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V M Katoch

Publications and source records attributed to V M Katoch.

At least 55 records · Page 3Linked to original sources
PubMed1999

Detection of viable organisms in leprosy patients treated with multidrug therapy.

Cutaneous biopsies were collected from multibacillary leprosy patients who attended the out-patient department of Jalma Institute for treatment at different time intervals, i.e. 6 months, 12 months, 18 months, 24 months, 30 months, 36 months and 42 months after starting multidrug therapy (MDT) when they were still skin smear positive. Biopsies were processed for inoculation into mouse foot pad (MFP) and estimation of bacillary ATP levels by bioluminescent assay (ATP assay) by earlier established procedures. Viable bacilli were detectable after 1 year (25% cases by MFP and 31% cases by ATP assay), 2 years (8% cases by MFP and 12% cases by ATP assay) and 3 years (4% cases by both MFP and ATP assays). Overall, the percentage of the persisters was 10% by MFP and 13% by ATP assay. It would be important to carry out surveillance studies in larger number of BL/LL cases to know the trends and also the resultant relapses.

Adult↗
PubMed1999

Histology and immuno-histology of lesions clinically suspicious of leprosy.

Forty-six patients presenting with lesions clinically suspicious of leprosy were selected among patients attending the outpatient department (OPD) of our Institute. The lesions were biopsied deeply for histological analysis. The cases chosen commonly belonged to the 20-35 years age group, were predominantly males, with rare cases of leprosy within the family (2/46, 4.35%). The clinical presentation in most of the cases was that of a solitary lesion, (44/46, 95.65%) located in one of the extremities (40/46, 86.95%). A positive Mitsuda reaction could be elicited in 40% of the cases. Routine histopathologic analysis using defined criteriae, has established the diagnosis of leprosy in 16/46 (34.78%) cases with positivity for acid-fast bacilli in 4 cases. The remaining cases (25/46, 54.35%) exhibited a non-specific histopathology with a perivascular/periadnexal mononuclear cell infiltrate, few (5/46, 10.86%) exhibited minimal or no histopathological features. The sections with nonspecific or minimal pathology when immunostained for the presence of mycobacterial antigen exhibited positivity in 11/30 (36.6%) cases. Presuming the features observed to be consequent to the presence of antigens nearby, the diagnosis of leprosy was significantly enhanced compared to the diagnosis achieved with routine histopathology alone.

Adolescent↗
PubMed1999

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.

Adenosine Triphosphate↗
PubMed1998

Effect of trifluoperazine on in vitro ATP synthesis by Mycobacterium leprae.

The effect of trifluoperazine (TFP), a calmodulin antagonist, was investigated on in vitro ATP levels of human derived Mycobacterium leprae. M. leprae were obtained from biopsies from multi-bacillary forms of leprosy and were incubated in a modified Dubos medium system which supports limited in vitro synthesis of M. leprae. This incubation was carried out in the absence and presence of different concentrations of trifluoperazine. Samples for estimation of bacillary ATP levels were taken at day 0 and at 14 days of incubation. TFP inhibited ATP levels in M. leprae and this inhibitory effect was marginal at 2.5 microg ml(-1) (35% inhibition), highly significant at 5 microg ml(-1) (87% inhibition) and almost total at 10 microg ml(-1) (98.5% inhibition). This compound appears to have potential as an anti-leprotic drug and also as a broad spectrum anti-mycobacterial agent in view of its anti-tubercular activity reported earlier.

Adenosine Triphosphate↗
PubMed1997

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.

Adenosine Triphosphate↗
PubMed1997

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.

Autoradiography↗
PubMed1996

Determination of mycobacterial phylogeny on the basis of immunological relatedness of superoxide dismutases.

Sixteen strains of cultivable mycobacteria were grown in Sauton's medium, and Mycobacterium leprae was purified from armadillo liver. Cell extracts were prepared from log-phase growths of each of the cultivable mycobacterial strains. Superoxide dismutase (SOD) enzyme was purified from all cultivable mycobacterial strains included in the study, and antibodies against purified SOD enzyme were raised in rabbits. Immunological distances (ImDs) between these anti-SOD antibodies and SOD antigens were determined by a previously described immunoprecipitation method and by a recently developed enzyme-linked immunosorbent assay (ELISA) technique. The reciprocal ImDs among mycobacterial strains were constant, reproducible and consistent by these two methods. An evolutionary tree was constructed on the basis of estimated ImDs. Except for M. duvalii and M. terrae, slowly and rapidly growing mycobacterial species appeared to be separately grouped by this analysis. Rapid growers clustered into a group which is near that of some slow-growing mycobacteria. M. avium falls almost in the middle of the evolutionary tree and the position of M. leprae was found to be between those of M. avium and M. bovis BCG. Measurement of immunological relatedness of SODs provides an alternative system with which to study the taxonomical relatedness among mycobacteria.

Animals↗
PubMed1996

Development of a SOD ELISA to determine the immunological relatedness among mycobacteria.

This study reports on the standardization of an enzyme-linked immunosorbent assay (ELISA) system for the measurement of immunological distances (ImDs) of the superoxide dismutase (SOD) molecule among the cultivable mycobacteria, namely, Mycobacterium vaccae, M. phlei, M. smegmatis, M. avium, M. scrofulaceum, M. tuberculosis H37Ra, M. tuberculosis H37Rv, and M. bovis BCG, and M. leprae. SODs from cultivable mycobacteria were purified, antibodies were raised against these molecules, and ImDs between these anti-SOD antibodies and antigen (SODs) were determined by an immunoprecipitation technique standardized earlier and by the ELISA technique developed in this study. The ELISA system developed in this study showed higher sensitivity and consistent and reproducible ImDs among various mycobacteria, including pathogens such as M. tuberculosis, M. leprae and M. avium. These values were comparable with the values derived by the immunoprecipitation technique. Our ELISA technique appears to be a sensitive and rapidly reproducible method with the additional advantage of the stability of reagents, and holds promise in the taxonomy as well as in the development of diagnostics for leprosy and other mycobacterial infections.

Animals↗
PubMed1996

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.

Biopsy↗
PubMed1995

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.

Adenosine Triphosphate↗
PubMed1995

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.

Adolescent↗
PubMed1994

Evaluation of PCR based test for the detection of Mycobacterium tuberculosis in coded sputum specimens.

A test based on amplification of 169 bp DNA specific to Mycobacterium tuberculosis was evaluated in a trial, on 50 coded samples which included 25 sputum specimens from radiologically, clinically and bacteriologically proven patients of pulmonary tuberculosis and 25 control specimens. At the end of the trial the code was broken and the results of PCR test were compared with those obtained with Ziehl Neelsen staining and culture test. The test appeared highly sensitive reacting 100 per cent in either of the hands with concordance rate of 76 per cent; 3 of 25 control samples gave false positive results.

Evaluation Studies as Topic↗
PubMed1994

Diagnostic value of gene probes and its correlation with clinical profile of leprosy in children.

Clinico-bacteriological profile of 73 leprosy patients below 16 years of age was studied. Majority of the patients were males and fell in 11-16 years age group (p < 0.05). Skin lesions were present in all cases on both exposed as well as unexposed areas and their number increased with advancing age. Cutaneous sensations were affected in most of the patients while nerve thickening was observed in 41. As age increased, the disease moved from the tuberculoid end of spectrum towards the lepromatous end (p < 0.05) and the positivity of the skin smears increased (p < 0.05). Majority of the paucibacillary cases were lepromin positive while most multibacillary cases were lepromin negative (p < 0.01). Two M. leprae specific gene probes were applied in 42 cases to assess their diagnostic value. Eighty one per cent cases were picked up by the probes indicating presence of active bacilli. These included all lepromin positive cases, all smear positive cases, and most of smear negative cases (p < 0.05). Seven children with inconclusive histology were also positive. Drug treatment and inadequate size of biopsy sample could explain the negative probe results in 19% cases. This study highlights the immense potential of gene probes in diagnosing leprosy in children.

Adolescent↗
PubMed1992

Histological changes with combined chemotherapy and immunotherapy in highly bacillated lepromatous leprosy.

Highly bacillated untreated lepromatous cases with an initial BI 4+ of to 6+ were treated with combined multidrug treatment (MDT) and immunotherapy with heat killed Mycobacterium w or BCG. The vaccines were administered intradermally every six months. It was observed that majority of cases on immunotherapy showed increased lymphocytic infiltration (both at local and distant sites) and some cases showed epithelioid cells as well. The lymphocytic infiltration was (slightly) more vigorous in those vaccinated with Mycobacterium w. Such changes were not seen in the patients on MDT alone. Also, the granuloma fraction reduced much faster in cases who were on additional immunotherapy as compared to those on MDT alone. These changes along with evidence of clinical and bacteriological improvements suggest that immunotherapy may have an important supportive role specially in the therapy of anergic lepromatous cases.

Antitubercular Agents↗
PubMed1992

A preliminary study of correlation of immuno-histological and ultrastructural characteristics of neural granuloma in leprosy patients.

With an aim to better understand the pathogenesis of nerve damage in leprosy, peripheral nerve biopsies from six untreated leprosy cases (3 BT/TT and 3 BL/LL) were studied by electronmicroscopy and immuno-histology. In addition to routine histopathology for diagnosis, infiltrating cells of granuloma were characterized after preparation of single cell suspension. The lymphocytes in the lesion were characterized by E and EAC rosetting and macrophage phagocytic system (MPS) cells were studied using histochemical markers like esterase and peroxidase. The results indicate that the lymphocyte content was significantly greater in tuberculoid neural granuloma compared to lepromatous nerves and these formed rosettes with sheep erythrocytes (E) and expressed HLA-DR antigen suggesting that they are activated T cells. Infiltrating macrophages in both the tuberculoid and lepromatous neural granuloma were esterase positive, peroxidase negative and did not form rosettes with sheep erythrocytes or EAC. Ultrathin sections of tuberculoid granuloma showed lymphocytes clearly associated to epithelioid macrophages having well developed Golgi apparatus and rough endoplasmic reticulum. Correlation of these immunological and ultrastructural characters suggests that hypersensitivity mechanisms are possibly responsible for nerve damage in tuberculoid leprosy. Ultrastructural examination of lepromatous nerves, on the other hand, showed the predominance of macrophages with large nucleus, heavily bacillated Schwann cells, and a few lymphocytes. The correlation of immuno-histological and ultrastructural characters indicates that the mechanism(s) of nerve damage in lepromatous leprosy are basically different wherein hypersensitivity appears to play a very limited role.

Academies and Institutes↗
PubMed1991

Rapid characterization of Mycobacterium fortuitum-chelonei complex by restriction fragment length polymorphism of ribosomal RNA genes.

Using labelled, gamma-32P rRNA of mycobacteria as a probe restriction fragment length polymorphism (RFLP) of rRNA genes of strains belonging to the Mycobacterium fortuitum-chelonei complex was analysed. Each DNA sample was cleaved with EcoRI restriction endonuclease, the fragments were separated by agarose gel electrophoresis and transferred to nitrocellulose membrane. Fragments of DNA containing rRNA genes were identified by hybridization with gamma-32P-labelled rRNA. Patterns were found to be species specific and both the species were distinguishable from each other. Results indicate that this approach can be used for rapid genomic characterization of the Mycobacterium fortuitum-chelonei complex.

DNA, Bacterial↗
PubMed1991

Clinical and bacteriological progress of highly bacillated BL-LL patients discontinuing treatment after different periods of MDT.

Highly bacillated lepromatous patients (BL/LL) with an initial bacterial index (BI) of 4 to 6+ are being treated with a modified World Health Organization-recommended multiple-drug therapy (WHO/MDT) regimen consisting of rifampin 600 mg once a month, clofazimine 100 mg on alternate days, and dapsone 100 mg daily. The clinical and bacteriological profiles of the patients who had discontinued treatment at different durations have been compared with patients who took the same treatment until attainment of smear negativity. All six of the patients who had discontinued treatment at 12-18 months had worsened clinically and bacteriologically, and viable bacilli could be demonstrated in those tested for ATP. In four patients who had stopped treatment at 24-30 months, the BI continued to fall and there was no clinical or bacteriological worsening in 1 to 2 years of follow-up. The fall in the BI in five cases who had discontinued treatment at 36-44 months was comparable to those on continuous treatment, and there was no worsening. These observations indicate that with the conventional MDT regimen it is not advisable to stop treatment at 12 and 18 months. It appears that treatment should be continued for at least 2 years, and longer in the untreated highly bacillated cases. Prospective clinical trials with a sufficient number of cases and long-term follow-up need to be carried out to ascertain the optimum duration.

Adolescent↗