UFs of Brazil.

UF 2001 recidiva % 2002 recidiva %Amazonas 1201 51 4,25 1437 57 3,97Pará 3149 136 4,32 6123 168 2,74Ceará 2092 87 4,16 2570 83 3,23Pernambuco 2414 127 5,26 3391 128 3,77Espírito Santo 1238 19 1,53 1716 25 1,46Rio de Janeiro 1576 64 4,06 3521 106 3,01Total 11670 484 18758 567

Source: www.portal saúde.gov.br/portal/svs/SINAN, 19/07/2005.

Page 44

The occurrence of clinical signals of leprosy activity, after discharge by cure, removing the possibility of leprosy reactions because of the non response to corticotherapy).

•Ministry of Health (Brasil, 2002)

Clinical characteristics of 155 patients re-treated as relapses in Pernambuco,

34% of them presented reaction post-discharge 54% were re-treated in the first three years after discharge, mainly based on clinical diagnosis criteria. Brito (2005)

Relapse definition criteria Objectives

Specific•To know the magnitude of relapses for each therapeutic scheme in each of the studied states;

•To validate the relapse cases, identified by the input way in the active record-SINAN, of the database of the respective State Secretariats of Health;

•To characterize the recent relapse cases regarding to the dermatological/neurological examination, baciloscopy, histopathology and serology;

•To support the identification of mutations associated with the M. leprae drug resistance to rifampicin and dapsone;

•To implement a protocol for diagnosis and handling of relapse cases and cohorts of relapse patients in the respective reference units, aiming the long term follow up and analysis of the cumulative risk of relapse

Type of study: Transversal study using clinical and epidemic parameters followed by a posterior longitudinal study.

Sampling:Size: Notified relapse cases among new ones :

~ Sample size of 370 cases in the 6 states of the study in a period of 2006 – 2007. 1.53-5.26% in 2001, ↓ in 2002, highest in Manaus 3.97.

Study sites: States: AM, PA, ES, PE, CE, RJ) : high (RJ) , hyperendemic (PA, ES).Reference ambulatories: Reference outpatient units (total of 8) where complex cases such as relapses are sent (AM:2; PA:1; ES:1;RJ:2; PE:1) .

Methodology

PB and MB leprosy patients with clinical diagnosis of relapses after discharge by cure

PB: nerve pain, new sensibility alterations, new lesions and/or exacerbation of previous, with no response to corticoids.

MB:1) Appearance of cutaneous lesions, exacerbation of old lesions or new neurological

alterations, after discharge by cure and that do not respond to the corticoid treatment and/or thalidomide;

2) (BI) with intact bacilli (revaluated by the reference unit); 3) Increase of BI in 2+ in any site in relation to the BI in the discharge by cure, if it is

available. Clinical-histopatological and serological correlation. 4) Patient that regularly completed the DNDS and MDT schemes and had therapeutic

discharge.

Inclusion criteria

Page 45

PCR amplification

↓

•177 bp fragment of folP1•178 bp fragment of rpoB gene

↓

SSCP

↓

Sequence of variants and controls

Resequencing

Samples: SSS/biopsyCostConvenienceCoverage (targets DR and others ~50 kb)Volume

Other assays:

RR assays: 107/ml, 1 ml per assay33oC for 7 days (flushed on 1,4,6 days with 2.5% O2-10%CO2-87.5%N2

Day 7: 1uCi [14C Palmitic acid]Read [14C CO2] at 1,3,5 and 7 days

ATP assays: 3.1 x 107/ml , 1ml per assay3 weeks

Mouse foot pad assays: 5 x 103 percoll purified Ml30 days no drug in diet+ 60 days drug in diet

Franzblau and Hastings: AAC 1988

Page 46

Molecular methods for detection of drug resistance in leprosy : Need

• Mouse footpad (MFP) is the accepted method for drug susceptibility test in leprosy.

• Besides the long time taken by MFP method, multibacillary cases are becoming fewer.

• In a recent study by JALMA at Ghatampur, multibacillarycases have decreased from 12% (2003) to 3% (2006).

• Molecular methods will be of special interest for detection of drug resistance in such paucibacillary cases.

Some reports of drug resistance in leprosy from India

• Sekar etal (2002): 16% dapsone resistance between 1983 to 1997, no case of rifampicin resistance, only one case of combined dapsone and unconfirmed clofazimine resistance.

• Ebenezer etal (2002): During 1987 to 1997 : 6.23% resistant to one or more drug (4.88%,dapsone resistance, 1.36% resistant to rifampicin and 2.44% resistance to low concentration of clofazimine, only one strain resistant to all drugs)

• Shetty etal (1996): one strain resistant to dapsone, rifampicin and clofazimine.• Shetty et al (2003): Two out of the 37 relapsed cases had resistance to DDS and

rifampicin, one strain resistance to dapsone, rifampicin and clofazimine.• Resistance to dapsone and rifampicin observe in 4 & 7 out of 77 isolates

respectively from relapsed cases/suspected cases of drug resistance during 1991 to 2002 at JALMA.

• No MDR (dapsone and rifampicin) observed in more recent data from JALMA.

NRYesMDR strains resistant to rifampicin, sulphones and quinolonesreported.

MFP & sequencing

Maeda et al2001,Matsuoka et al 2000 & 2003.

Japan

NDnil6.8 (PR)47.1(SR)

157MFPButlin et al 1996

Nepal

NDND16 cases of dapsoneresistance:6/6 strains HR3/4 IR1/6 LR

38MFPCambau et al 2005

France

2.4430(PR)13(SR)

1.3617.4(PR)4.35(SR)

4.8830(PR)48(SR)

265 (1987-97)

MFP Ebenzer et al 2002

South India

ClofazimineRifampicinresistance

Sulphoneresistance

Resistance (%)Samples

Methods used

AuthorCountry Molecular methods for detection of drug resistance in leprosy

• PCR- SSCP (Honore et al 1993)•PCR and sequencing (Ramasoota et al 2000, Zhang et al 2005)•DNA Heteroduplex analysis (Williams et al 2001)•Touch down PCR- SSCP/ sequencing (Kim et al 2003, You et al 2005)•Reverse line probe assay (Sapkota et al 2006)•DNA Microarray (Suzuki and Matsuoka 2006)

Page 47

Touch down PCR-SSCP and sequencing

Kim et al 2003: Korean strains• Analyses of folP, rpoA, B and gyr A,B genes

were done.• Mutations were identified by PCR-SSCP

and DNA sequencing. • Mutations were observed in the particular

region of folP, rpoB and gyrB.

PCR Single Strand Conformation Polymorphism

Honore et al 1993:In 8 cases missense mutations Ser-425 was observed and in the remaining mutants a small insertion was found.You et al 2005: Korean strains by Touch down PCR- SSCP19.2% mutations- folP2.89% mutations- folP+rpoB1.92% mutations- folP+gyr0.96% double mutations

DNA Heteroduplex analysis(HD-DDS-ML assay)

Williams et al 2001:PCR amplification of 231 bp of folP1 containing codons 53 and 55

PCR product anneal to a universal heteroduplex generator

PAGE

Observed 93% correlation with DDS susceptibility as determined by both DNA sequencing of folP1 and MFP

PCR and Sequencing

Ramasoota et al 2000: Thai strains from cases unresponsive to therapy:

• Observed multiple mutations (Mutations at two, three, six, seven, eight and nine positions)in rpoB gene of 9 M.lepraestrains.

• Most of were point mutation substitutions (few were silent) and some insertions.Zhang et al 2004:Relapsed lepromatous patient were included in the studyMissense mutation at 516 in the rpoB gene of M.leprae

Page 48

Results of JALMA/SLRTC/BLP/GRECALTIS STUDY

48

16

11

10

11

(Sample No)

Total

*NA(21)*NA(13) Amplified (27)Amplified (35)

No Mutation(9)

No Mutation (6)CCC→TC;Pro55Leu(1)

No Mutation (5)

No Mutation(5)ACC→ATC;Thr53Ile

(1)

folP1 GenerpoB Gene

73No Mutation(13)

JALMA

41No Mutation(10)

Karigari

54No Mutation(6)

Kolkata

55No Mutation(6)

Mumbai

Mutation DetectionResigns

*NA/WA=Not Amplified/Week Amplified

Amplification in PB and MB specimens

9

6

5

5

NM

5

3

1

1

NRS

Mutation

2

1

NRSNM

5

1

1

-

-ive

2

1

-

-

-ive+ive+ive

6

9

8

9

MB

3

1

2

PB

48

16

11

10

11

(Sample No)

Total

NA(11)

NA(10)

Amplified (37)Amplified (38)

CCC→CTC;Pro55Leu

(1)

ACC→ATC;Thr53Ile

(1)

folP1 GenerpoB Gene

2113JALMA

1110Karigari

446Kolkata

446Mumbai

Mutation DetectionResigns

NA=Not Amplified; NRS= Not Readable Sequence

ACC →ATC, (Thr53Ile)Mutation

CCC →CTC, (Pro55Leu)Mutation

Panel A: Sequence showing no mutation (normal sequence) of folP1 gene region in Dapsone susceptible M.leprae sample

Panel B: Sequence showing the of the folP1 gene, ACC53ATC(Thr53Ile) mutation and

Panel C: Showing the sequence of the folP1 gene region, CCC55CTC(Pro55Leu)In Dapsone resistant M. leprae samples

B C

A

Dapsone sensitive sequence (no mutation)

A B

Figure A- The electopherograms showing the sequence of the rpoB region TCG(Ser) at codon 531 in susceptible strain M.tuberculosis H37Rv.

B- The electopherograms showing the sequence of the rpoB region mutant codon in resistant isolate TTG (Leu) at codon 531. The actual nucleotide changes are marked bellow.

TCG(Ser) at codon 531

(H37Rv – no mutation)

TCG TTG

Ser 531 Leu

Page 49

Reverse line probe assaySapkota et al 2006:

• Mutations in rpoB gene of 40 strains of M.lepraeanalysed by reverse hybridization based line probe assay after PCR.

• Nine distinct single nucleotide substitutionin drug resistant strains.

72

2-nucleotide substitution

3 nucleotide substitution

JALMA Experience with INNO-LiPA

30/35 (85.7%)30/35Definite RIF resistant (Gp II)

7/10 (70%)3/10Intermediary (Sub Gp ii, iii)

10/10 (100%)0/10Complete sensitive (Sub Gp I)

Microbiologically RIF sensitive (Gp II)

INNO-LiPAconcordance with sensitivity profile

Mutation detection by INNO-LiPA

Sensitivity profile groups

Detection of mutations in rpoB gene region of M.tuberculosis/ mycobacteria

with indigenously designed probes

Oligonucleotides targeting variable regions blotted and hybridized against PCRamplicons generated encoding rpoB gene region of M tuberculosis

RFMresistant

RFMsensitive

Fig.: Assessment of probe system targeting rpoB at CJIL

75

80

85

90

95

100

1st evaluation 2nd evaluation 3rd evaluation 4th evaluation

culture clinical samples

Page 50

2.223.413.6329.5421.5988PCR Seq

Maeda et al 2001

Japan, Haiti, Indonesia, Pakistan, Philippines

NDNilNilMultiple mutations

ND9PCR SeqRamasootaet al 2000

Thailand

NDNilNil1/1010

4/1822

PCR seqKai et al 2004

Pakistan

nil1.922.89119.2104Touch down PCR, SSCP

You et al 2005

Korea

23S rRNAfol P+gyr A

folP+rpoBrpoBfol P

Strains showing Mutations(%)SamplesMethods used

Author

CountryCorrelation of resistance with mutations in M.tuberculosis

52%gyrA, gyrBOfloxacin

48%rspL, rrnStreptomycin

72%katG, inhA, ahpC

INH

97%rpoBRifampicin

PercentageMutationsDrug

DNA Microarray

Suzuki and Matsuoka 2006:Used low density oligonucleotide array that enables the detection of base substitutions involved in resistance against anti-leprosy drugs on a single slide.

Molecular methods for other drugs

• Mutations in gyrA shown to be useful for detection of quinolone resistance (Cambauet al 2002). While it will be tempting to include this target in future molecular assay, the experience of M.tuberculosisneeds to be kept in mind.

• For clofazimine and other drugs the scene is quite hazy.

Page 51

Contributors (Molecular mechanisms) V.M.Katoch, D.S.Chauhan, R.Das, V.D.Sharma, U.D.Gupta,

K.Katoch, M.Natrajan, GP Singh, Anuj Gupta, P.Singh, M. Singh, Mallika Lavania, J. Fauzdar, P.Upadhya, K.Srivastava, and others National JALMA Institute for Leprosy & Other Mycobacterial Diseases

(ICMR) , Taj Ganj, Agra-282001and Groups of S.K.Sharma, Sarman Singh, G.C.Khilnani (AIIMS,

New Delhi)J.K.Agarwal & M.Hanif (NDTBC, New Delhi) ; S.E.Hasnain &

colleagues (CDFD, Hyderabad)Groups of R. Ganapati/V. Pai (BLP), G.Norman( SLTRC),

GRECALTES etcFinancial support : DBT, ICMR

Page 52

Trends in Sulphone and Rifampicin Resistance in MDT Era

: JALMA Experience

Sreevasta et al 1984: Parallel dapsone and rifampicin resistance in three out of five refractory cases observed.

Strains tested between 1985-90 and 1990-2002 analysed for the trends (Gupta et al)

Materials and Methods Patients attending the OPD of JALMA,Agra from 1985-

2002 were the subjects of present study.Skin biopsies from the patients suspected to have drug

resistance ( poor responders/ relapses etc) were taken , homogenized and inoculated in to mouse (BALB/C)foot pads by standard method (Shepard 1960).Harvesting was done after 6 and 8months.

For determining dapsone resistance ,mice were divided in to various groups : (I). Controls with normal diet(II). Treatment group : medium levels –diet containing 0.0001% dapsone ;high levels ; diet containing 0.01% dapsone)

For rifampicin: 0.01% concentration taken.

4(5.19%)23

(27.4%)

(3.9%)(1.3%) (19.1%)(8.3%)Percentage

317716784

High ResMed ResNo. of patients

High Res

Med ResNo. of patients

Post 1990Pre 1990

Results : Trends in Trends in Sulphoneresistance in patients attending JALMA OPD

Rifampicin resistance in patients attending OPD of JALMA (1990-2002)

• No. of patients Resistance (0.01%)77 7(10%)

In 4/7, parallel resistance to rifampicin also existed.

*In none of trial cases, any drug resistance in specimens from relapse cases observed.

Page 53

Contributors

• Dr. Sreevatsa , Dr. Desikan and Dr.U.D. Gupta for MFP analysis.

• Groups of Dr. G.Ramu, Dr. B.K.Girdharand Dr. Kiran Katoch for providing the biopsy specimens.

4 (2.4%)1 (3.17%)3 (2.9%)HES

1 (0.6%)Nil 1 (0.9%)HRS

2 (1.2%)1 (1.53%) 1(0.9%)ES

12 (7.2%)6 (9.23%)6 (5.9%)HS2 (1.2%)Nil2 (1.9%)HE4 (2.4%)Nil4 (3.96%)HRSE

NilNilNilHR11 (6.6%)4 (6.1%)7 (6.9%)Streptomycin (S)1 (0.6%)1 (1.53%)NilEthambutol (E)13 (7.8%)4 (6.15%)9 (8.9%)Isoniazid (H)NilNilNilRifampicin (R)

Mono resistance116 (69.8%)48 (73.8%)68 (67.3%)Fully sensitive16665101Total TestedKanpur DistrictKanpur RuralKanpur Urban

Patterns of antiPatterns of anti--tuberculosis drug resistance in Kanpurtuberculosis drug resistance in Kanpur

JALMA (ICMR)

MultiMulti-- drug resistance in Agra and drug resistance in Agra and KanpurKanpur

0

12

345

678

Perc

enta

ge

AGR2004 AGR2005 Kanpur2005 Kapur2006

Rural Urban Total

JALMA (ICMR)

Multicentric Study for Drug Resistance Surveillance in Leprosy

• An ICMR Task Force Project• Study initiated in January 2005.• 59 biopsies by mouse foot pad and 48

by molecular methods targeting rpoBand folP 1 studied.

• Interim results available.

Page 54

Surveillance on rifampicin resistance in tuberculosis and available tools for

diagnosis

C.N.ParamasivanFoundation for Innovative New Diagnostics

Global MDR TB Burden in New Cases

Global MDR TB Burden in Previously Treated Cases

Burden of Multidrug-Resistant Tuberculosis • JID 2006:194 (15 August) • 483

Page 55

Estimates of MDR TB Cases in New Cases in India

2.4 (1.0–5.0)

44,653 (13,547–85,068)

1,824,395India

% (95% CI)No (95% CI)

MDR-TB casesNew casesCategory

Burden of Multidrug-Resistant Tuberculosis • JID 2006:194 (15 August) • Table 2

Estimates of MDR TB Cases in Previously Treated Cases in India

14.7 (2.1–56.9)

42,760 (6068–171,774)

290,019 India

% (95% CI)No (95% CI)

MDR-TBPreviously Treated cases

Category

Burden of Multidrug-Resistant Tuberculosis • JID 2006:194 (15 August) • Table 4

Prevalence of Primary Drug ResistanceTuberculosis Research Centre Studies – (1974 -2001)

0

5

10

15

20

XII 74-77

XIII 77-80

XIV80-85

XV 85-86

XVI 86-90

XVII90-95

XVIII95-98

XIX 98-00

XXI2001

perc

enta

ge

MDRRIFSTREPINH

After the introduction of rifampicin in Controlled Clinical Trial at TRC

Page 56

DRS sites of India (1985-2003)

North Arcot – 1985-89 (2%), 1989-90 (1.7%), 1999 (3%)

Pondicherry - 1985 (0.9%)

Tamil Nadu - 1995 (3.3%)

AFMS – 1995 -1999 (2.7%)

Bangalore - 2002 (2.2%)

Mysore - 2001 (1.2%)

Raichur - 1989(3.2%), 1999 (2.5%)

Wardha - 2001 (0.5%)

Jabalpur - 2002 (1%)

Mayurbhanj - 2002 (0.7%)

Hoogli - 2003 (3%)

Nawgong - 2003 (7.2%)

Population covered = 8.1%

Level of MDR in ‘New’ in Different Sites in India(Population Covered 8.1%)

7.2%

3.3%

3.2%

3.0%

3.0%

2.7%

2.5%

2.2%

2.0%

1.7%

1.2%

1.0%

0.9%

0.7%

0.5%

0.0% 1.0% 2.0% 3.0% 4.0% 5.0% 6.0% 7.0% 8.0%

NAWGONG (350) (2003)

TAMILNADU (389) (1995)

RAICHUR (244) (1989)

HOOGLY (350) (2003)

NORTH ARCOT (282) (1999)

AFMS (2562) (1995-99)

RAICHUR (278) (1999)

BANGALORE (366) (2002)

NORTH ARCOT (2779) (1985-89)

NORTH ARCOT (350) (1989-90)

MYSORE (203) (2001)

JABALPUR (273) (2002)

PONDICHERRY (2127) (1985)

MAYURBHANJ (343) (2002)

WARDHA (197) (2001)

SITE

S

MDR – TB% amongst New cases

85

73

15

27

10 13

1.7 2.20

102030405060708090

U n T re a te d

fully sens.Any Res.Any HHR

Drug resistance among newly diagnosed casesDrug resistance among newly diagnosed cases

MDP area N=1603

Population:500 000

TB Res. Centre, Chennai

1999 - 2003

Bangalore city N=271NTI, Bangalore

2000-2001

59 60

41 4037

27

12 13

0

10

20

30

40

50

60

70

R e tre a tme n t c a se s

Fully densitiveAny Res.Any HHR

Drug susceptibility among previously treated casesDrug susceptibility among previously treated cases

MDP area N=443

Population: 500 000

TB Res.Centre, Chennai

1999-2003

Bangalore city N=226NTI, Bangalore

2000 - 01

Page 57

Drug Resistance in Patients With HIV / TB in South India

62.2

13.6

36.8

13.2

27

4.2

13.5

84.4

0

10

20

30

40

50

60

70

80

90%

of P

atie

nts

suseptible to all Any resis.

Res. To H MDR

New cases-167 Treated cases-37

Swaminathan S et al IJTLD 2004

Fig Zoning for DRS surveys

- Maharashtra & Gujarat- Andhra Pradesh & Kerala- West Bengal & Orissa- Uttar Pradesh & Delhi

Zones &Selected States ( )

Year 2005Year 2005--8 DRS sites of India 8 DRS sites of India

Maharashtra, 102.8 millions (9.4%), IV Qrt 2005

Gujarat, 53.8 millions (4.9%), I Qrt 2005

Orissa, 38.2 millions (3.5%), 2007- 8

Andhra Pradesh, 78.7 millions (7.2%), 2007 - 8

Population being covered = 25%

Resurvey – TamilnaduDRS-Sikkim,2005-06

Gujarat DRS DataInterim analysis

DRUS SUSCEPTIBILITY RESULTS (GUJARAT)

56.0%

21.2%

44.0%

14.9%

25.6%

10.5%

34.7%

2.8%

15.9%

78.8%

17.0%

2.4%

0.0%

10.0%

20.0%

30.0%

40.0%

50.0%

60.0%

70.0%

80.0%

NEW CASES TREATED CASES

Perc

enta

ge

Fully sensitive Any resistance Any S resistance Any H resistanceAny R resistance Any HR resistance

Page 58

Extensively resistant (XDR) tuberculosis

• XDR-TB is defined as cases in persons with TB whose isolates were resistant to isoniazid and rifampin and at least three of the six main classes of second line drugs (SLDs) such as aminoglycosides, polypeptides, fluoroquinolones, thioamides, cycloserine, and para-aminosalicyclic acid.

CDC, MMWR. 2006; 55: 301 - 305

Revised XDR TB - Definition

• Resistance to:– At least rifampicin and isoniazid– A fluoroquinolone– One or more of the following injectable drugs:

• kanamycin• amikacin• capreomycin

M.tuberculosis with extensive resistance to 2nd

line drugs- World wide,2000-04

(The extent to which resistance to 2nd line drugs emerged among MDR-TB isolates)

No.of SRLs participated : 25No. of countries studied : 49No. of isolates analysed : 17,690Prevalence of MDR TB : 20%Prevalence of XDR TB : 2%XDR in South Korea : 15%XDR in E. Europe / W.Asia : 14%

(Limitations: Variations in testing methods; XDR definitions described & sample bias)

MMWR 2006; 55(11): 301 - 305

Drug Resistance pattern of referred samples to TRC, Chennai, 2001-04

Susc. 32.5%Res. 1 or more 67.5%

(n: 2816 patients)(n: 2816 patients)

Page 59

Drug Resistance pattern of referred samples to TRC, Chennai, 2001-04

(n : 2816 patients)

43.2

67.5

1

29.9

6.5

24.6

9.7

53.2

0

20

40

60

80

100

S H R E K Th Ofl. HR

Drugs

( % )

XDR-TB among referred samples to TRC, Chennai , 2001- 2004

• XDR-TB to be approximately 4% of 1498 MDR TB isolates during the period 2001 to 2004 from chronically ill patients ( highly selective group) with a prolonged and

varying period of treatment history.

DRUG SUSCEPTIBILITY TESTS

It is one of the most demanding procedures to perform &to standardize in the Mycobacteriology lab. Proficiency in susceptibility tests demands an understanding of the origin of drug resistanceThe variation in stability of drugs subjected to different conditions of filtration, heat or storageThe alteration in the activity of certain drugs when incorporated into different kinds of mediaThe type of susceptibility test performedThe reading and reporting of test resultsThe criteria of resistance

Most common DST techniques

• Proportion method

• Absolute concentration

• Resistance ratio

• Broth (or liquid) methods

• Detection of metabolic changes

• Mycobacteriophage - based tests

• Molecular methods

World Health Organization. Guidelines for the programmatic management of drug-resistant tuberculosis. 2006. WHO/HTM/TB/2006.361

Page 60

Other Phenotypic Methods

E TestMicro Well Alamar Blue Assay (MABA) Micro plate tetrazolium reduction assayNitrate reductase assayMycolic acid index susceptibilty testingMODSMicro colony detectionPha B AssayLuciferase reporter phage assay

LINE PROBE ASSAY : A meta analysis

• LiPA is a highly sensitive and specific test for the detection of rifampicin resistance in culture isolates.

• The test appears to have relatively lower sensitivity when used directly on clinical specimens.

• More evidence is needed before LiPA can be used to detect MDR-TB among populations at risk in clinical practice

Morgan M et al. BMC Infectious Diseases 2005, 5:62

TB bacilli

ActiphageTREATMENT WITHVIRUSOL

INFECTION

NEUTRALISATIONOF VIRUSOL ANDADDITION OFSENSOR CELLS

PLATING OFMIXTURE IN APETRI DISH ANDOVERNIGHTINCUBATION

Phage start toreplicate in

cells

Sensorcells

Sensorcells

Sensorcells

Sensorcells

Sensorcells

Phage replication assay for detection or DST

POSIT

IVE

NE

GA

TIV

E

FIND/TDR Trial in PeruPreliminary Results (2)

Performance Characteristics for RIF-R*Test Sens Spec PPV NPVINNO 93% 99% 93% 99%FP-1 95% 96% 79% 99%FP-2** 94% 100% 100% 99%D-LJ 95% 99% 95% 99%

*Subject to change pending results of discrepant analysis**Based on results 135 tests with complete results

Page 61

Characteristics of several drug susceptibility testing methods

Most drugsNo Low>0.95Nitrate reduction assay

Most drugsNoLow >0.95Colorimetric methods

R, H, E, SYes High >0.93ESP System II

R, H, E, SYes High >0.95MB/BacT

R, HSome skill Low/medium >0.9Fast Plaque TB

RYesHigh >0.95Solid-phase hybridization test

Several drugsYes High >0.9 (for R)DNA sequencing

R, H, E, SNo Medium >0.9E-Test

R, H, E, SYes High >0.9Automated MGIT 960

Several drugsNo Medium >0.9Manual MGIT

Most drugsYes High >0.95BACTEC radiometric

All drugsSomeLow >0.95Proportion method

CoverageSkills/ equipmentCost Accuracy

%Method

MGIT: mycobacteria growth indicator tube; TB: tuberculosis; R: rifampicin; H: isoniazid; E: ethambutol; S: streptomycin.

J.C. Palomino, Eur Respir J 2005; 26: 339–350

Extraction and purification DNA/RNA

Amplification

Detection

Conventional NAAT Cepheid

Extraction and purification DNA/RNA

Amplification

&

Detection (automated)

Workflow• sputum• simple 1-step external sample prep.

procedure• time-to-result < 2 h • throughput: > 16 tests / day / module• no need for biosafety cabinet• integrated controls

Performance• specific for MTB• sensitivity similar to culture• detection of rif-resistance via rpoB gene

Product and system design• test cartridges for GeneXpert System• modular expansion and swap replacement of

detection unit • ~1 day technician training for non-

mycobacteriologists

GeneXpert

Sample Prep

<10 minutesAmplification and Detection

< 1 hour

MTB / RifMTB / Rif--resistance testresistance test

Cartridge Bodywith Fluid Reservoirs

Cap

PCR Reaction

Tube

Syringe Barrel

Ultrasonic Interface

Rotary Valve

Cartridge Foot

Valve Body Assembly

Page 62

What are the next step ?• Strengthen and expand the ongoing TB control programme

• Establishing quality assured laboratories in high burden countries and net

working of these labs

• Rapid detection of rifampicin resistance by retooling the available quicker

methods

• Application of cost effective tools in different settings with a low turn around

time

• Rapid surveys in high MDR and XDR settings

• Suitable treatment strategies to contain the spread of MDR TB by

establishing a close link with the HIV programme

Page 63

OPERATIONAL CONSIDERATIONS OF MONITORING THE PREVALENCE OF

RIFAMPICIN (RMP)-RESISTANT LEPROSY IN A POPULATION

Baohong Ji, M.D.

TWO MAJOR FACTORS FOR EMERGENCE OF DRUG RESISTANCE

• Exposure of patient to monotherapywith an active drug, and

• Pre-existence of drug-resistant mutantsdue to spontaneous, independent andchromosomal mutations.

WHY MONITORING RMP-RESISTANCE (1)? BECAUSE MB/MDT REGIMEN IS…..

• Dapsone (DDS) and clofazimine (CLO) mayeliminate spontaneously occurring RMP-resistant mutants, thus preventing selectivemultiplication of RMP-resistant mutants, but both drugs are self-administered, shouldthe patients fail to comply with self-administration of daily DDS-CLO, they are treated virtually with RMP-monotherapy. Hence, MB/MDT is not resistance-proof.

WHY MONITORING RMP-RESISTANCE (2)? BECAUSE RMP IS…..

• Among the three components of MDT, RMP is far more bactericidal againstM.leprae than DDS or CLO, and istherefore the backbone of MDT regimen.

• Emergence of RMP-resistance would creategreat difficulties for treatment of individualpatients; its widespread disseminationwould pose a serious threat for leprosycontrol.

Page 64

WHY MONITORING RMP-RESISTANCE (3)? BECAUSE LACK OF INFORMATION

• Because no post-MDT surveillance; lack of manpower, resources & skills of general healthservices; no skin-smear service in the field; anddrug susceptibility rarely been tested, the claim of very low relapse rate or no RMP-resistance are only statements of ‘absence of evidence’, but not ‘evidence of absence’. The magnitude of RMP-resistant leprosy remains unknown, clearly definethe magnitude is urgently needed.

•

OBJECTIVE FOR MONITORING RMP-RESISTANT LEPROSY

• Define the minimal point-prevalence of RMP-resistant leprosy.

TWO ESSENTIAL COMPONENTS FOR MONITORING PREVALENCE OF RMP-

RESISTANT LEPROSY• Field activities: identifying the suspects

among targeted subjects; and• Laboratory activities: testing RMP-

susceptibility of the M.leprae isolatedfrom the suspects. Visions, leadership, collaboration & coordination are needed.

CREATION OF AN INTERNATIONAL NETWORK

• The survey should be conducted by an international network, which includesthe national authorities, national leprosy program, national & international experts, NGOs andWHO.

• A coordination body is crucial to ensure adequate operation of thenetwork.

Page 65

RESPONSIBILITIES OF THE COORDINATION BODY

• Creation of international network,• Identification of study areas,• Development of protocol,• Negotiations with various partners,

particularly donor agencies and preparationof budget,

• Organizing Standardisation Workshops,• Closely monitoring the progress of the

survey through regular meetings and site visits, and

• Preparation of Progress Report and Final Report of the survey.

REQUIREMENTS FOR A STUDY AREA (ENTIRE COUNTRY OR A SUB-

NATIONAL UNIT OF LARGE COUNTRY)• Strong political will of the authorities, • Reasonable health infrastructures,• Functioning leprosy program with

appropriate recording system, and• Existence of at least 1000 retrievable eligible

subjects. Careful feasibility assessment through site visits must be conducted before makingdecision on study areas.

NECESSARY PREPARATIONS OF FIELD ACTIVITIES

• Creating a national specialized team whichconsists of doctors, nurses and labtechnicians; nominating an experienced, dynamic and devoted team leader is crucial.

• Training team members with standardizedtechniques;

• Reintroducing skin smear service; and• Recreating the registers of eligible subjects.

CRITERIA OF ELIGIBLE SUBJECTS

• A history of smear-positive MB leprosy, regardless the degree of positivity, and

• Had completed a full course of MDT, either 24 or 12 months, at least 5 yearsearlier.

Page 66

VERY DIFFICULT TO RETRIEVE GREAT MAJORITY OF ELIGIBLE SUBJECTS

ACCORDING TO THE CRITERIA

• Patients’ records are often incomplete, andare removed from register after completionof MDT,

• Quality of skin-smears was less thandesirable before early 90s, and no longer available afterward.

READY TO BE COMPROMISED, BUT TO WHAT EXTENT?

•Reliability is doubtful,•Missing MB diagnosedwithout smear results

Only choose ex-MB fromera when leprosy classi-fied by smear results

Include many PB as denominator, under-estimate real prevalence

Choosing all ex-MB andignore smear-positiverequirement

Mostly from areas withbetter adherence, lesslikely develop resistance

Only examine readilyretrievable subjects

Possible consequenceOption

IS IT WORTHWHILE TO CONDUCT A SURVEY IF THE ELIGIBLE

SUBJECTS CANNOT BE RETRIEVED PROPERLY?

SAMPLE SIZE OF ELIGIBLE SUBJECTS

• Difficult to predict, and different fromcountry to country. Hopefully, in mostendemic countries, between 500 to 2,000eligible subjects would be retrieved, and in major countries like Brazil or India,between 4,000 to 8,000 eligible subjects may be retrieved from 2 or 3 previously hyper-endemic states.

Page 67

EXAMINATIONS OF THE ELIGIBLE SUBJECTS

• Each of the subjects will be examined bothclinically and bacteriologically (skin smears) by the national specialized team.

• Skin smears must be taken from at least four sites; to facilitate the later selection of biopsy site, at least half of the smears shouldbe taken from sites other than on the face.

CRITIRION FOR SUSPICION OF MB RELAPSE

• An ‘ex-patient’ with a history of skin smear positive MB leprosy and hadcompleted a full course of either 24- or 12-month MDT at least 5 years earlier;

• Now, the skin-smears reveal a confirmed BI of ≥ 2+ in at least one site, with or without obvious skin lesions.

RECORDING SYSTEM AND QUALITY CONTROL

• Individual file must be established for each eligiblepatient. The file should include identityinformation, history of leprosy (including smearresults) and MDT treatment, results of currentexamination.

• All skin-smear positive slides and 10% negativeslides must be preserved and confirmed.

• Biopsies must be taken from the same sites withsmear-positive results.

• Quality of clinical examination and skin smearsshould be randomly controlled by national andinternational experts.

RMP-SUSCEPTIBILITY TESTING OF M.leprae(1): BY MOUSE FOOTPAD TECHNIQUE?

• Constraints of MFP technique: difficult to optimize the technique, highly demandingfor storage and shipment conditions of biopsies, labor intensive, time consumingand rather expensive.

• Not enough qualified laboratories & expertise.

• Due to administrative, financial andtechnical reasons, it is no longer feasible to undertake a meaningful scale survey of RMP-resistant leprosy by MFP technique.

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RMP-SUSCEPTIBILITY TESTING (2):PCR-based, DNA SEQUENCE ANALYSIS OF rpoB GENE IS A

COST-EFFECTIVE TECHNIQUE• Results were in full concordance with those

carried out by MFP technique.• Conditions for storage and shipment of

biopsies are less demanding, and is the onlymethod for testing M.leprae unable to multiply in MFP.

• Results become available only a few days, and costs only a fraction as compared withMFP technique.

• (Question: Is there a need of quality controlby simultaneously tested with MFP technique?)

DIAGNOSIS OF RMP-RESISTANT LEPROSY

RMP-resistance will be diagnosed if therpoB mutation with amino acid substitution of Ser531 or His526 is identified. (Question: what to do if the mutation isidentified at a position other than Ser531 or His526?)

CAPACITY BUILDING OF GENETIC ANALYSIS TECHNIQUE AMONG

NATIONAL LABORATORIES• Most of the genetic analysis will be carried out by

national reference lab of countries where thesurvey will be conducted, or by major lab whichalready possess certain expertise and facilities, with the technical supports from leadinginternational experts.

• Once the national laboratories are identified, a Standardisation Workshop will be held to shareexperience, standardise the technique and finalizethe protocol.

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Department of Public HealthUniversity of Aberdeen

Reliable tools and procedures for surveillance

of rifampicin resistance

Cairns Smith

Components

• Reliable tools• Reliable field procedures• Use of reliable tools and

field procedures together

Reliable Tools

• Agreed standard methods• Standardised operating procedures

- comparable results• Quality assurance• Reproducible• Validity

Purpose

• Assess threat to leprosy control• Management of relapsed cases or

those not responding to MDT– Relapsed case respond to MDT– Difficult to assess clinical

response to MDT (speed, reactions, expectations)

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Reliable Field Procedures

• Relapsed cases - demonstrate resistance exists- rare events (1/1000/year)- cannot assess size of problem- issue of transmission key

• New Cases- real threat to leprosy control

Relapsed Cases – secondary resistance

• Indicator of existence of resistance• Cannot estimate scale• Definition (see Guidelines)

– Completed MDT (not ‘defaulter’)– Not reaction (PB Relapse)– MB relapse (increase in BI of 2 or more)– Need good quality smears– Correct original Classification

– Need good clinical details

New cases – Primary Resistance

• Smear positive cases• Previously untreated• Sampling

- representative (defined area)- size (precision of estimate)

Which Control Programmes?

• Not just good ones!• Defined areas• Ability of take smears at diagnosis• 100 + previously untreated, smear

+ve cases• Link to lab (national/international)

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Surveillance of Trends

• Snap shot• Trends of time• Retrospective

– 1991-1995, 1996 – 2000, 2001 - 2005• Prospective

– Annual survey for next 5 – 10 years

Conclusions

• Relapsed Case (on-going)– Existence of resistance– Individual patient management– Relationship to MDT

• New Cases (essential)– Threat to future leprosy control– Retrospective and prospective trends– Include Asia, Africa, Americas

Documents

Presentations made during the Informal Consultation on ...Molecular basis of rifampicin resistance and methods for detection Stewart Cole Nadine Honoré 3-D structure of RNA Polymerase