P Joseph, J Ponnaiya, M Das, VS Chaitanya, S Arumugam, M Ebenezer

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1 Indian J Lepr 2016, 88 : Hind Kusht Nivaran Sangh, New Delhi Original Article Evaluation of anti-bacterial activity of Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin and eir combinations in Murine Model of Rifampicin Resistant Leprosy P Joseph, J Ponnaiya, M Das, VS Chaitanya, S Arumugam, M Ebenezer Received : Accepted : Leprosy, a debilitating disease of e skin and peripheral nerves is caused by Mycobacterium leprae (M. leprae) and is treated by multidrug erapy (MDT) comprising of Dapsone, Rifampicin and Clofazimine. Resistance to any of ese drugs poses a reat to e current disease control strategies. Wi e emergence of Rifampicin resistance in leprosy, it is important at alternative drugs need to be tested to develop a treatment strategy to combat drug resistant leprosy. In e current study, we have investigated WHO MDT, Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin and eir combinations in intermittent and daily dose regimens in rifampicin resistant strains of M. leprae rough mouse foot pad experiments in order to determine e loss in viability of M. leprae in response to ese drugs and eir combinations. Our findings suggest at WHO MDT is still e best combination in Rifampicin resistance cases. Combination of Moxifloxacin wi Minocycline and Clariromycin may also be taken up for clinical trials in cases wi Rifampicin resistant leprosy. Rifapentine and Moxifloxacin can be effective alternative drugs to replace Rifampicin where required eier in daily dose shorter duration regimens or intermittent dose longer regimen to treat resistant strains. Key words : Multidrug Therapy, Mycobacterium leprae, Rifampicin Resistant Leprosy, Bactericidal Drugs Introduction Leprosy caused by Mycobacterium leprae (M. leprae) is treated wi WHO regimen of Multidrug Therapy (MDT) containing Dapsone, Rifampicin and Clofazimine; however, new case detection rate remains steady. While e modes of entry, point of exit, demonstration of attenuation of bacterial activity wi treatment and complete cure in leprosy are unclear, emergence of drug resistant strains poses a much greater reat of resurgence of e disease wi no appropriate treatment strategies to combat e same. (1 Dr Priya Joseph, MBBS, MD Derm, Dermatologist 2 Dr Joyce Ponnaiya, MBBS, MD Pa, Consultant Paologist 3 Dr Sundeep Chaitanya V, MSc, PhD, Research Officer 4 Mrs Shana Arumugam, BSc, Animal House Technician 5 Dr Mannam Ebenezer, MBBS, MS Oro, Director Schieffelin Institute of Heal Research & Leprosy Centre (SIH R & LC), Karigiri, Vellore District, Tamil Nadu , India. Corresponding auor : Dr Mannam Ebenezer directorate@karigiri.org

2 148 Joseph et al Cure rates are difficult to estimate as ere is no infallible test for residual disease and histopaological changes resolve after varying periods of time. The diagnosis of relapse or detection of persistent bacilli becomes difficult due to non-availability of techniques in e field/patient settings. Drug resistance is one of e major causes of relapse in leprosy. The most common drug resistance reported in leprosy is to Dapsone. This was found to be due to mutations at positions 157, 158 and 164 in folp1 gene of M. leprae, resulting in changes in codon positions 53 and 55 of dihydropteroate synase (DHPS). Drug resistance to rifampicin has been found to be emerging due to various mutations in e rpob gene of M. leprae in e rifampicin resistance determining region (RRDR). This gene encodes e beta subunit of RNA polymerase on which rifampicin acts. However it has been found at rifampicin resistance demonstrated by e mouse foot pad assay has not always been confirmed by mutations in e rpob gene. Maeda et al suggested oer mechanisms such as acquired changes in membrane permeability and efflux pump functioning. In a recent study, Singh et al have identified new SNPs in e multi-drug resistant Airaku-3 stain where e mechanism of resistance to rifampicin has not yet been found. In 2013, Williams et al reported a case of multidrug resistant leprosy, resistant to dapsone and rifampicin, who showed clinical response (clearing of skin lesions) to daily Dapsone, Clofazimine and Rifampicin for 44 mons, but relapsed after 6 years. This illustrates at e Rifampicin-Dapsone-Clofazimine may work in a setting of drug resistance, if e strain of M. leprae is resistant to only one of e drugs, where e oer drugs compensate for e resistant one and shows a temporary response, but does not kill e bacilli completely. Quinolones are e ird group of drugs to which M. leprae has become resistant. The molecular mechanism involves mutations of e gyra gene which results in M. leprae resistant to quinolones. As most of e mutations lie between amino acid 67 and 106 of e gyra gene, is region is denoted as quinolone resistance determining region (QRDR). In e current study, we have investigated e efficacy of WHO MDT, Rifapentine (RPT), Clariromycin (CLARI), Minocycline (MINO), Moxifloxacin (MOXI), Ofloxacin (OFLO) and eir combinations in intermittent and daily dose regimens in rifampicin resistant strain of M. leprae rough mouse foot pad experiments, determining e loss in viability of M. leprae in response to ese drugs and eir combinations. The alternative drugs CLARI, RPT, MINO MOXI and OFLO were chosen based on e earlier reports in murine models (Single-lesion Multicentre Trial Group 1997, Ji and Grosset 2000, Ji et al 1991) which proved as effective alternatives to MDT in leprosy. Oer clinical trials performed wi different patient settings as well as mice model experiments testing bacteriostatic and bactericidal effects of alternate drugs to WHO MDT, were also taken into consideration. (Colston et al 1978, Grosset et al 1990, Ji et al 1994 & 1998, Pattyn and Saerens 1974). Meodology The study was conducted in accordance wi e eical guidelines of Indian Council of Medical Research and was approved by e Institutional Eical Committee of Schieffelin Institute of Heal-Research & Leprosy Centre and e Animal Eical Committee. 1. Selection of patient wi leprosy relapse The Rifampicin resistant strain was obtained from a patient who was diagnosed clinically as a leprosy relapse. This patient was

3 Evaluation of anti-bacterial activity of Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin diagnosed as a case of relapse wi new skin lesions and bacteriological index of 3+ at e ear lobes and at skin lesions, ree years after completing MB MDT at SIH-R&LC Karigiri. Informed consent for participation was sought from e chosen patient and biopsy was procured from e site of active lesion. 2. Confirmation of Rifampicin Resistance by Mouse Foot pad and Molecular Meods a. Mouse Foot Pad Technique: Bacilli were extracted from e skin biopsy by manual homogenization (Shepard 1960) and resistance to rifampicin in varying concentrations was determined using technique described by Levy and Ji, b. Molecular Technique: DNA was extracted from e skin biopsy using DNeasy Blood and Tissue Kit (Cat No: 69506, Qiagen Inc. USA) and primers corresponding to e rifampicin resistance determining region (RRDR) of M. leprae were used to amplify rpob gene rough PCR as described earlier by Matsuoka ( 2010). The results indicated at rpob gene region of M. leprae showed a mutation at cod on position 441 where Asp was replaced by Tyr. This mutation was reported earlier (Maeda et al 2001) and was identified to demonstrate a strong pattern of rifampicin resistance in leprosy. 3. Mice used in e Experiments: i. Normal Mice: Cross bred albino (CBA mice) were used to multiply e identified strains of M. leprae to prepare sufficient inoculum for e experiments. ii. Immunocompromised (Thymectomized Irradiated (TR)) Mice: The mice were ymectomized at 6-8 weeks and en subjected to radiation of 900 rads after a furer 3 weeks. The mice were en inducted into e experiments after a furer period of 6 weeks. 4. Preparation of Initial Inoculum: M. leprae strains were extracted from e biopsy tissues of relapse patient using manual homogenization protocol in normal saline and injected into foot pads of 5 TR mice. Nine mons later e TR mice were euanized and M. leprae was extracted from e hind footpads. Suspensions were en pooled and an inoculum prepared to yield bacilli per ml wi a solid ratio of 1%. 5. Inoculation of TR Mice and Induction into treatment schedules: i. Inoculation into experimental TR mice: The inoculum us prepared above was 5 en diluted to 1 X 10 bacilli per 0.03ml and was injected into hind foot pads of 108 TR mice. The number of mice was calculated based on e treatment regimens in Table 1 taking into account 10% possibility of failure to develop swollen footpads and 20% mortality rate during experiments. The mice were grown on normal diet for 9 mons for e footpads to be swollen wi M. leprae. ii. Treatment Schedules: After e end of 9 mons, 93 mice survived wi swollen foot pads out of e 108 TR mice (86.11%). The count was estimated in a representative set of two mice and was identified to be on an 6 average of 3 10 bacilli per each hind foot pad. These mice were en allocated into e treatment groups as shown in Table 1. The treatment schedule was divided into intermittent

4 150 Joseph et al iii. and daily dose wi a control group. The intermittent and e daily dose regimens were followed as per e earlier reports (Ji B et al 1996). a. Intermittent Dosage: In e intermittent dose, single drugs Rifapentine (RPT) and Moxifloxacin (MOXI) and drug combinations Clariromycin, Moxifloxacin, Minocycline (CMM) and Clariromycin, Ofloxacin, Minocycline (COM) were administered as per e dosage shown in Table 1. The group wi WHO MB MDT served as positive control and e control group which was untreated served as a negative control. The dosage was administered for 24 weeks (once every 4 weeks). b. Daily Dosage The daily dose regimens included administration of a single bactericidal drugs (RPT or MOXI) as well as drug combinations CLARI + MOXI + MINO (CMM) and CLARI + OFLO + MINO (COM). Untreated mice were used as controls. The treatment schedules erefore were for a maximum period of 6 days in daily dose regimen. Harvest post treatment: At e end of designated period of treatment, e mice were sacrificed and hind foot pads were harvested for M. leprae from each group under e intermittent and daily regimens. The bacterial counts were enumerated and solid ratios estimated. A solid bacillus is defined as e organism at was stained adequately by Ziehl Nielsen staining and whose leng is approximately four times its wid. The solid ratio was estimated in all suspensions (Ridley 1960). 6. Sub-inoculations: The bacterial harvests mentioned in e above section were serially diluted 10-fold to obtain 10, 10, 10 and >10 bacilli/ml (undiluted) suspensions. These dilution was sub-inoculated into 2 normal mice and 1 TR Mice except for undiluted suspensions which were inoculated into 2 normal and 2 TR mice as mentioned in e Table 1. The mice were maintained on normal diet for 12 mons. At e end of 12 mons M. leprae were harvested from e hind foot pads of sub inoculated mice, enumerated (Levy and Ji 2006) and e proportion % of viable bacilli were counted based on Spearman and Karber calculations (Shepard 1982). The p value for statistical significance was calculated using z test of proportions. 7. Statistical Meods and Assessment of treatment efficacy: The proportional bactericidal technique was employed in e establishment of efficacy of drugs and drug regimens based on e dosage (Colston et al 1978). The bactericidal activity in each of e regimens was estimated by measuring and comparing e proportion of viable organisms in each groups using Spearman and Karber calculations (Shepard 1982). The calculations employ a median infective dose (ID50) and e percentage of viable M. leprae organisms remaining after treatment was derived from e equation: % viable M. leprae = 0.69/50% infectious dose. A two tailed p value of <0.05 was considered statistically significant. Results A pre-treatment bacterial load in e experimental TR Mice (n=93) at e end of 9 mons

5 Evaluation of anti-bacterial activity of Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin Table 1 : Schedule of Experiments : Rifampicin Resistant Strain Groups Drugs* No of Duration Frequency Number of Mice in sub (mg/kg/dose) Mice of of inoculations*** treatment harvests** At each Total # Harvest Intermittent Dosage: N TR N TR 1 MOXI (150) + CLARI (100) Wks. W4, W12, W MINO (50) (CMM) 2 CLARI (100) + MINO (50) Wks. W4, W12, W OFLOX (150) (COM) 3 WHO MDT 9 24 Wks. W4, W12, W RPT (10) 9 24 Wks. W4, W12, W MOXI (150) 9 24 Wks. W4, W12, W Daily Dosage: N TR N TR 6 RPT (10) 6 6 Days D1,D3,D MOXI (150) 6 6 Days D1,D3,D MOXI (150) + CLARI (50) Wks. D1, D3, W4,W MINO (25) (CMMD) 9 CLARI (50) + MINO (25) Days D1,D3,D OFLOX (150) (COMD) Untreated Control: N TR N TR 10 Control Mice Wks. D0,D1,D3,D6, on Normal Diet W4,W8,W12, W24 *Moxi = Moxifloxacin, Mino = Minocycline, Clari= Clariromycin, Oflox= Ofloxacin, RPT= Rifapentine, **W= Week, D= Day. ***N = normal mice; TR =Thymectomized radiated mice =RMP at 10 mg/kg of body weight once every 4 weeks plus 0.01% DDS (Dapsone) % CLO (Clofazimine) in daily diet. # = At each harvest, e inoculum was diluted into 10, 10, 10, and undiluted (>10 ) and injected into 2 normal mice and 1 TR mice per dilution totaling to 8 normal mice and 5 TR mice (one additional TR mice was inoculated 5 wi >10 ). and before induction in e treatment groups was (mean Log ). This increased to at e end of week 4 and decreased to at e end of 24 weeks in all e treatment groups. The solid ratios were in e range of 1-3%. Hence, ere was no perceptible change indicated in e bacterial populations wi bo intermittent and daily dose regimens at 4 weeks or at 24 weeks when compared to e pre-treatment levels. (Tables 2 & 3). This indicates at e infection is well established and follow-up

6 152 Joseph et al Table 2 : Initial Harvest Counts from normal mice in e intermittent regimen before titration and subinoculation (Mean log 10 + Standard Deviation) Drugs Harvest Week 4 Week 12 Week 24 Weeks A1* A2 A1 A2 A1 A2 CMM W4 W12 W COM W4 W12 W RPT W4 W12 W MOXI W4 W12 W WHO MDT W4 W12 W CONTROL W4 W12 W *A1, A2 indicate Mouse1 and Mouse 2 respectively. Table 3 : Initial Harvest Counts from normal mice in e daily dose regimen before titration and subinoculation (Mean log + Standard Deviation) 10 Drugs Harvest Day 0 Day 1 Day 3 Day 6 Week 4 Week 8 Days A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 A1 A2 Control D0 D D3 D RPT D1 D D MOXI D1 D D CMM D1 D NA** W4 W COM D1 D D ** NA = Not able to enumerate e suspension observations in sub-inoculations are feasible. These observations were in concordance wi e similar studies reported earlier (Ji et al 1996). 1. Intermittent dose: a. Intermittent Dose - Normal Mice: (Table 4) At e end of 4 weeks, RPT showed antibacterial activity wi 68.37% proportion killed. The rest of e drugs, combinations and WHO MDT did not show bactericidal activity at e end of 4 weeks. At e end of 12 weeks, in addition to RPT, MOXI and WHO MDT also showed bactericidal effect. The bactericidal effect of RPT reduced from e effect at e end of 4 weeks. At e end of 24 weeks, all drug combinations and individual drugs showed antibacterial activity wi WHO MDT showing highest bactericidal

7 Evaluation of anti-bacterial activity of Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin Table 4 : Rifampicin Resistant Strain - Intermittent Dosage Regimen Proportion % of Viable Proportion of viable M. leprae M. leprae killed in % Normal Mice: 4 week 12 week 24 week 4 week 12 week 24 week COM CMM RPT MOXI * WHO MDT * TR Mice: COM CMM RPT MOXI WHO MDT *p<0.05 (Statistically Significant differences) (Z Test of Proportions) activity (99.90%) followed by MOXI (99.68%) and CMM (82.21%). At is point, e bactericidal effect of bo MOXI and WHO MDT are statistically significant (p<0.05) among e intermittent group when compared to COM, CMM and RPT in normal mice. b. Intermittent Dose - TR Mice: (Table 4) At e end of 4 weeks, Rifapentine showed bactericidal activity (98.99%). Moxifloxacin, CMM, COM and WHO MDT did not show bactericidal activity. At e end of 12 weeks, all combinations and individual drugs showed bactericidal activity wi CMM (98.99%) showing e highest bactericidal effect. At e end of 24 weeks, all combinations and individual drugs showed bactericidal activity wi WHO MDT (99.68%) having a superior effect. At e end of 24 weeks, WHO MDT, CMM and MOXI demonstrated 98-99% bacterial killing in TR mice. However, e statistical analysis revealed at ere is no significant difference at exists across various drug combinations and individual drugs at e end of 24 weeks. Wi e intermittent dose among single drugs Moxifloxacin had e best bactericidal activity comparable to WHO MDT bo in normal and TR mice. Among drug combinations WHO MDT had e best bactericidal activity bo in normal and TR mice. In bo normal and TR mice, as an alternative to WHO MDT e next best available combination is CMM followed by COM. 2. Daily dose a. Daily dose - Normal Mice: (Table 5) At e end of Day 1, Moxifloxacin showed antibacterial activity wi 98.22% proportion killed. The rest of e

8 154 Joseph et al Table 5 : Rifampicin Resistant Strain - Daily Dosage Regimen Proportion % of Proportion of viable (dose Viable M. leprae M. leprae killed [mg/kg]) Day 1 Day 3 Day 6 Wk 4 Wk 8 Day 1 Day 3 Day 6 Wk 4 Wk 8 Normal Mice: CMM COM RPT MOXI * ND TR Mice: CMMD COMD RPT MOXI * *p<0.05 (Statistically Significant differences) (Z Test of Proportions), ND= Data not available drugs, combinations did not show bactericidal activity. At e end of Day 3, none of e drug combinations or drugs had bactericidal effect including Moxifloxacin. At e end of day 6, all drug combinations and individual drugs showed equal bactericidal activity. Statistical analysis revealed at ere is no significant difference between e bactericidal activities of various drug combinations at e end 6 days of treatment. At e end of 4 week and 8 week, e bactericidal effect of CMM increased from day 6 and e effect was sustained into e 4 and 8 week. b. Daily Dose - TR Mice: (Table 5) At e end of day 1, all drug combinations and single drugs showed antibacterial activity wi Moxifloxacin showing significant (99.68%) bactericidal effect. At e end of day 3, antibacterial activity of all combinations and single drugs increased from day 1, except for Moxifloxacin where it decreased (89.99%). At e end of day 6, all combinations and single drugs showed bactericidal activity wi CMM being e highest (99.99%). Statistical analysis revealed at ere is no significant difference between e bactericidal activities of various drugs and combinations at e end of 6 days of treatment. At e end of 4 and 8 week, e bactericidal effect of CMM at day 6, continued to be sustained into e 4 week and 8 week. Wi daily dose regimen as far as single drugs are concerned Rifapentine and Moxifloxacin have shown bactericidal activity at Day 3 and Day 6

9 Evaluation of anti-bacterial activity of Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin wi bo having e same degree of effect in normal and TR mice. Wi daily dose drug combinations, CMM had e best bactericidal activity bo in normal and TR mice when compared to COM. 3. Comparison of daily dose wi inter-mittent dose Normal mice (Table 6) At e end of 4 weeks, when daily dose regimen of CMM was compared to intermittent dose of CMM, COM and WHO MDT, daily dose CMM showed bactericidal activity (99.98%) whereas intermittent dose of CMM or COM or WHO MDT did not show any activity. The bactericidal effect of daily dose CMM (99.98%) continued into e 8 week. At e end of 12 weeks, CMM and COM did not show any bactericidal activity. WHO MDT showed weak bactericidal activity (43.27%). At e end of 24 weeks, WHO MDT (99.90%), intermittent dose of CMM (82.21%) and COM (68.37%) showed bactericidal activity. WHO MDT showed e best bactericidal activity. The bactericidal activity of daily dose of CMM at e end of 8 weeks (99.98%) is higher an e bactericidal effect of intermittent regimens of CMM, COM and WHO MDT at e end of 24 weeks. This comparison was statistically significant (p<0.05). In normal mice, daily dose activity of CMM showed early onset of bactericidal activity which sustained into e 8 week and probably would have sustained e effect into e 24 week if continued, being comparable to WHO MDT. 4. Comparison of daily dose wi intermittent dose Tr mice (Table 7) At e end of 4 weeks, when daily dose of CMM was compared to intermittent dose of CMM, COM and WHO MDT, daily dose CMM showed bactericidal activity (99.98%) while oer combinations including WHO MDT not showing bactericidal activity. The bactericidal activity of daily dose CMM continued to e end of 8 week (99.98%). Table 6 : Comparison of Daily Dose wi Intermittent Dose - Normal Mice Regimen 4 week 8 week 12 week 24 week CMM 98.99(D)* 99.98(D)* (I) - 0(I) 82.21(I) COM 0 (I) - 0(I) 68.37(I) WHO MDT 0 (I) (I) 99.90(I)* *p<0.05 (Statistically Significant differences) (Z Test of Proportions) Table 7 : Comparison of Daily Dose wi Intermittent Dose - TR Mice Regimen 4 week 8 week 12 week 24 week CMM 98.99(D)* 99.98(D)* (I) (I) 98.99(I) COM 0 (I) (I) 96.83(I) WHO MDT 0 (I) (I) 99.68(I) *p<0.05 (Statistically Significant differences) (Z Test of Proportions)

10 156 Joseph et al At e end of 12 weeks, intermittent dose of CMM (89.99%), COM (96.83%) and WHO MDT (96.83%) showed bactericidal activity. At e end of 24 weeks, WHO MDT, CMM and COM showed bactericidal activity wi WHO MDT (99.90%) being e best. The bactericidal activity of daily dose of CMM at e end of 8 weeks is higher an e bactericidal effect of intermittent regimens of CMM, COM and WHO MDT at e end of 24 weeks. Daily dose CMM indicated statistically signi ficant (p<0.05) bacterial killing at 4 week when compared to WHO MDT. The effect of daily dose CMM at 8 weeks was comparable to WHO MDT at 12 and 24 weeks. As in normal mice, in TR mice also Daily dose CMM showed early onset of bactericidal activity which sustained into e 8 week and probably would have continued into e 24 week being comparable to WHO MDT. Discussion In is study, nine regimens of single drugs and drug combinations bo in intermittent dose and daily dose and WHO MDT were tested in normal and TR mice infected wi Rifampicin resistant strain. Two single drugs, RPT and MOXI were tested for individual efficacy against e resistant organism. Two drug regimes, CLARI/MOXI/MINO (CMM) and CLARI/OFLO/MINO (COM) were tested against WHO MDT for efficacy against rifampicin resistant strain. Single Drugs Intermittent dose and daily dose of Moxifloxacin exhibited bactericidal effect in normal and TR mice in Rifampicin resistant strain. The bactericidal results of daily and intermittent Moxifloxacin regimes are comparable wi WHO MDT at different time intervals. Intermittent dose Rifapentine showed better bactericidal effect in TR mice an normal mice. This effect was less an at of Moxifloxacin. Daily dose Rifapentine showed bactericidal effects comparable to Moxifloxacin bo in normal and TR mice. From ese results it seems at Moxifloxacin is e drug of choice if Rifampicin has to be replaced in e present WHO MB MDT. Drug Combinations Clariromycin, Ofloxacin and Minocycline (COM) in normal mice in intermittent dose showed no bactericidal effect at e end of 4 and 12 week. At e end of 24 week e bactericidal effect of COM (68.37%) was not very good. Even in TR mice, COM did not fare well when compared to CMM or WHO MDT. Wi daily dose, even ough e bactericidal effect of COM was comparable wi CMM and WHO MDT in normal mice, in TR mice it was less. Based on ese results it seems at COM may not be considered as an effective drug combination for rifampicin resistant cases. The intermittent dose of Clariromycin, Moxifloxacin and Minocycline (CMM) in normal mice showed no bactericidal effect at e end of 4 and 12 week. Even at e end of 24 week e bactericidal effect of CMM (82.21%) was not comparable wi WHO MDT in normal mice. However in TR mice, intermittent dose CMM bactericidal effect was comparable to WHO MDT at 24 week. Wi daily dose CMM, bactericidal effect of CMM on 6 day, 4 week and 8 week was very good in normal mice and TR mice. When daily dose and intermittent dose were compared, e bactericidal activity of daily dose of CMM at e end of 8 weeks is higher an e bactericidal effect of intermittent regimens of CMM, COM at e end of 24 weeks and comparable to WHO MDT in bo normal and TR mice. The results show at among e drug combinations tested, WHO MDT is still e best combination to be used in Rifampicin resistance

11 Evaluation of anti-bacterial activity of Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin cases. If for some reason WHO MDT has to be replaced in Rifampicin resistance cases, CMM may be e choice of combination preferably as a daily dose regimen. It is paradoxical at WHO MDT is still e best drug combination in e presence of resistance to Rifampicin. One of e explanations could be at in e presence of Rifampicin resistance, DDS and Clofazimine compensate and treatment wi WHO MDT shows clinical response. Even in MFP studies DDS and Clofazimine combination have been shown to be effective antibacterial drugs in earlier studies. As per e present clinical protocol in rifampicin resistance proven cases a furer trial of WHO MDT is tried. If is furer trial of WHO MB MDT in patients wi Rifampicin resistance proven by molecular meods does not show clinical response, e recommendation will be to treat e patient wi daily or intermittent CMM combination because merely replacing Rifampicin in WHO MDT wi Moxifloxacin alone may not cover for co-existing DDS resistance. In e field setting where relapses are diagnosed on e basis of clinical criteria alone wi no smear or molecular biology support it is prudent to start e patient on MB MDT because a number of patients would not have completed MDT and treatment history is not reliable. If no clinical improvement is seen en a combination of CMM can be started preferably daily dose. Acknowledgments The auors acknowledge e all e staff and students of e department of laboratories SIH- R&LC Karigiri for eir help in e animal experiments and e entire project. Special anks to e administration of SIH-R&LC Karigiri for e infrastructural support roughout e study and for e financial support from e Indian Council of Medical Research (ICMR) Grant No: 5/8/3(6)2009-ECD-1. References 1. Colston MJ, Hilson GR, Banerjee DK (1978). The 'proportional bactericidal test': a meod for assessing bactericidal activity in drugs against Mycobacterium leprae in mice. Lepr Rev. 49: Efficacy of single dose multidrug erapy for e treatment of single-lesion paucibacillary leprosy. Single-lesion Multicentre Trial Group. (1997). Indian J Lepr. 69: Emmanuelle C, Bonnafous P, Evelyne P et al (2002). Molecular Detection of Rifampin and Ofloxacin Resistance for Patients Who Experience Relapse of Multibacillary Leprosy. Clin Infect Dis 34: Grosset JH, Ji BH, Guelpa-Lauras CC et al (1990). Clinical trial of pefloxacin and ofloxacin in e treatment of lepromatous leprosy. Int J Lepr and Oer Mycobact Dis. 58: Ji B, Grosset J (2000). Combination of rifapentinemoxifloxacin-minocycline (PMM) for e treatment of leprosy. Lepr Rev. 71 Suppl: S Ji B, Perani EG, Grosset JH (1991). Effectiveness of clariromycin and minocycline alone and in combination against experimental Mycobacterium leprae infection in mice. Antimicrob Agents Chemoer. 35: Ji B, Perani EG, Petinom C et al (1994). Clinical trial of ofloxacin alone and in combination wi dapsone plus clofazimine for treatment of lepromatous leprosy. Antimicrobial Agents Chemoer. 38: Ji B, Perani EG, Petinom C et al (1996). Bactericidal activities of combinations of new drugs against Mycobacterium leprae in nude mice. Antimicrob Agents Chemoer. 40: Ji B, Sow S, Perani E et al (1998). Bactericidal activity of a single-dose combination of ofloxacin plus minocycline, wi or wiout rifampin, against Mycobacterium leprae in mice and in lepromatous patients. Antimicrob Agents Chemoer. 42: Joshi R (2011). Clues to Histopaological Diagnosis of Treated Leprosy. Indian J Dermatol 56:

12 158 Joseph et al 11. Levy L, Ji B (2006). The mouse foot-pad technique for cultivation of Mycobacterium leprae. Lepr Rev. 77: Maeda S, Matsuoka M, Nakata N et al (2001). Multidrug Resistant Mycobacterium leprae from Patients wi Leprosy. Antimicrob Agents Chemoer. 45: Matsuoka M (2010). Drug resistance in leprosy. Japanese J Infect Dis. 63: Pattyn SR and Saerens EJ (1974). Results of intermittent treatment wi dapsone and rifampicin of mice inoculated wi Mycobacterium leprae. Ann Soc Belge de Med Trop. 54: Rees RJW and Weddell AGM (1968). Experimental Models for Studying Leprosy. Ann New York Acad Sci. 154: Shepard CC (1982). Statistical analysis of results obtained by two meods for testing drug activity against Mycobacterium leprae. Int J Lepr Oer Mycobact Dis. 50: Shepard CC and Mcrae DH (1965). Mycobacterium leprae in Mice: Minimal Infectious Dose, Relationship between Staining Quality and Infectivity, and Effect of Cortisone. J Bacteriol. 89: Singh P, Benjak A, Carat S et al (2014). Genomewide re-sequencing of multidrug-resistant Mycobacterium leprae Airaku-3. Clin Microbiol Infect. 20: Shepard CC (1960). The Experimental Disease That Follows e Injection of Human Leprosy Bacilli into Foot-Pads of Mice. J Exp Med. 112: WHO 30 August 2013, vol. 88, 35 (pp ). (n.d.). Retrieved 24 February 2015, from Williams DL and Gillis TP (2012). Drug-resistant leprosy: monitoring and current status. Lepr Rev. 83: Williams DL, Hagino T, Sharma R et al (2013). Primary Multidrug-Resistant Leprosy, United States. Emerg Infect Dis. 19: Williams DL, Spring L, Harris E et al (2000). Dihydropteroate Synase of Mycobacterium leprae and Dapsone Resistance. Antimicrob Agents Chemoer. 44: How to cite is article : Joseph P, Ponnaiya J, Das M et al (2016). Evaluation of anti-bacterial activity of Rifapentine, Clariromycin, Minocycline, Moxifloxacin, Ofloxacin and eir combinations in Murine Model of Rifampicin Resistant Leprosy. Indian J Lepr. 88 :