Clinical and histopathological evaluation of the effect of addition of immunotherapy with Mw vaccine to standard chemotherapy in borderline leprosy.

This study reports detailed analysis of clinical parameters and clearance of granuloma in borderline leprosy patients treated with immunotherapy and chemotherapy. It aims to assess the additive effect of immunotherapy (Mwvaccine) with standard MDT on clinical status of untreated borderline leprosy cases and on granuloma fraction of untreated borderline leprosy cases. Patients attending the OPD were serially recruited in two groups. A total of 150 cases in one treatment (trial) group (Mw vaccine plus MDT) and 120 cases in another treatment (control) group (MDT only) of border line leprosy have been included. After the formal written consent, detailed clinical examination, charting, smear examination of all untreated borderline patients of both groups was done, biopsies were taken from the active lesions of all patients of both groups at start of therapy and every six month thereafter till the completion of therapy. The same procedure was repeated every six months during the follow-up period. Standard MDT was given to all the patients of both groups according to type of disease. Mw vaccine 0.1 ml (0.5 x 10(9) bacilli) was injected intra-dermally at the start of therapy and every six months in addition to chemotherapy to the treatment group. The BT cases were followed up after 6 doses of MDT and 2 doses of Mw vaccine, and, the BB, BL cases were followed up after 24 doses of MDT plus 5 doses of Mw vaccine. Clinically, greater and faster improvement was observed in all the clinical parameters, faster attainment of smear negativity and two episodes of lepra reaction occurred in cases treated with combined chemotherapy and immunotherapy, as compared to controls (chemotherapy alone) wherein clinical improvement was slower in all parameters, slower attainment of smear negativity in bacillary index and seven showed the occurrence of reactions, histipathologically in addition to more rapid clearance of granuloma in immunotherapy treated group, a significant finding was an increase in the epithelioid cells population in this group. This suggests a possible immunoactivation of the macrophages especially in BB/BL immunotherapy group. Overall comparison of regression induced by chemotherapy alone with that induced by combined chemotherapy and immunotherapy shows a greater reduction in clinical parameters as well as granuloma fraction in BT cases as well as in BB/BL cases. This trial shows the potential usefulness of this approach of addition of immunotherapy to standard chemotherapy in borderline leprosy cases which leads to in faster recovery from disease reduced chances of reactions and faster granuloma clearance. Such information is expected to be useful in improving the immunotherapeutic approaches for treatinggranulomatous conditions in general and in leprosy in particular.

Evaluation of diagnostic role of in situ PCR on slit-skin smears in pediatric leprosy.

A large proportion of early cases of leprosy in children remain AFB negative in skin smears. Such cases required additional techniques to confirm the diagnosis. In situ PCR on slit- skin smears is minimally invasive and less cumbersome as compared to skin biopsies. This study was initiated in our institute with the objective to evaluate the diagnostic value of in situ PCR on slit- skin smears in pediatric leprosy. A total of 25 cases of leprosy below 16 years of age were included in the study. After detailed history and thorough clinical examination, informed consent was obtained from the parents of children for slit- skin smears from lesion sites for AFB staining and for in situ PCR technique. Cases were clinically categorized according to IAL classification into indeterminate (I), tuberculoid tuberculoid (TT), borderline tuberculoid (BT), borderline borderline (BB), borderline lepromatous (BL) and lepromatous (LL). Most of the patients (76%) were between 9-16 years of age and 64% of the cases had history of contact with leprosy patients within the family. Skin smears were positive for AFB in only 20% of the cases. On applying in situ PCR, it was observed that 62.5% cases of I/TT/BT/BB category and 88.8% of BL/LL category gave positive signals. Overall in situ PCR confirmed the diagnosis in 72% cases while by slit smears diagnosis was confirmed in only 20% of cases. Further, out of 20 skin smear negative cases, 13 were positive by in situ PCR. Specificity of the signals of in situ PCR was established by demonstrating the absence of signals in nonleprosy dermatological conditions of vitiligo and P.alba. This study supports the potential usefulness of in situ PCR on slit- skin smears of early pediatric leprosy cases. This strategy will be especially useful in cases where skin smears are negative and in those cases where skin biopsy can not be done either because of unusual locations of lesions or because of sensitive age of the patients.

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.

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.

Diagnostic value of in situ Polymerase Chain Reaction in leprosy.

OBJECTIVE: This prospective study was carried out to assess the diagnostic value of in situ Polymerase Chain Reaction in leprosy, particularly in enhancing the histopathological diagnosis. METHOD: Clinical examination of 20 patients (< 16 yr) was done and skin smear for AFB was prepared. Biopsy of lesion site was taken for histopathological examination and in situ PCR testing. RESULTS: The histopathological examination confirmed the clinical diagnosis in 45% cases only; non-specific histopathology was reported in the remaining 55% cases. In situ PCR showed a positivity of 57.1% in early/localized form of leprosy (IIBT) and 61.5% in (BB/BL) group. When compared to histopathology examination, a significant enhancement of 15% in diagnosis was seen. With in situ PCR, the diagnosis could be confirmed in 4/11 (36.3%) cases with non-specific histopathological features, (which is common in early disease) in addition to confirmation of 8/9 (88.8%) histopathologically-confirmed tissue sections. Histopathology and in situ PCR, combined together, confirmed the diagnosis in 13/20 cases (65% of total cases). CONCLUSION: Thus, in situ PCR is an important diagnostic tool especially in early and doubtful cases of 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.

Microdensitometric scanning procedure for quantitative assessment of hybridization of rRNA targeting probes in leprosy.

In order to develop an objective criteria of grading of positivity of hybridization signals of gene probes targeting rRNA, a microdensitometric scanning procedure was standardised. Ribosomal RNA was extracted from the bacilli harvested from biopsies of leprosy cases across the spectrum and blotted on nitro-cellulose membranes. M. leprae specific rRNA targeting oligonucleotide probes were end-labelled and hybridization was done by the technique standardised and published earlier. The autoradiographs were developed and microdensitometric scanning was done by altering different parameters. Positivity was graded in 5 grades and compared with visual positivity. Microdensitometric scanning procedure and 5 grade system appear to be useful and reproducible. Signals in paucibacillary specimens were in 2+ to 3+ grading range whereas those in multibacillary specimens varied in grades from 2+ to 5+. This approach appears to have potential usefulness for assessing the bacillary load (possibly viable) in the clinical specimens from leprosy cases.

Detection of M. leprae by gene amplification; combined ethidium-bromide staining and probe hybridization.

Biopsy and skin-scraping specimens from 130 leprosy cases across the disease spectrum (56 TT/BT/I, 73 BB/BL/LL, and 1 neuritic case) and 50 healthy contacts were studied to assess the application of gene amplification. The nucleic acids from these clinical specimens were extracted by an integrated freeze-thawing--optimized lysozyme-/proteinase-k treatment-purification and fractionation procedure. The nucleic acids from cultured organisms were isolated by the stepwise procedure earlier standardized at this laboratory. Gene amplification for a 360-bp fragment of the 18-kDa protein gene was carried out using primer and the procedure described by its developers, and a 360-bp fragment on Southern blot was taken as the yardstick of positivity. The polymerase chain reaction product was analyzed by electrophoresis, ethidium-bromide (EB) staining, and blot (B) hybridization. Overall sensitivity ranged from 71% in specimens with undetectable acid-fast organisms to 100% in specimens with demonstrable acid-fast bacilli. A positivity of 73% in TT/BT/I specimens and 93% in BB/BL/LL specimens was observed. Four combinations were discerned: EB+, B+ (71%); EB-suspicious, B+ (14%); EB-, B+ (3%) and EB-, B- (12%). By combining the blot hybridization with EB staining, the sensitivity could be significantly improved as compared to EB staining alone. The test was found to be absolutely specific by the absence of any false positivity in control specimens as well as with purified DNAs from mycobacterial as well as non-mycobacterial organisms, grown from these specimens. It is recommended that for optimum sensitivity and specificity both EB staining and blot hybridization should be done.

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.

Enhancement in the histological diagnosis of indeterminate leprosy by demonstration of mycobacterial antigens.

In a clinico-pathological study of Indeterminate leprosy, fifty-six cases were chosen based on specified clinical criteria. Their clinical features were noted, the smears for acid fast bacilli (AFB) were prepared from lesions, lepromin inoculation and biopsies were performed from the lesional edges. They were subsequently treated with a modified extended WHO regimen for paucibacillary leprosy. On routine hematoxylin eosin (HE) and Fite-Faraco staining of paraffin embedded sections, histopathological confirmation of Indeterminate leprosy was observed in only 17/56 (31%) of the clinically diagnosed cases whereas the remaining were labelled as non-specific pathology. Histometric analysis of all HE stained sections did not show any characteristic finding which could be considered as characteristic and discriminatory for Indeterminate leprosy. Immunoperoxidase staining for demonstration of mycobacterial antigen by direct staining procedure using conjugated rabbit anti-BCG and indirect three step procedure using primary rabbit anti-BCG and avidin biotin complex, was next performed on the sections exhibiting non-specific pathology. With the direct immunoperoxidase method, antigen was demonstrable in (11/35) 31% of the cases. The more sensitive indirect method could demonstrate the presence of antigen in (21/35) 60% of the cases. This study thus shows that demonstration of mycobacterial antigen by simple and inexpensive immunoperoxidase techniques enhances the histopathologic diagnosis of Indeterminate leprosy.

Immunotherapy of treated BL/LL cases with BCG: histopathological, immunohistological and bacteriological assessments.

The persistence of dead as well as viable bacteria is an important therapeutic problem in multibacillary leprosy. In highly bacillated patients, viable bacteria are detectable in 10-15% of cases after 2 years of treatment and none of these cases became smear negative by 2 years of recommended multidrug therapy (MTD). Immunotherapy trials using BCG (0.1 mg) by intradermal route have been undertaken in cases who had MDT for 2 years and who had viable bacilli by ATP photometry and/or FDA-EB staining. Biopsies and smears were taken from local as well as distant sites at 0.4 weeks and 6 months after BCG vaccination. Biopsies were processed for ATP counts, FDA-EB staining, histopathology and immunohistology for cell types at 0.4 weeks. There was transient effect on BI, ATP counts, FDA-EB staining at local as well as distant sites in some cases. Histopathology and immunohistological findings suggest that there is tendency to form epithelioid cell granuloma at local site in all cases and at distal sites in some. There was infiltration of subepidermal zone in one case, 4 weeks after vaccination. BCG may be of use as potential immunotherapeutic agent but its usefulness needs to be investigated in depth preferably in the beginning or early phases of chemotherapy and with also repeated inoculations.