Abstracts of Congress for Papers and Posters
MICROBIOLOGY & MOLECULAR BIOLOGY
PM & BM 1
A HISTOLOGICAL AND BACTERIOLOGICAL ASSESSMENT OF LEPROSY PATIENTS WITH < 5 LESION
Sujai Suneetha *. Narasimha Rao P., T.S.S. Lakshmi
Department of Dermatology, Osmania General Hospital, Hyderabad.
* LEPRA India - Blue Peter Research Centre, Cherlapally, Hyderabad - 501301
Aim: To study the histological and bacteriological features of leprosy patients with 5 or less than 5 lesions and relate it to clinical features.
Methods: 76 consecutive leprosy patients (M 57 F 19) who had 5 lesions were included in the study. Clinical features were recorded, slit skin smears and skin biopsies were done on all patients. A nerve biopsy was performed (radial cutaneous or Sural nerve) in 18 patients who had a clinically thickened cutaneous nerve.
Results: Out of the 76 patients, 28 patients had single skin lesions, 17 had 2 lesions, 13 had 3 lesions, 5 had 4 lesions and 2 had 5 lesions. The clinical diagnosis was TT leprosy in 4, BT in 68 and indeterminate in 4. Slit skin smears were positive in only 1 BT leprosy patient. Histological examination revealed features of TT leprosy in 2 patients (2.6%), BT leprosy in 42 patients (55.3%), BL in 4 patients (5.3%). indeterminate leprosy in 16 (21%) and non-specific inflammation in 12( 15.8%). Acid fast bacilli ranging from a bacterial index of granuloma (BIG) of 1+ to 4+ were present in 10 of the skin biopsies (13.2%). The cutaneous nerve biopsies in 16 of the 18 patients (88.8%) revealed features of BT leprosy consisting chiefly of lympho-epithelioid granuloma. 12 of these nerve (66.7%) revealed AFB in them with a BI ranging from l+to4+.
Conclusion: The findings from the study indicate that the number of lesions does not determine the type or extent of the disease.
PM & BM 2
A HISTOPATHOLOGICAL STUDY OF TYPE II (ENL) REACTION IN LEPROSY
Sujai Suneetha , Desikan, K.V.
LEPRA India - Blue Peter Research Centre, Cherlapally, Hyderabad - 501301
Type II lepra reaction produces a defined clinical picture of painful and tender erythematous nodules which are described as 'Erythema Nodosum Leprosum'. The histology of these lesions have been variously described. The aim of this study was to document the different components that constitute a histological diagnosis of ENL and their consistency of occurrence in each lesion.
A detailed study was made of 22 skin biopsies from ENL lesions. A histological diagnosis of 'LL in ENL' was made in 11 biopsies (50%). The most consistent feature noticed in these 11 biopsies was the presence of foamy macrophage granulomas in a pale oedematous dermis. The oedema was more prominent in the upper dermis and was associated with dilated vascular channels. Neutrophilic infiltrate was a consistent finding in 9 biopsies and vasculitis in 8. Plasma cells were present in 5 and panniculitis was noticed only in 1 biopsy.
Acid-fast stain revealed predominantly beaded and granular bacilli in the macrophages, nerves, smooth muscle and in the sub epidermal zone. Bacilli were also seen in the endothelial cells in 2 biopsies and in the wall or lumen of die blood vessels in 2 biopsies.
In the remaining 11 biopsies although the patient was clinically diagnosed as LL in ENL the histology did not reveal reatares that were sufficient to lahel as ENL. The tindings in these biopsies were of lepromatous leprosy with macrophage grantikimas andacici fast hacilli. Oedema, vasculitis and neutrophilicintiltrate were absent in these lesions.
PM & BM 3
ALTERED PROTEIN PHOSPHORYLATION IN LEPROSY LYMPHOCYTES A PRELIMINARY STUDY
Karuna Devi, Lavanya M. Suneetha and Sujai Suneetha
Lepra India - Blue Peter Research centre, Cherlapally. Hyderabad - 501 301
Protein phosphorylation is a post-translational modification that modulates the specific functions of various effector proteins and is a major biochemical mechanism by which cells integrate extracellular signals and respond to it. Recently, a detailed picture of protein kinases involved in the regulation of immune cells has been reported. cAMP dependent kinases. Calcium/Calmodulin dependent kinases and Protein Kinase C (PKC) have been shown to be involved in B and T cell responses to antigens. Leprosy is a disease in which varied types of immune responses to M. leprae are observed.
To understand the molecular basis of immune response, we carried out protein phosphorylation of lymphocytes from leprosy patients in the presence and absence of modulators - cAMP, cGMP and Phoshotidylinositol. A wide range of proteins were phosphorylated in lymphocytes of normal and leprosy patients. The modulators had a similar effect on both normal and leprosy lymphocyte phosphorylation patterns, except for the 20 and 29 kDa proteins which showed a decreased phosphorylation. The pattern and significance of the phosphorylation in leprosy lymphocytes is presented.
PM & BM 4
ARMADILLO-DERIVED Mycobacterium leprae PRODUCES A HEPARIN-BINDING HEMAGGLUTININ ADHESIN (HBHA)
Marques. M.A.M.1, Pessolani, M.C.V.2, Brennan, P.J.1. Locht, C.3 and Menozzi, F.D.3
1Dept. of Microbiology, Colorado State University, Fort Collins, CO, USA:
2Laboratório de Hanseníase, Instituto Oswaldo Cruz, FIOCRUZ. Rio de Janeiro. Brazil;
3INSERM U447, Mécanismes Moléculaires de la Pathogénie Microbienne. Institut Pasteur de Lille, Lille, France.
As described for several bacterial pathogens. Mycobacterium tuberculosis expresses a surface-exposed heparin-binding hemagglutinin adhesin (HBHA). which is specifically involved in epithelial adherence through interactions with heparan sullate-conlaining proteoglycans. Recent data showed that the disruption of the hbhA gene impaired M. tuberculsis dissemination from the lungs after intranasal infection of mice, indicating thai HBHA plays an important role in the pathogenesis of tuberculosis. The aim of this study is to investigate the role of the M. leprae HBHA homologue in leprosy. Indeed, the recent conclusion of the M. leprae genome revealed the presence of a hbhA gene coding for a protein of 199 amino acids and sharing 81.4 % identity with the M. tuberculosis homologue. To demonstrate the expression of this adhesin in armadillo-derived M. leprae. the bacilli were sonicated and subcellular fractions were isolated and analyzed by western blot developped with a panel of anti-M. tuberculosis HBHA antibodies. A reactive band with the expected apparent molecular weight was detected in the cell wall and soluble fractions, suggesting that the adhesin is present on the bacterial surface. As observed for M. tuberculosis, the HBHA expressed by M. leprae is also posttranslationally modified by methylations of the lysine residues present in the carboxy-terminal heparin-binding domain of the adhesin. Investigations are currently in progress to determine the role of HBHA in the interaction of M. leprae with Schwann cells.
Supported by NIH. WHO. INSERM. and Institute Pasteur de Lille.
PM & BM 5
ASSOCIATION OE NRAMPI GENE POLYMORPHISM WITH GENETC SUSCEPTIBILITY TO LEPROSY
Ferreira, ER., Goulart, IMB., Cunha, G., Nishioka, A.S., Goulart, L.R.
Centro de Referência Estadual em Hanseníase/Dermatologia Sanitária Faculdade de Medicina / Universidade Federal de Uberlândia Av. Pará 1720, CEP 38400-902 - Uberlândia-MG, Brasil. Fax: +55-32182349; E-mail; imbgoular@ufu.br.
Mitsuda test is an intradermic lepromin test, which measure the specific immune response to heat-killed leprosy bacilli that have a high prognostic value, meaning susceptibility to the lepromatous form when negative. Linkage analyses have confirmed association of NRAMP1 gene with susceptibility to tuberculosis and leprosy. However, case-control studies could not found association of NRAMP alleles with leprosy status. This study aimed the association of the (GT)n repeats at the NRAMP1 gene promoter region with susceptibility to leprosy and also to the positive Mitsuda lest on high endemic Brazilian population assisted by Sanitary Dermatology/Leprosy Reference Center of Uberlândia, Federal University of Uberlândia (UFU). Leprosy patients (69) were diagnosed by WHO requirements, classified in sub-clinical forms, and submitted to the Mitsuda Test and BCG scar evaluation. Statistical analysis has clustered patients in paucibacillary-PB (36) and mullibacillary-MB (33) forms. The control group consisted of 34 healthy non-consanguine household contacts of leprosy patients. Genotypes were obtained by the polymerase chain reaction (PCR), followed by detection through LISSSCP (14% PAGE, 49:1 acrylamide:bis. for 20h,10V/cm. at room temperature). There were no significant differences among 2, 3 and 4 allele frequencies with Mitsuda test average and leprosy status. The allele 3 frequency (0.666) has shown a slight increase in MB patients compared to PB (0.611) and to the control group (0.573). The NRAMP1 gene may be associated to Leprosy resistance; however, our results do not agree with this affirmative, probably due to others factors, such as bacillus exposition. BCG status and genetic heterogeneity.
Support: EAPEMIG
PM & BM 6
ASSOCIATION OF VITAMIN D RECEPTOR GEN (VDR) Taq1 POLYMORPHISM WITH SUSCEPTIBILITY TO LEPROSY
Ferreira, F.R.; Goulart, I.M.B.; Borges, D.S.; Pinheiro, C.A.; Goulart L.R.
Centro de Referência Estadual em Hanseníase/Dermatologia Sanitária Faculdade de Medicina / Universidade Federal de Uberlândia. Av. Pará 1720, CEP 38400-902 - Uberlândia-MG. Brasil. Fax: +55¬ 32182349; E-mail: imbgoular@ufu.hr
Leprosy is a chronic disease caused by Mycobacterium leprae, with a wide spectrum of clinical manifestations. This spectrum of clinical/histological characteristics ranging from the polar paucibacillary (PB) form, which corresponds to tuberculoid leprosy (TT). exhibiting strong cellular immunity and a predominance of TH 1-cytokine pattern, to the multibacillary (MB) form, which corresponds to lepromatose leprosy (LL) and a TH2-cytokine pattern. The mechanism of THI/TH2 shift remains unclear but early studies of the leprosy treatment with medications containing vitamin D (VD) analogs are consistent with a possible immunomodulatory effect of VD on bacteriostasis. Also, the VDR gene polymorphism has been implicated with susceptibility to M. malmoense, M. tuberculosis and with clinical types of leprosy. This case-control study inquired the association of VDR with susceptibility to leprosy per se and also lo leprosy types on high endemic Brazilian population assisted by Sanitary Dermatology/Leprosy Reference Center of Uberlândia. Federal University of Uberlândia (UFU). Leprosy patients (67) were diagnosed by WHO requirements and classified in sub-clinical forms as described by Ridley and .lopling ( 1966). Statistical analysis has clustered patients in paucibacillary (36) and multibacillary (31) forms. The control group consisted of 34 healthy non-consanguine household contacts of leprosy patients. The genotypes were obtained by the polymerase chain reaction (PCR), previously described by Roy et al (1999). and followed by Taql restriction. Frequency distribution of Taql T/t polymorphism (p = 0.5967: q = 0.4033) for MB patients was significantly different from general population as delected by logistic regression. The MB group exhibits a lower frequency of "T" allele when compared lo the control and PB groups, for which frequencies were: T = 0.7352; t = 0.2648 and T = 0.7083 and I = 0.2917, respectively. This study suggests thai VDR polymorphism modulate susceptibility to leprosy development probably by affecting lhe THI/TH2 differentiation of the host immune response.
Support: FAPEMIG
PM & BM 7
CHANGES IN THE PREVALENCE OF DAPSONE RESISTANT LEPROSY SINCE THE IMPLEMENTATION OF MDT
Kapil Dev Neupane, Sarah Failbus, Ruth Butlin, Paul Roche and Murdo Macdonald
Mycobacterial Research Laboratory, Anandaban Leprosy Hospital, PO Box 151, Kathmandu, NEPAL. E-mail:anandaban@mail.com.np
Aim: To screen all new previously untreated multibacillary leprosy cases and relapses presenting at our leprosy referral hospital for dapsone resistance using MFP culture.
Methods: Skin biopsies were taken from all appropriate consenting patients presenting at Anandaban clinics. These were homogenised and injected into the hind footpads of Swiss Albino mice. Test drugs were included in mouse feed throughout the course of the experiment. When control group mice showed two logs of growth, experimental mice were sacrificed, and numbers of bacteria estimated.
Results: During the period 1987- 2000 a total of 348 samples were lesled in our system. Twenty-three of 266 tested for primary dapsone resistance (0.09%) showed resistance at low dose (0.0001%, equivalent to 0.1 mg/kg in humans): only one showed resistance at high dose (0.01%, ?10mg/kg). Levels of DDS resistance in patients treated with DDS monotherapy prior to MDT decreased over the period of monitoring. In 11 patients treated with MDT only, none had secondary dapsone resistance. While there was evidence that some secondary dapsone resistant strains were resistant at high dose, only a single case of primary resistance to high dose dapsone was observed within our population.
Conclusion: Our studies indicate: i) that dapsone resistance has almost entirely disappeared as the remaining dapsone monotherapy patients have died or been treated with MDT. and ii) that secondary dapsone resistance does not develop in MDT regimens.
PM & BM 8
CHANGES IN VIABILITY OF INTERDERMAL M . leprae ASSOCIATED WITH THE HISTOPATHOLOGICAL RESPONSE OF SUSCEPTIBLE AND RESISTANT ARMADILLOS
Richard Truman Ph.D., and James L. Krahenbuhl Ph.D.
Laboratory Research Branch, Division of National Hansen's Programs, HRSA, Baton Rouge, La. 70894, USA.
Leprosy manifests over a broad clinical and histopathological spectrum associated with Th1/Th2 immunological responses. Other than man, nine banded armadillos are the only hosts which develop the full clinical spectrum of leprosy. The CMI that they can manifest towards M. leprae has been indexed only with heat killed M. leprae (Lepromin) in a Mitsuda reaction. While the form of leprosy that a host may develop is generally consistent with the type of granuloma that they manifest with Lepromin, the Mitsuda reaction is known to be a poor indicator of susceptibility for leprosy. Several Mitsuda (-) armadillos resist experimental infection with M. leprae. To better understand the differences between granuloma formation and resistance, we examined the granulomas formed in the skin of armadillos in response to intradermal inoculation of highly viable M. leprae and to killed leprosy bacilli. We found that the granulomas formed in response to live M. leprae were significantly larger than those produced to M. leprae killed by heat, gamma irradiation or by freeze/thaw. Among Mitsuda(-) animals (n=20) granulomas involving viable bacilli ranged 2-12 times larger in size than those made to killed M. leprae, but their cellular composition was little changed and the bacillary number remained high. Mitsuda (+) animals showed similar enhancement with little qualitative difference in cellular composition. We used Radiorespirometry (RR) and conventional mouse foot pad technique (MFP) to examine the viability of M. leprae recovered from these intradermal inoculations. M. leprae viabilities fell markedly after initial inoculation but then stabilized. Bacilli recovered from living Mitsuda reactions showed a broad range of viabilities and varied by the Ridley-Jopling classification of the animal (n=8). Highest M. leprae viabilities were found among multibacillary hosts and lowest among BT's. Over a six week period, intradermal M. leprae viabilities among most multibacillary animals tended to increase, while they decreased or remained very low among BT animals and other paucibacillary hosts. The pattern for intradermal M. leprae viability among leprosy resistant Mitsuda (-) animals (n=4) resembled that seen among BT hosts, with the higher initial viabilities waning over time. The histopathological response of these animals to Lepromin remained the same. The trends in viability seen for intradermal M. leprae generally correlated with the outcome of systemic infections. Within 15 months after intravenous challenge with 1x109 M. leprae, the LL-spectrum animals that had accommodated high intradermal M. leprae viabilities developed signs of fully disseminated disease, while the resistant Mitsuda(-) and paucibacillary spectrum animals remained free of leprosy. Actively metabolizing bacilli may produce antigens that are not present among killed bacillary preparations, and they secrete them to the host over a long period of time. Histopathology is likely too insensitive to reveal the full range of variable resistance across the leprosy spectrum. A better understanding of the M. leprae antigens involved in resistance to leprosy by armadillos, and the specific cytokine profile of their responses, would be useful in our efforts at in vivo propagation, and could significantly benefit our ability to identify disease susceptible and resistant individuals in human populations
PM & BM 9
DETECTION OF M . leprae AND ITS SUSCEPTIBILITY TO DAPSONE USING DNA HETERODUPLEX ANALYSIS
Diana L. Williams and Thomas P. Gillis
Molecular Biology Research Dept. Laboratory Research Branch, National Hansen's Disease Programs at LSU-SVM, Skip Bertman Dr. Baton Rouge, LA, USA
Current recommended MDT for leprosy should control the spread of drug-resistant leprosy; however, dapsone resistance continues to be reported. Comprehensive estimates of dapsone-resistant leprosy are difficult to obtain clue to the cumbersome nature of the conventional drug susceptibility testing methods using mouse foot pad inoculation, which requires at least 6 months to obtain results. Recently we have determined that dapsone-resistant strains contain mutations in codons 53 and 55 of thefolP1 gene encoding the dihydopteroate synthase, a key enzyme in the folate biosynthetic pathway, and used this information to design a. PCR-based heteroduplex assay for rapid detection of M. leprae and dapsone susceptibility from clinical specimens. PCR was used to amplify a 231-bpfolP1 fragment from crude cell lysates of biopsy homogenates. The PCR products were annealed to a universal heteroduplex generator and the resultant DNA duplexes were separated on a PAGE mini-gel. This assay took 6 hrs to perform, correctly detected the presence of M. leprae from eight biopsy specimens and from 14 separate M. leprae strains harvested from either armadillos or mice. It addition, this assay demonstrated a 93% correlation with dapsone susceptibility results as determined by both DNA sequencing of folP1 and mouse footpad susceptibility testing and was sensitive enough to detect 103 bacteria. Therefore these results demonstrate that a new tool has been developed for rapid detection of dapsone resistance. This tool should be useful for drug resistance surveillance in leprosy control programs when combined with similar molecular tests developed of other drug resistance markers