Comparative cytomorphology of skin, lymph node, liver and bone marrow in patients with lepromatous leprosy.

OBJECTIVE: The aim of this study was to compare the cytological changes in skin, lymph nodes, liver and bone marrow in patients with lepromatous leprosy. METHODS: Skin lesion, lymph node, liver and bone marrow aspirates were analysed. May-Grunwald-Giemsa (MGG) and Ziehl-Neelsen (Z-N) stains were employed. Comparative cytomorphology was studied. RESULTS: Twenty patients with lepromatous leprosy were studied. Lepra cells (LC) predominated in the skin aspirates of 12 patients with lepromatous leprosy (LL), lymphocytes accompanied LC in eight patients with borderline-lepromatous (BL) leprosy. Three patients of LL leprosy and two of BL leprosy in type 2 reaction additionally had numerous neutrophils. Two patterns of lymph node aspirates were seen: partial replacement with few LC in a reactive lymphoid background (10), complete replacement with either only LC or LC in a background of degenerating neutrophils (10), the latter a feature of type 2 reaction. Liver aspiration was performed in seven patients and of bone marrow in eight patients. Occasional LC were present in five liver-aspirated patients, steatosis and Kupffer cell hyperplasia in four patients, and myelopoiesis in two patients. Bone marrow smears invariably had occasional LC and a relative increase in mature plasma cells; sea-blue histiocytes were seen in six patients. CONCLUSION: Lepra cells predominated in skin and lymph node aspirates with complete replacement. In comparison, liver, bone marrow and lymph node aspirates with partial replacement were dominated by a preponderance of cells native to these organs with only few or occasional LC.

Topical phenytoin suspension and normal saline in the treatment of leprosy trophic ulcers: a randomized, double-blind, comparative study.

OBJECTIVE: To evaluate and compare two strengths of topical phenytoin sodium suspension (2% and 4%) with normal saline in the healing of acute trophic ulcers in leprosy patients. METHODS: A prospective, parallel, double-blind, randomized study was conducted in 45 leprosy inpatients with acute trophic ulcers. Patients were randomized to receive 2%, 4% or normal saline dressing on their ulcers once daily for 4 weeks. Efficacy parameters such as a reduction in the surface area of the ulcer, bacterial culture of the ulcer swab, appearance of healthy granulation tissue, cessation of ulcer discharge and overall gradation of clinical healing and safety were assessed at weekly intervals. RESULTS: The ulcer area reduction was greater in the 2% and 4% phenytoin groups compared with the normal saline group (p<0.001). Appearance of healthy granulation tissue and cessation of ulcer discharge was also observed earlier in the two phenytoin groups. At the end of 4 weeks, 11 ulcers each had healed completely in both the 2% and 4% phenytoin groups compared with none in the control group. There were no statistical differences between the 2% and 4% phenytoin groups. No side effects were reported by any patient. CONCLUSION: Topical phenytoin appears to be an effective, safe and cheap therapeutic option for the healing of trophic ulcers in leprosy patients.

Histological assessment of dermal nerve damage occurring during multidrug therapy for leprosy.

This is a prospective histomorphological assessment of dermal innervation in biopsies taken before and after multidrug therapy (MDT) from 41 leprosy patients: 35 borderline tuberculoid (BT), 3 borderline lepromatous (BL), 3 lepromatous (LL). Biopsies of the same lesions taken before commencement (diagnostic therapy) and at the end of therapy (check biopsy) were compared. Hematoxylin and eosin, immunoperoxidase stain for S-100 protein, and the Holmes' silver impregnation method for nerve cells and fibers were used. Skin biopsies were classified as having detectable or undetectable nerves. Of 35 patients with BT leprosy, 17 had no detectable nerves in their diagnostic biopsies; in the check biopsies of 13 of these 17, dermal nerves remained undetectable, in 2 they were S-100 positive but were Holmes negative. Identifiable dermal nerves were present in diagnostic biopsies from 18 patients; in the check biopsies 5 of these 18 had no detectable nerves while in the remaining 13 nerve branches could be detected. The study provides histological documentation of complete damage to dermal innervation in 62.85% (22/35) of patients with BT leprosy, of which 14.28% (5/35) occurred during MDT. Of the patients with detectable dermal innervation at the onset of MDT, 27.7% (5/18) suffered continuing damage during MDT.

Fine needle aspiration cytology of reactions in leprosy.

OBJECTIVE: To define diagnostic cytomorphologic features of reactions in leprosy. STUDY DESIGN: Part-retrospective, part-prospective, single-blind, controlled study of the applicability of fine needle aspiration cytology in the diagnosis of reactions in leprosy. Cytomorphologic features were compared in 42 clinically diagnosed patients with reactions in leprosy with those in a control group of patients with nonreactional leprosy. The study groups included type 1 and type 2 reactions in 35 and 9 patients, respectively. May-Grünwald-Giemsa and Ziehl-Neelsen staining methods were employed. RESULTS: Statistically significant (P < .01) cytomorphologic features of type 1 reaction were the presence of fragments of collagen and elastin; giant cells; giant cells exhibiting elastin phagocytosis; loose, epithelioid cell granulomas; and fibroblasts. Type 2 reaction was characterized in aspirates by the presence of an abundance of neutrophils in a background of lepromatous leprosy (P < .01). CONCLUSION: Criteria that are used in histopathology for the diagnosis of leprosy reactions can be applied satisfactorily to cytologic smears. A good correlation between clinical diagnosis and cytomorphology can be achieved. Multiple-site aspirates from the skin, nerve and lymph nodes are helpful in substantiating the diagnosis.

Chemotherapy trial in paucibacillary leprosy using clofazimine.

In a double blind study, 300 PB patients (smear negative, indeterminate, tuberculoid and borderline tuberculoid) were randomly allotted to two regimens, the control subjects (150 patients) receiving the standard WHO multidrug regimen of six doses of once a month rifampicin with daily dapsone therapy for six months, while the study group (150 patients) receiving 50 mg of clofazimine daily for six months in addition to the WHO regimen. After stoppage of therapy all the patients were followed up on placebo. The regimens were well tolerated. In 7.5% of patients on clofazimine containing regimen, the lesions showed persisting activity at the time of stoppage of therapy, compared with 16% on the control regimen. This activity subsided spontaneously, more rapidly, in the study group (80% compared with 30% in the control group) in six months. Two patients in the control group and one patient in the study group developed late reaction. There were no relapses in the study group, whereas, two patients have relapsed in the control group during a follow-up of 2.5 to 3.5 years.

Fine-needle aspiration cytology of lepromatous leprosy.

A prospective study correlating cytopathology with clinical morphology and histopathology in 22 patients with lepromatous leprosy was performed. Aspirates were taken from skin lesions in all patients. Lymph node aspirates were also performed in four patients with lymphadenopathy. Fine-needle aspirates yielded sufficient cellular material with excellent preservation of morphological detail. Diagnosis and correlation with bacillary index, clinical and histopathological findings was possible in all patients. In addition, the two patterns, partial and diffuse, of lymph node involvement could be recognized. Fine-needle aspiration cytology is a simple method for the laboratory assessment of leprosy.

Nodular post-kala-azar dermal leishmaniasis: a distinct histopathological entity.

Post-kala-azar dermal leishmaniasis (PKDL) is an infrequently occurring sequel to treated visceral leishmaniasis. Diagnosis, particularly in non-endemic areas, is difficult because the clinical appearances may be subtle and simulate lepromatous leprosy. The histopathology of the condition has been a neglected subject. Nodular lesions constitute one of the large variety of lesions that can be seen in PKDL. This paper describes the histopathology of such lesions in 26 patients seen over a period of approximately 8 years in a non-endemic setting. All the biopsies had strikingly similar light microscopic features with characteristic findings: a dense lymphohistiocytic infiltrate beneath an atrophic epidermis, pronounced follicular plugging, vascular hyalinization and collagen changes and negative Fite stain. These allow a definite diagnosis of PKDL even in the absence of demonstrable Leishman-Donovan (L-D) bodies.

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.

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.

Cytomorphology of leprosy across the Ridley-Jopling spectrum.

OBJECTIVE: To evaluate the possible role of cytology in classifying leprosy lesions on the Ridley-Jopling scale. STUDY DESIGN: A double-blind, prospective study comparing cytologic assessment of 30 clinically diagnosed cases of leprosy with their histopathology. May-Grünwald-Giemsa and Ziehl-Neelsen stain were done on slit skin smears and fine needle aspiration material. RESULTS: Cytologic subclassification was possible in 23 cases as tuberculoid leprosy (11), midborderline (3), borderline lepromatous (5) and lepromatous leprosy (4). These correlated with histologic subtypes. CONCLUSION: May-Grünwald-Giemsa complements Ziehl-Neelsen stain, yielding information almost comparable to that from histologic examination of skin biopsies.

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.

Response of leprosy patients with single lesions to MDT.

This study reports the clinical profile and therapeutic response of seventy-two mono-lesions leprosy cases. These 72 cases were among 578 paucibacillary (PB) cases classified according to WHO (1982) and were followed-up on multidrug therapy (MDT). Of these 72 mono-lesion cases, 46 (64%) were tuberculoid (TT) cases, 24 (33%) were Indeterminate (Ind) cases and 2 (3%) were of borderline tuberculoid (BT) types. While 37.5% of these cases presented as macular patches, the remaining 62.5% had raised erythematous lesions. In majority of cases (94%), the lesions were present on the exposed parts like legs and feet, arms and hands, face, whereas only 6% presented on covered areas of trunk and buttocks. These cases were treated with dapsone 100 mg daily for 12 months and rifampicin 600 mg once a month for 6 months. After 6 months of MDT, lesions in 81% of the patients regressed clinically and by one year of therapy 96% of cases had regressed. Treatment was stopped in all cases by one year of therapy. There were no relapse or late reaction in the 5 years of post treatment follow-up. The response of mono-lesion PB cases was better than the multi-lesions PB cases at 6 months and during the post treatment follow-up period.

An evaluation of the S-100 stain in the histological diagnosis of tuberculoid leprosy and other granulomatous dermatoses.

Forty biopsies of granulomatous dermatoses, 12 of which were tuberculoid leprosy (TL), were studied for patterns of nerve twig distribution using an immunoperoxidase technique for S-100 protein. Four distinct patterns of nerve twigs were identified: 1) within granulomas, 2) between granulomas, 3) within and between granulomas, and 4) undetectable nerve twigs in an adequate biopsy. Pattern 4 was seen exclusively in TL (p < 0.05). The other patterns occurred in nonleprosy dermatoses as well, suggesting that pattern 4 is the best indicator toward a diagnosis of TL. The granules of mycetoma and Mycobacterium leprae also stained positively with the S-100 stain.

Clinical and histopathological correlation in the classification of leprosy.

This study reports our observations on the correlation between clinical and histopathological diagnoses of the classification of leprosy. The histopathological classification of leprosy in 1351 cases was done per Ridley-Jopling criteria and was compared with the clinical diagnoses of the same cases. These 1351 cases included 79 cases diagnosed clinically as having a "reaction." However, the histopathologists could not detect any evidence of reaction in 16 of these 79 cases (20%). Of the remaining 1272 cases, 68 (5%) were reported as "no evidence of leprosy" by the histopathologists; 37 of these 68 were found to be from the clinically indeterminate type of leprosy. Histopathological and clinical diagnoses of the classification of leprosy coincided in 69% of the cases. Concordance between the clinical and histopathological diagnoses for different types of leprosy was: indeterminate (I) = 36%, tuberculoid (TT) = 50%, borderline tuberculoid (BT) = 77%, borderline (BB) = 26%, borderline lepromatous (BL) = 43%, and lepromatous (LL) = 91%. When some of the types were combined (BT with TT, BL with LL), the overall concordance figure was 76%; concordance for the TT/BT group was 80%, for the BL/LL group it was 93%. Since both TT and BT are considered paucibacillary and LL or BL are considered multibacillary for treatment purposes, differentiating TT from BT or BL from LL is, perhaps, therapeutically irrelevant. However, for classification purposes it appears that the weight given to different signs and/or histopathological parameters for classifying leprosy cases (especially TT, BB and I) needs to be reassessed.

Treatment of paucibacillary leprosy with a regimen containing rifampicin, dapsone and prothionamide.

Ninety paucibacillary leprosy patients having indeterminate (I), tuberculoid (TT) and borderline tuberculoid (BT) type of leprosy with bacterial index (BI) of less than two on the Ridley scale were treated with rifampicin (RFM) 600 mg once a month, dapsone (DDS) 100 mg daily and prothionamide (PTH) 250 mg daily. Treatment was stopped at the end of six months. The patients tolerated the drugs fairly well and in only two patients the drugs had to be stopped (in one due to jaundice and in the other due to gastric intolerance). About 6% of patients had early reactions which subsided with additional steroid therapy. The inactivity rate was 60% at six months and this improved to 96% at 12 months. No cases of late reactions and relapses were encountered in the limited follow-up period of six months; and a longer follow-up is necessary for ascertaining the relapse rates. The preliminary results however suggest that the addition of prothionamide to the standard WHO paucibacillary regimen is well-tolerated with increased inactivity rate and fewer instances of late reactions.

Association of mycobacterial-specific and Mycobacterium leprae specific antibody levels with clinical activity in tuberculoid leprosy: a comparative study of three serological enzyme-immunoassays.

The ELISAs for polyclonal antibodies against Mycobacterium leprae (ML-ELISA) and specific antibodies against epitopes on 35 kDa protein (SACT-ELISA) and phenolic glycolipid I (PG-ELISA) of M. leprae were evaluated comparatively in a group of 88 tuberculoid leprosy patients. The overall seropositivity rate with a battery of 3 tests (68%) was not significantly higher than that obtained with ML-ELISA alone (55%) for IgG class of antibodies. Seropositivities for SACT-ELISA and PG-ELISA were, respectively, 38% and 26%. ML-ELISA for IgM class of antibodies was least sensitive, showing only 8% positivity. A significant correlation was noted between individual values of the three assays, but the positive proportions overlapped maximally in the case of ML-ELISA (IgG) and SACT-ELISA. Further, positivity for the latter two assays, particularly SACT-ELISA, showed significant associations with the extent of 'active' (largely untreated) infection. Immunoblotting revealed that the main antibody response was directed towards M. leprae antigens in the molecular weight range of 20-40 kDa and the densitometry results of this zone correlated significantly with corresponding SACT-ELISA and ML-ELISA (IgG) values.

Activation of complement by circulating immune complexes isolated from leprosy patients.

Circulating immune complexes isolated from different types of leprosy sera as polyethylene glycol (PEG) precipitates were found to be efficient activators of the alternative pathway of complement. PEG precipitates from BL/LL leprosy patients and those with erythema nodosum leprosum were found to activate both the classical pathway and the alternative pathway of complement efficiently, while PEG precipitates from TT/BT leprosy patients and borderline tuberculoid patients in reaction were found to active the alternative pathway of complement but not the classical pathway. No significant differences were observed between the PEG precipitates from reactional and nonreactional TT/BT and BL/LL patients in their complement activating ability.

Relapses in paucibacillary patients after treatment with three short-term regimens containing rifampin.

Three multidrug regimens all containing rifampin and dapsone have been tried for the treatment of 278 cases of paucibacillary leprosy. Regimen I was the one recommended by the WHO Study Group. Regimen II was the same as Regimen I with depsone alone continued for a further 6 months. Regimen III was the same as Regimen II but rifampin was given daily for the first 7 days. The patients were comparable with regard to disease classification, lepromin status, bacteriological status, and number of lesions. As reported earlier, the disease inactivity rates by 1 year of treatment were much greater with Regimens II and III than with Regimen I (94% and 97% vs 76%). Early reaction was seen in 6% of those in Regimen III and in none in Regimens I and II. Late reaction was observed in 9% of those in Regimen I and none in Regimens II and III. During 3 1/2 years of follow up, 13% of the cases in Regimen I, 1% in Regimen II, and 2% in Regimen III relapsed. Since the patients in the three regimens were otherwise comparable, it is concluded that the high inactivity rate, low relapse rate (1%-2%), and no early or late reaction as observed in Regimen II patients were because of adequate treatment.