Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009-15.

OBJECTIVES: Antimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network. METHODS: From 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA. RESULTS: Among 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases. CONCLUSIONS: This is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.

Molecular mimicry between HSP 65 of Mycobacterium leprae and cytokeratin 10 of the host keratin; role in pathogenesis of leprosy.

Mycobacteria are known to induce autoimmune response in the host. Anti-host keratrin antibodies (AkAbs) might be responsible for the autoimmune phenomena in leprosy patients as majority of leprosy lesions are manifested in the skin and occurrence of keratosis is not an uncommon feature. The aim of this study was to find out the level of AkAbs in leprosy patients across the spectrum and to explore its correlation with the clinical manifestation of the disease. Further, mimicking epitopes of keratin and Mycobacterium leprae components were characterized. We screened 140 leprosy patients (27 BT, 28 BL, 41 LL, 25 T1R, 19 ENL), 74 healthy controls (HC) and 3 psoriasis patients as positive control. Highest AkAbs level was observed in the psoriasis patients followed by T1R, LL, BL, ENL, TT/BT. AkAbs level was significantly (p<0.05) higher in all the groups of leprosy patients except TT/BT in comparison to HC. Significant positive correlation was found between number of lesions and level of AkAbs in leprosy patients. Highest lympho-proliferation for keratin protein was observed in T1R, followed by BL/LL, TT/BT, ENL. Lympho-proliferation was significantly (p<0.05) higher in all groups of leprosy patients except ENL in comparison to HC. Interestingly, it was noted that hyperimmunization of inbred strains of female BALB/c mice and rabbit with M. leprae soluble antigen (MLSA) induce higher level of AkAbs. The percentage of FoxP3(+) expressing Treg cells (total CD4(+)CD25(+)FoxP3(+) andCD4(+)CD25(+hi)FoxP3(+)) in splenocytes and lymph nodes of hyperimmunized mice were declined in comparison to control mice. Further, it was found that this autoimmune response can be adoptively transferred in naïve mice by splenocytes and lymph node cells as well as T cells. Comparative molecular characterization between keratin and MLSA noted a cross-reactivity/similarity between these two antigens. The cross-reactive protein of keratin was found to be in molecular weight range ≈74-51kDa and at pI 4.5 while the cross-reactive protein of MLSA was found to be in molecular weight ≈65kDa and at pI 4-4.5. Cross-reactive protein of keratin and MLSA was identified and characterized by MALDI-TOF/TOF analysis and Mascot software. It was found that the keratin (host protein) which reacted with anti-M. leprae sera is cytokeratin-10 and MLSA which reacted with anti-keratin sera is heat shock protein 65 (HSP 65). Seven B-cell epitopes of cytokeratin-10 and HSP 65 was found to be similar by multiple sequence alignment using ClustalW server and out of which 6 B-cell epitopes were found to be on the surface of HSP 65. In conclusion, our study provides evidence for the existence of molecular mimicry between cytokeratin-10 of keratin (host protein) and 65kDa HSP (groEL2) of M. leprae. Presence of heightened CMI response of leprosy patients to keratin and positive correlation of AkAbs level with number of lesions of leprosy patients showed the clinical evidence for its role in the pathogenesis in leprosy.

Protective efficacy of Mycobacterium indicus pranii against tuberculosis and underlying local lung immune responses in guinea pig model.

Tuberculosis kills two million people each year. As the current vaccine BCG fails to prevent adult cases of TB, an improved vaccine and/or vaccination strategy is urgently needed to combat TB. Previously we reported the higher protective efficacy of Mycobacterium indicus pranii (MIP), formerly known as Mycobacterium w (M.w) as compared to BCG in murine model of TB. In this study we further evaluated the protective efficacy of MIP in guinea pig model of TB. Modulation of post infection immune response was analyzed in the lungs of MIP immunized and control groups. We found reduced bacterial loads, improved pathology and organized granulomatous response at different post infection time points in the MIP-immunized group as compared to the BCG-immunized group. Combined results suggest that MIP-immunization results in heightened protective Th1 response as compared to BCG group, early after infection with M.tb and a balanced Th1 versus immunosuppressive response at late chronic stage of infection. The study demonstrates the higher antigen presenting cells function both inside the granuloma as well as in the single cell suspension of the lung in the MIP-immunized group. We further demonstrate that live MIP is safe to use in vivo as we observed quick clearance of MIP from the body and no untoward reaction was found. Aerosol route of immunization provided higher protection. Further this study provides evidence that MIP-immunization gives significantly better long term protection as compared to BCG against TB.

Gelatin nanocarriers as potential vectors for effective management of tuberculosis.

The aim of the research work was to develop and characterize rifampicin (RIF) loaded gelatin nanoparticulate delivery system for the effective management of tuberculosis. Gelatin nanoparticles (GPs) containing RIF were prepared using two-step desolvation method. Formulations were characterized through transmission electron microscopy (TEM), atomic force microscopy (AFM), size and size distribution analysis, polydispersity index (PDI), zeta potential, percent drug entrapment, percent nanoparticulate yield and in vitro drug release. Formulations were further characterized for in vitro cytotoxicity, in vivo biodistribution, and antitubercular activity. The nanoparticles were found to be spherical in shape. The size of nanoparticles was found to be 264+/-11.2 nm with low PDI suggesting the narrow particle size distribution. The drug release showed the biphasic pattern of release i.e. initial burst followed by a sustained release pattern. The cytotoxicity studies revealed that nanoparticles are safe, non toxic as compared to free drug. In vivo biodistribution study showed higher localization of RIF loaded GPs in various organs, as compared to plain RIF solution in PBS (pH 7.4). In contrast to free drug, the nanoparticles not only sustained the plasma level but also enhanced the AUC and mean residence time (MRT) of the drug, suggesting improved pharmacokinetics of drug. RIF GPs additionally resulted in significant reduction in bacterial counts in the lungs and spleen of TB-infected mice. Hence, GPs hold promising potential for increasing drug targetability vis a vis reducing dosing frequency with the interception of minimal side effects, for efficient management of tuberculosis.

Immunogenicity and protective efficacy of "Mycobacterium w" against Mycobacterium tuberculosis in mice immunized with live versus heat-killed M. w by the aerosol or parenteral route.

As the disease caused by Mycobacterium tuberculosis continues to be a burden, there is a concerted effort to find new vaccines to combat this problem. One of the important vaccine strategies is whole bacterial vaccines. This approach relies on multiple antigens and built-in adjuvanticity. Other mycobacterial strains which share cross-reactive antigens with M. tuberculosis have been considered as alternatives to M. bovis for vaccine use. One such strain, "Mycobacterium w", had been evaluated for its immunomodulatory properties in leprosy. A vaccine against leprosy based on killed M. w is approved for human use, where it has resulted in clinical improvement, accelerated bacterial clearance, and increased immune responses to Mycobacterium leprae antigens. M. w shares antigens not only with M. leprae but also with M. tuberculosis, and initial studies have shown that vaccination with killed M. w induces protection against tuberculosis in Mycobacterium bovis BCG responder, as well as BCG nonresponder, strains of mice. Hence, we further studied the protective potential of M. w and the underlying immune responses in the mouse model of tuberculosis. We analyzed the protective efficacy of M. w immunization in both live and killed forms through the parenteral route and by aerosol immunization, compared with that of BCG. Our findings provide evidence that M. w has potential protective efficacy against M. tuberculosis. M. w activates macrophage activity, as well as lymphocytes. M. w immunization by both the parenteral route and aerosol administration gives higher protection than BCG given by the parenteral route in the mouse model of tuberculosis.

Development and evaluation of real-time RT-PCR assay for quantitative estimation of viable Mycobacterium leprae in clinical samples.

Detection of live organisms by molecular methods has special significance in leprosy where causative organism can not be cultivated in vitro. Such techniques would be especially important for monitoring the progress of the disease. While real-time RT- PCR technology will be appropriate for this purpose, there is very little experience of use of such tools in leprosy. This study describes the development of a quantitative RT-PCR targeting 16S rRNA based on primers used in a semi quantitative RT-PCR and its application on clinical samples including slit scraping and biopsies. RNA was extracted from biopsies from 3 lepromatous leprosy (LL) cases and standard curve was generated by plotting crossing over point against the dilutions of input RNA quantity (number of bacilli used for RNA extraction). Real-time RT-PCR was performed for quantitative detection of live M. leprae in 28 slit (13/28 smear positive) scrappings and 32 biopsies (22/32 smear positive). Number of viable bacteria as estimated by solid stained bacilli and real-time PCR correlated (no difference p>0.05). The test achieved a theoretical analytical sensitivity limit of up to single live bacillus even considering 11.3% efficiency of RNA preparation which was calculated by spiking of known number of leprosy bacilli in non leprosy skin biopsies (PCR negative). All smear positive cases were positive by this assay. This assay appears to be a promising tool for detection and quantification of viable bacilli in selected clinical situations and should be of use even in smear negative cases also.

Long term follow-up results of 1 year MDT in MB leprosy patients treated with standard MDT + once a month Minocycline and Ofloxacin.

BACKGROUND: This study was initiated in consultation with the National Leprosy Eradication Programme (NLEP) in mid nineties to try new treatment regimens for leprosy which were more robust in terms of control of reactions, long term relapses, operationally easier to undertake and feasible in field conditions. It was also envisaged to see if the addition of newer bactericidal drugs would be beneficial. OBJECTIVES: (i) To test the feasibility, safety and response of the patients to the new regimen. (ii) To observe the incidence of reactions during and after stoppage of therapy, for a period of 8-10 years after release from treatment. MATERIALS AND METHODS: A total of one hundred skin smear positive MB patients (15 LL, 35 BL and 50 BB) patients were included in this study. All the patients received the standard MDT + once a month supervised 100 mg of Minocycline and 400 mg of Ofloxacin for 12 months during the treatment phase. Thereafter, the treatment was stopped in all the patients which were followed-up on placebo (B complex tablets). Of these, 70 patients completed the treatment schedule of one year therapy and the post treatment follow-up of 9 to 10 years. RESULTS: All the patients tolerated the drugs well. The clinical response of the patients to the treatment was very good of which 32.85% of cases had history of reactions before starting treatment. During treatment, the incidence of reactions increased marginally to 38.5%, but these were easily controlled with concurrent administration of steroids. After completion of treatment the incidence was much less i.e. 10% and 3% after 1 and 2 years of post treatment follow-up respectively. The overall relapse rate is 5.7% (4/70) with an incidence density of 0.05/100 patient years. Relapses were confirmed by clinical, bacteriological, molecular biological (rRNA probes and 36 kD targeting PCR) as well as ATP bioluminescence. The relapsed patients presented with the appearance of new lesions, slit-skin smears were again found to become positive after becoming negative. Three of the four cases who relapsed had the initial mean BI of 2 to 2.9+ whereas one had the initial mean BI of 1.5+. Also, 2 of the 4 relapsed patients had positive PCR signals at the time of stoppage of treatment. CONCLUSION: The addition of Minocycline and Ofloxacin to the standard FDT has been observed to be a well tolerated. Overall as of now, the incidence of reactions observed with the newer treatment regimen is found to be significantly lower than that of 2 years fixed duration MB-MDT. The efficacy of this regimen regarding bacteriological clearance and relapse rates could not be compared due to non-availability of the results of experience with standard 1 year MDT regimen. However, this regimen appears to be operationally feasible and safe for the users.

Persister studies in leprosy patients after multi-drug treatment.

Cutaneous biopsies were collected from leprosy patients who attended the out-patient department of the Institute for treatment at different intervals, i.e., 12 months, 18 months, 24 months, 36 months, and more after beginning the multi-drug treatment therapy (M.D.T.). The patients belonged to the two drug regimens; (i) standard multibacillary (MB) M.D.T. after 12, 24, and 36 months; or (ii) standard M.D.T. + Minocycline 100 mg once a month (supervised) + Ofloxacin 400 mg once a month supervised for 12 months Biopsies were processed for mouse footpad inoculation and for estimating ATP levels by bioluminescence assay as per established methods. Viable bacilli were observed in 23.5% up to 1 year, 7.1% at 2 years, and in 3.84% at 3 years of M.D.T. by MFP and 29.4%, 10.7%, and 3.84% by ATP assay in the M.D.T. group at the same time period, respectively, but not in M.D.T. + Minocycline + Ofloxacin group after one year. The overall percentage of persisters was 5.55% by MFP and 7.14% by ATP assay up to 3 years of treatment.

Analysis of quantitative relationship between viability determination in leprosy by MFP, ATP bioluminescence and gene amplification assay.

Two hundred twenty-one untreated, borderline lepromatous/lepromatous (BL/LL) leprosy patients have been investigated for viability by the mouse foot pad method (MFP), adenosine triphosphate (ATP) and polymerase chain reaction (PCR). The biopsies were collected at the beginning of and 12/24 months after treatment. The patient group was treated with a) immunotherapy (BCG/Mw) + MDT; b) MDT + pyrazinamide; c) control MDT; d) MDT + minocycline 100 mg once a month supervised + ofloxacin 400 mg once a month supervised. Biopsies were divided in three parts for use in the mouse foot pad, molecular and ATP investigations. In untreated and treated patients (at 12 and 24 months), there was a general agreement among all three techniques, and PCR and ATP showed higher positivity as compared to MFP. Further, there was good correlation among the viable biomass estimated by bacillary ATP levels, PCR assay and growth in mouse foot pads. The positivity was observed by MFP as well as PCR assay (18-kDa and 36-kDa) from all of the specimens when the ATP content was more than 3.6 pg/million. When the ATP content was below 3.5 pg/million, the positive takes in MFP decreased but the PCR positivity correlated with ATP bioluminescence up to 0.04 pg/million. When the ATP content was even lower, the uptake in the MFP was possibly a matter of chance, while PCR positivity was observed in 96% of the cases. For specimens with undetectable ATP, positivity was seen in 1% of the cases, showing the inability of ATP bioluminescence method to detect low background due to host ATP. PCR signals in some cases could be due to the higher sensitivity of the method or persistence of DNA after bacterial death in some cases. On the whole, the PCR methods even though targeting DNA have shown good correlations with biomass which confirm their usefulness in monitoring therapeutic responses in leprosy.

Assessment of viability by normal mouse foot-pad and bacillary ATP bioluminescence assay in multibacillary cases treated with an MDT regimen using conventional as well as newer drugs like minocycline and ofloxacin.

The therapeutic effect of a drug regimen of conventional drugs as well as newer drugs like ofloxacin and minocycline in smear-positive multibacillary (MB) leprosy cases was assessed by mouse foot-pad and ATP bioluminiscence methods. Biopsies were taken before starting treatment and after one year of treatment. They were processed for viability assessment by normal mouse foot-pad inoculation and bacillary ATP assay techniques. The test regimen was quite effective in its anti-bacterial effect as it was found to result in loss of bacillary viability in all the cases, as assessed by both methods.

Chemotherapy trials in MB leprosy using conventional and newer drugs pefloxacin and minocycline.

One hundred, untreated, smear positive BB, BL and LL patients were treated with a regimen comprising of once a month, supervised, 600 mg of Rifampicin+ 400 mg Ofloxacin + 100 mg of Minocycline in addition to self administered 100 mg dapsone and 50 mg of clofazimine daily for twelve months.The treatment was then stopped and patients were followed up on placebo. This study reports the preliminary results after 2.5 to 3.5 years of post treatment follow-up. The drugs were well tolerated, the clinical response to the treatment was very good, and there was no case of treatment failure. Bacteriologically 25 out of the total 70 patients available for follow- up were still positive at the end of one year of treatment. These patients continued to progress satisfactorily and four patients were still positive at the end of 2 years. No growth was observed in the normal mouse foot pad after one year of therapy. No bacillary ATP was detected in the biopsy tissues after one year. While no M. leprae specific rRNA was detectable in any of the specimens after one year of treatment, weak PCR signals were detectable in 3/57 specimens at that period. In the follow up available no patient has relapsed. The patients are being followed up on placebo and longer follow-up is required to draw firm conclusions.

Effect of treatment on PCR positivity in multibacillary leprosy patients treated with conventional and newer drugs ofloxacin and minocycline.

In order to develop objective criteria to monitor trends of therapeutic responses positivity of PCR signals and ATP assay methods has been compared in multibacillary (MB) leprosy patients. Biopsies from lesions of 95 BL/LL patients before and after one year of treatment with a new drug regimen comprising of conventional and newer drugs ofloxacin and minocycline have been studied. These biopsies were processed for bacillary ATP assay and PCR positivity for a 36 kDa gene target by earlier published methods. In the untreated patients bacillary ATP levels were detectable in all specimens and ranged from 0.02 to more than 36 pg/millions organisms. After one year of treatment ATP levels were not detectable in any of the 57 biopsies specimens available for analysis. However, PCR signals were detectable in 3 out of 57 biopsies. In two specimens signals were very weak detectable only by hybridization. It may be concluded that DNA based PCR assay may be useful in monitoring the trends of therapeutic responses in MB patients under treatment.

Viability determination of M.leprae: comparison of normal mouse foot pad and bacillary ATP bioluminescence assay.

Correlation between viability assessment by mouse foot pad and ATP bioluminescence was studied in biopsy specimens from multibacillary leprosy cases. Biopsies were processed for inoculation into mouse foot pad and estimation of bacillary ATP levels by bioluminescent assay by earlier established procedures. ATP content as pg/million bacilli was estimated and correlation was assessed with growth in the mouse foot pad. It was observed that when the ATP content was > 36 pg/million bacterial cells, (> 1% probable viables) there was growth in the mouse foot pad from all the specimens. Similar results were observed when the ATP content was in the range of 3.6 to 35.99 pg/million cells (0.1 to 1% probable viables). The positivity rates in the mouse foot pad decreased when the ATP content decreased further. No positive growth in the specimens below 0.04 pg/million bacilli (< 0.001% viable organisms) was observed. These findings show an overall correlation between viability assessed by mouse foot pad and ATP bioluminescence. These observations validate the concept of ATP content of viable unit of M.leprae being in the order of 10(-15) g/live cell which is in the same order of magnitude as a colony forming unit of cultivable mycobacteria.

Treatment of bacilliferous BL/LL cases with combined chemotherapy and immunotherapy.

Thirty-six, untreated borderline lepromatous/lepromatous (BL/LL) leprosy patients with an initial bacterial index (BI) of 4+ to 6+ were serially allocated to three treatment groups. Group I patients received a slightly modified WHO regimen (rifampin once a month, clofazimine and dapsone daily) and BCG intradermally (i.d.) (0.1 mg/per dose). Group II patients were administered the same MDT and Mycobacterium w (2 x 10(8)) killed bacilli/dose i.d., and Group III received the same MDT with 0.1 ml of distilled water i.d. Vaccination was repeated every 6 months. Biopsies were taken from the local site of vaccination and from a distant site, i.e., the back. The progress was monitored periodically by clinical, histopathological and bacterial (BI, mouse foot pad, ATP) parameters. Twenty-five patients had completed a follow up of more than 2 years. These included: 7 in Group I, 10 in Group II, and 8 in Group III. One patient of the MDT + BCG group who was progressing well dropped out after 28 months. In cases on combined chemotherapy and immunotherapy, no viable bacilli were demonstrable by mouse foot pad and ATP measurement after 6 months (at 12 months or afterward). However, in come of the control cases on MDT alone, viable bacilli could be detected even up to 18 months (by mouse foot pad) and 2 years (by ATP estimation). With 36 months of treatment, the mean BI decreased from 4.64+ to 1.66+ in the group on MDT alone (controls), 4.9+ to 0.08+ in the MDT + BCG group, and 4.75+ to 0 in the MDT+Mycobacterium w group. Compared with the MDT and MDT + BCG groups, the fall in the BI was significantly more in the MDT + Mycobacterium w group at 12, 18, and 24 months. While all of the cases in the Mycobacterium w groups became smear negative by 36 months, it took 42 months for all of the BCG group to achieve negativity. Immunotherapy appears to have a significant effect on the killing and clearance of bacilli and should be considered as an adjunct to chemotherapy, especially in bacilliferous lepromatous cases.