- Volume 70 , Number 4
- Page: 3–372
Abstracts of Congress for Papers and Posters
IMMUNOLOGY
OI 1
A NON-INVASIVE METHOD FOR DIAGNOSIS OF LEPROSY BASED ON DETECTION OF SPECIFIC ANTI-MYCOBACTERIAL ANTIBODIES IN SALIVA
M. Yushin; M.N. Dyachina; A.A. Juscenko; V.A. Bochanovsky
Leprosy Research Institute. Astrakhan, Russian Federation.
The aim of the present work was to improve methods of diagnosis of leprosy through the development of immune test system for anti-M. leprae antibodies (Ab) in saliva. Samples of saliva and blood sera from 116 leprosy patients at different stages of their disease and 23 healthy donors (control group) were studied with using indirect enzyme labeled immune assay. As test-antigen for detection of anti-M. leprae Abs, preparations of M. lufu, a value of which for leprosy serodiagnosis was proved by us earlier, were used. Rabbit Abs against human peroxidase-labeled immunoglobulins (IgG, IgA. IgM) were used as conjugate. The investigations showed that Ab titers in saliva and blood sera were comparable both in patient and control groups. Correlation analysis indicated interrelations between Ab liters in saliva and serum samples. Dynamic investigations showed high tilers of Abs both in saliva and blood sera in patients with active leprosy. Inactive leprosy patients giving seronegative results for 1-2 and more years, showed no specific anti-mycobacterial Abs in their samples of saliva. In the periods of activation of the disease (relapses, exacerbations of leprosy neuritis) Ab levels against M. leprae in saliva were increasing in parallel with increasing anti-M. leprae Abs in blood sera. Thus, a test-system was developed, high diagnostic value and reliability of which was achieved owing to using a new antigen from M. lufu and conjugate of peroxidase-labeled rabbit Abs against human immunoglobulins of IgG. IgA, IgM classes. Detection of anti-M. leprae Abs in saliva opens possibilities for early diagnosis of leprosy infection in leprosy contacts and general population of leprosy endemic areas. Monitoring of Ab levels in saliva of leprosy patients under treatment allows estimating effectiveness of antileprosy therapy.
OI2
ANTIGENIC SPECIFICITY OF THE Mycobacterium leprae HOMOLOGUES OF ESAT-6 AND CFP-10
John S. Spencer; Hee Jin Kim; Maria A.M. Marques; Patrick J. Brennan
Department of Microbiology, Colorado State University, Ft. Collins, CO, USA.
The recent completion of the sequencing of the genomes of M. tuberculosis and M. leprae provides the opportunity to identify leprosy-specific antigens. An analogous approach applied to M. bovis BCG allowed the identification of deleted genes and the development of antigens that can distinguish between M. tuberculosis infection and vaccination with BCG. Among those antigens which have shown promise are two low-molecular weight M. tuberculosis culture filtrate proteins, ESAT-6 (esat-6) and CFP10 (lhp), both encoded by genes in the RD1 region, a genetic segment that has been deleted from all strains of BCG. Because the M. leprae ESAT-6 (ML0049) and CFP-10 (ML0050) proteins have only 36% and 40% identity, respectively, to their homologues in M. tuberculosis (Rv3875 and Rv3874), we decided to analyze the immunologic cross-reactivity of these proteins in mice by characterizing the B and T cell epitopes recognized. We had previously reported this analysis of the ESAT-6 homologues, and found that the dominant B and T cell epitopes recognized in H-2d haplotype (BALB/c) strain mice for the M. tuberculosis and M. leprae proteins were in different regions. In addition, polyclonal antisera against the two forms of ESAT-6 did not cross-react at the level of the whole protein or with any of the heterologous peptides. We have since performed a similar immunological analysis of cross-reactivity with the CFP-10 homologues, and found that polyclonal antiserum raised against ML0050 did not cross-react with the M. tuberculosis homologue, and vice versa. We are currently in the process of analyzing antibody and T cell immune responses against members of the ESAT-6 family of proteins and other unique proteins discovered in the analysis of the M. leprae genome.
OI3
CELL MEDIATED IMMUNITY IN LEPROSY PATIENTS WITH ERYTHEMA NODOSUM LEPROSUM (ENL)
Murdo Macdonald; Niraj Shrestha; Patrick Haslett; Rakesh Manandhar; Matthew Albert; Steven Lubinsky; Paul Roche; Gilla Kaplan
Mycobacterial Research Laboratory, Anandaban Leprosy Hospital, PO Box 151, Kathmandu, Nepal. E-mail: anandaban@mail.com.np.
The dramatic resolution of erythema nodosum leprosum (ENL) following therapy with thalidomide suggests that immunologic changes associated with this treatment may afford insights into the pathogenesis of ENL. It has been reported recently that thalidomide may promote Th-1 immunity. However, it is unknown if thalidomide acts in this way in patients with ENL.
Aim: To study cell-mediated immune responses in Nepali leprosy patients with ENL undergoing thalidomide treatment, and to compare their response to those of lepromatous patients without ENL.
Methods: Venous blood was obtained from appropriate (LL) patients: 20 with and 20 without ENL. Plasma levels of interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α) and interleukin 12 (IL-12), and of soluble IL-2 receptor (sIL-2R) were measured using a standard immunoassay. Leprosy-specific and mitogen-induced IFN-γ producing cells in the peripheral blood were measured by ELISPOT and flow cytometry, respectively on days 0, 7 and 21 of a 21 day course of thalidomide.
Results: Thalidomide-induced activation of Th-1 immunity was suggested by an increase in numbers of T cells induced ex-vivo to produce interferon IFN-γ as assessed by both ELISPOT and flow cytometric assays (p > 0.01). This activation was transient, however, observed on day 7 of thalidomide treatment. Although in is difficult to discern obvious trends in the plasma cytokine levels, there is some correlation between the patterns in TNF-α levels and those for IL-.12.
Conclusions: Our results suggest transient T cell activation following thalidomide treatment, and may give some clues to the pathological processes underlying ENL as well as to new treatment strategies.
OI 4
CYTOKINE LEVELS IN TYPE 1 REACTIONS: RELATION TO NERVE DAMAGE AND THE RECURRENCE OF REACTION
Murdo Macdonald; Niraj Shrestha; Rakesh Manand-har; Maria Jacob K; Paid Roche
Mycobacterial Research Laboratory. Anandaban Leprosy Hospital, PO Box 151. Kathmandu, NEPAL. E-mail: anandaban@mail.com.np
Aim: To elucidate the role of cytokines during and after steroid treatment of Type I Reaction (T1R), and their relation to recurrent reaction episodes
Methods: We enrolled 192 borderline (BT, BB, BE) leprosy patients, 96 with T1R and 96 without, in this study. Blood was collected from Tl R patients at various timepoints before, during and after prednisone treatment, and used in a standard 24 hour whole blood assay.
Results: Leprosy specific antigen-induced levels of IFN-γ, TNF-α and IL-10 were measured in a 24-hour whole blood assay in T1R patients. Cytokine levels were significantly increased when compared with appropriately matched borderline leprosy patients without T1R. Steroid treatment lowered levels of IFN-γ, but levels of TFN-α increased as the doses of steroids were lowered, IL-10 levels increased during steroid therapy. High TFN-α levels in untreated patients (higher than 75th percentile) was associated with a 5 times greater risk of reactivation of symptoms during treatment phase. High levels of TFN-α after treatment with 30mg of steroids was associated with a 3-5 times greater risk of nerve function impairment or failure to improve nerve function. The relationship between cytokine levels and subsequent reactions was investigated by follow up for up to three years after initial observations.
Conclusion: This study seeks to link cytokine levels with recurrent T1R reactions and nerve function impairment and offers a means to identify patients failing to respond adequately to steroid therapy.
OI5
DENDRITIC CELL-MEDIATED PRODUCTION OF IL-12 AND IFN-γ BY Mycobacterium leprae-DERIVED CELL MEMBRANE
Yumi Maeda; Masaichi Gidoh; Norihisa Ishii; Masahiko Makino
Department of Microbiology. Leprosy Research Center, National Institute of Infectious Diseases. Tokyo. Japan.
The development of reliable vaccination agents toward leprosy is currently desired. In order to identify molecules capable of inducing effective cellular immunity against Mycobacterium leprae, the antigenicity of M. leprae-derived cell membrane fraction was examined using human dendritic cells (DCs). Immature DCs internalized and processed the cell membrane components, and expressed Ags, which reacted to lipoarabinomannan mAb or to leprosy patient's sera. The expression of MHC class II. CD86 and CD83 Ags on surface DCs was up-regulated indicating that the DCs were potently stimulated by the membrane Ags. Moreover these stimulated DCs induced significantly higher proliferation of autologous CD4+ and CD8+ T cells and higher IFN-γ production by the T cells than those pulsed with equivalent doses of M. leprae-derived cytosol fraction or whole live M. leprae. The involvement of CD40 ligand signaling on membrane pulsed DCs enhanced the IFN-γ production. CD4+ and CD8+ T cells from tuberculoid leprosy patients produced marked and significantly higher IFN-γ than those from healthy donors, when they were stimulated by autologous cell membrane pulsed DCs. The CD8+ T cells stimulated for 10 days by DCs pulsed with the membrane and CD40L, produced intracellular perforin in the Ag dose or CD40L dependent manner, in 507 of lymphocytes donors. Furthermore, the M. leprae cell membrane was more efficient in the CD40L-associated IL-12 p70 production from DCs than the cytosol fraction, but was less efficient than cell membrane from M. smegmatis. Both hydrophobic and hydrophilic fractions of M. leprae cell membrane induced IL-12 p70. These results suggest that M. leprae cell membrane has pleural antigenic molecules that might be useful as the vaccinating agents against leprosy
OI6
EFFICACY OF SHORT TERM MULTIDRUG THERAPY ON THE CONTROL MULTIBACILLARY LEPROSY
Foss, N.T; Ramalho. E.M.; Nunes. T.L.; Andrade, J.L.; Ferreira. M.A.N.; Souza CS
Faculty of Medicine of Ribeirão Preto, São Paulo University.
The purpose of this study is verify whether the short term multidrugtherapy (MDT) on leprosy treatment can be efficient on the immune response and the consequent control of the evolution of the disease. To evaluate this hypothesis 67 multibacillaries leprosy patients (MB) (lepromatous leprosy-LL and borderline leprosy-BL) were randomized to receive 12 or 24 doses of MDT and 9 healthy controls were evaluated. All the patients were classilied by Ridley-Jopling criteria and the bacilloscopic index (BI) serum antibody and PGL-I (APGL-I), inflammatory cytokines and the co-stimulatory and adhesion molecules serum levels were measured before and after MDT. The APGL-1, IFN-γ, IL10, IL6 and TNF alpha serum levels were determined by ELISA assay. The results showed that MDT 12 and 24 doses can reduce the APGL-1 levels in a similar range. The decrease of BI and APGL-1 levels is followed by the augment of IFN-γ serum levels associated with enhancement of LAF/CD4+ molecules. The quantification of the LAF/CD8+ molecules is higher in MB patients before the treatment and after 24 doses of MDT the values are similar to the normal controls. Since IFN-γ is a cytokine able to induce an enhanced cellular immunity this results can suggest that 12 doses of MDT might be efficient on the control of MB leprosy specially in those patients with moderate bacillary index (below 3,0)
OI 7
ENUMERATION OF IFN-γ-PRODUCING CD4+T CELLS AS A TOOL FOR SELECTING HIGH-LEVEL IFN-γ-lNDUCING Mycobacterium leprae ANTIGENS
Márcia V. B. S. Martins2*; Monica C.B.S. Lima1,2,3*; John S. Spencer1; Maria A. M. Marques1; Heejin Kim1; Bruce C. Gregory1; Nadia C. Duppre1; José A. C. Nery1; Euzenir N. Sarno2; Patrick J. Brennan1; Maria C.V. Pessolani2; Geraldo M. B. Pereira2,3.
1. Department of Microbiology. Colorado State University, CO, USA.
2. Leprosy Laboratory, Oswaldo Cruz Institute. FIOCRUZ.
3. Laboratory of Immunopathology. School of Medical Sciences, State University of Rio de Janeiro. Rio de Janeiro, Brazil.
* These authors contributed equally to this work.
The sequencing of the genomes of M. leprae and other mycobacteria has afforded new opportunities for the development of pathogen-specific diagnostic tests for mycobacterial infections, critical in the context of leprosy eradication. Recently, antigen-specific IFN-γ production was used with success for the diagnosis of latent tuberculosis. We evaluated the IFN-γ inducing capacities of various fractions derived from M. leprae itself (e.g. ammonium sulfate or ethanol precipitated cytosolic proteins; cytosolic proteins fractionated by anion exchange chromatography or isoelectric focusing) and various recombinant proteins dictated by analysis of the genome (e.g. ESAT-6, CFP-10, 10kDa, Ag85B, H1p, EfTu, MMP-I, MMP-II) and genetically fused versions of some of these recombinant proteins (e.g. CFP10-ESAT-6; ESAT-6-Ag85B). The initial screening was done with blood samples from two untreated newly diagnosed leprosy patients (TT and BT; from Leprosy Lab Outpatient Unit, Rio de Janeiro). The presence of lFN-γ-producing CD4+ T cells (IFN-T) was detected by intracellular cytokine assay, using flow cytometry, in response to several of these antigens and, when observed, was associated with high-level IFN-γ in the culture supernatants as determined by ELISA. In particular, some of the native M. leprae fractions, as well as MMP-l. had high frequencies of IFN-T and induced high IFN-γ supernatant levels, comparable for instance to that of S. aureus enterotoxin-B. (Research was supported by NIAID, NIH and FAPERJ).
OI 8
EXPRESSION OF CHEMOKINES AND THEIR RECEPTORS IN LEPROSY SKIN LESIONS
Clement Alain Musonda; Alice Amanda Kirkaldy; Saroj Khanolkhar-Young; Sujai Suneetha; Diana N.J. Lockwood
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine. Keppel Street. London, WCIE 7HT. United Kingdom
BPRC, Hyderabad. India
Chemokines are small peptides that are potent activators and chemoattractants for leukocyte subpopulations and some nonhaemopoietic cells. Chemokines and their receptors have been associated with infectious diseases.
Study: We have investigated the expression of chemokines and their receptors in leprosy skin lesions using immunohistochemislry. Skin biopsies from 25 leprosy patients across the leprosy spectrum, 11 patients undergoing Type I reversal reactions and 4 normal donors were immunostained by ABC peroxidase method using antibodies against CC and CXC chemokines and their receptors. We have also investigated mRNA production for MCP-1, RANTES and IL-8 chemokines using an in situ hybridisation technique.
Results: Chemokine and receptor expression was detected in all leprosy skin biopsies. Expression of CC chemokines MCP-1 (p < 0.05), RANTES (p < 0.005) and CXC chemokine, IL-8 (p < 0.005) were significantly elevated in borderline tuberculoid leprosy in reversal reaction compared to borderline tuberculoid leprosy. However, the expression of CC (CCR2 and CCR5) and the CXC (CXCR2) chemokine receptors did not differ across the leprosy spectrum. Similarly, there was no significant difference in the expression of MCP-1 and IL-8 mRNA. Nevertheless, a small but significant elevation in RANTES mRNA (p < 0.05) was detectable in borderline lepromatous leprosy in reversal reaction compared to borderline lepromatous leprosy.
Conclusion: Surprisingly, we did not find any difference in the expression of chemokine receptors across leprosy spectrum. In addition, RANTES expresion was slightly elevated in borderline tuberculoid leprosy in reaction. The presence of a neutrophil chemoattractant IL-8 in leprosy lesions, which do not contain neutrophils, here strongly suggests a role of IL-8 as a monocyte and lymphocyte recruiter in leprosy lesions.
OI 9
HUMAN T CELL RECOGNITION OF FRACTIONATED ANTIGENS FROM Mycobacterium leprae: POTENTIAL AS DIAGNOSTIC REAGENTS.
H. M. Dockrell1; R. Hussain2; J. S. Spencer3; G.F. Black4, F. Shahid2; S. Zafar5; S. TerLouw3; S. Chagu-luka3; L. Sichali4; A. Crampuv4; P.E.M. Fine1; P.J. Brennan3
1. London School of Hygiene & Tropical Medicine, London WC1E 7HT, UK.
2. Aga Khan University, PO Box 3500, Karachi 74800. Pakistan.
3. Colorado State University, Fort Collins. CO 80523, USA.
4. Karonga Prevention Study, PO Box 46, Chilumba. Malawi.
5. Marie Adelaide Leprosy Centre, PO Box 8666, Karachi 74400, Pakistan.
One approach to the development of species-specific mycobacterial antigens is to progressively fractionate the antigens in whole bacteria until functional specificity is achieved. Equivalent preparations of Mycobacterium leprae and Mycobacterium tuberculosis cell wall and cytosolic antigens were used to test lymphocyte proliferation and IFN production in 6 day assays in leprosy and tuberculosis patients in Pakistan. The M. leprae antigenic preparations were less potent for T cells from tuberculoid leprosy patients than the M. tuberculosis antigens, and also induced T cell responses in tuberculosis patients. Further fractionation of M. leprae cytosolic antigens (MLSA) produced components of potentially greater specificity. The presence of M. leprae-specific antigens within MLSA was confirmed in healthy, non-BCG-vaccinated young adults in Malawi, where IFN-γ production to MLSA in diluted whole blood assays was more strongly associated with skin test indurations to Rees MLSA than to M. tuberculosis PPD. Thus fractionation of the cell wall or cytosolic proteins of M. leprae may yield specific diagnostic reagents for leprosy.
OI 10
IFN-γ DETECTION AND ABSENCE OF IL4 IN SITU UNDER NON STIMULATED CONDITIONS IN PAUCIBACILLARY SINGLE SKIN LESION LEPROSY
M. M. A. Stefani1; C.M.T. Martelli1; TP. Gillis2; J.L. Krahenbubl2; Brazilian Leprosy Study Group.
1. Federal University of Goiás, Goiânia- Go. Brazil.
2. National Hansen's Disease Programs, USA.
Objectives: Define in early leprosy lesions the Cell Mediated Immunity by cytokine profiling: IFNγ, IL12, IL10, IL4, TNFα and M1PI and assess M. leprae DNA.
Methods: 259 SSL-PB leprosy patients were enrolled (1997-98) for ROM therapy and were clinically monitored. Six cytokines- IFN-γ, IL10, IL12, IL4, TNFα and MIP1α had mRNA assessed by RT-PCR using Real Time PCR (ABI Prism 7700 Perkin Elmer) in skin biopsies from 39 patients with different clinical outcomes. M. leprae DNA-PCR was performed in skin biopsies using primers for the specific 18KDa protein gene.
Results: Highest values of IFN-γ were among the TT group (median= 1.77) with well-formed granulomas, followed by BT (1.08) and I group (0.02). IL10 values were similar for TT (0.79) and BT (0.72) groups. MIPlγ detection was higher in TT lesions followed by BT and I groups. IL4 values were zero for all specimens tested. Statistically significant correlation was observed between IL12 and IFN-γ (r=0.4. p=0.02) and between IFN-γ and IL10 (r =0.67, p<0.05), possibly reflecting regulatory measures related to macrophage activation. Also IL10 and IL12 correlation was observed (r=0.6, p<0.01) suggesting in situ relationship between induction and control mechanisms in early leprosy lesions. 48.6% M. leprae DNA-PCR positivity was observed.
Conclusions: Our results support the concept that SSL-PB leprosy patients are tuberculoid-like with reasonable strong CMI contributing to the good prognosis after early treatment with ROM. TDR/WHO grant 98100.
OI 11
IMMUNOPHENOTYPIC STUDY IN PAUCI-BACILLARY SINGLE SKIN LESION LEPROSY
M.M.A. Stefani1; F.C. Figueiredo2; C.M.T. Martelli1; M.B. Costa1; Sousa A.L.O.M.1; Sacchetim S.C1; T.P. Gillis3
1. Federal University of Goias/ Rua Delenda Rezende s/n. Setor Universitario. CEP 74605-050, Brazil.
2. University of Brasilia/Brazil.
3. National Hansen's Disease Programs/ USA.
Objectives: Assess the microanatomy of single skin lesion paucibacillary leprosy (SSL-PB) characterizing the phenotypes of different cell populations present in the cellular infiltrates.
Methods: 36 SSL-PB leprosy patients from Central Brazil, treated with ROM therapy were evaluated by immunostaining in skin biopsies collected before drug intake, lmmunophenotypic study was performed in deparaffinized skin biopsies using monoclonal antibodies and immunoperoxidase methods, after microwave antigen retrieval. The distribution, location and estimated proportion of seven cell populations were evaluated: T lymphocytes (CD3+), B lymphocytes (CD20+), T lymphocyte subpopulations (CD4+, CD8+) and NK cells, CD 68+ macrophages and mast cells. Samples were previously coded and all laboratory tests performed in independently, by different experts. Data were analyzed taking into account conventional histopathology and M. leprae DNA- PCR findings.
Results: 50% of SSL-PB were classified as BT, 27.8% TT and 22.2% I. M. leprae DNA was detected in 14/36 (45%). Cell phenotypes immunohistochemistry markers were observed in all preparations regardless of the morphological classification. Presence of neural aggression observed in histopathology was associated with positivity for M. leprae DNA by PCR (p<0.05). Detection of different cell phenotypes in early leprosy lesions, many of them with confirmed M. leprae DNA detection, provides indepth evaluation of the in vivo immune/inflammatory response in early paucibacillary leprosy. TDR/WHO grant 98100
OI 12
IMPROVING SUBUNIT DNA VACCINES AGAINST MYCOBACTERIAL INFECTIONS.
Britton W.J.1,*; Martin E.1,2; Kamath A.T.1
1. Centenary Institute of Cancer Medicine and Cell Biology, Locked Bag No 6, Newtown, NSW, 2042.
2. Cooperative Research Centre for Vaccine Technology, QIMR, P.O. Royal Brisbane Hospital, QLD, 4029, Australia.
Immunisation with Mycobacterium bovis (BCG) confers significant protection against leprosy and has contributed to the control of the disease. We have demonstrated that immunisation with a DNA vaccine expressing the immunodominant 35 kDa antigen of M. leprae causes equivalent protection to BCG in the mouse footpad model of M. leprae infection (1). We have investigated two ways of improving the efficacy of this approach, using DNA expressing the M. avium homologue of the 35 kDa protein (DNA-35), which shows 95% aa identity to the M. leprae protein, and infection with virulent M. avium. First, we co-immunised mice with DNA-35 and a plasmid producing both chains of IL-12 as a self-cleaving protein. This resulted in increased frequency of antigen-specific IFN-γ secreting T cells, and a reduction in specific IgG antibody responses. Moreover, following IVI infection with M. avium, these mice showed significantly reduced bacterial loads compared to mice immunised with DNA-35 alone or BCG. This increased protection was associated with a significantly stronger IFN-γ response to both the 35 kDa protein and M. avium sonicate after challenge. Second, we examined whether targeting the 35 kDa protein to the B7 molecules on antigen presenting cells increased the vaccine efficacy. The 35 kDa gene was fused to the gene for CTLA-4-Ig within the DNA vaccine. Mice immunised with this construct showed an increase in both specific IFN-γ T cell and IgG responses. However, this was not associated with increased protection against M. avium infection. Therefore plasmid IL-12 is an effective adjuvant to increase the protective effect of DNA vaccines against M. avium. We are testing whether this increases protection against M. leprae infection. Future subunit vaccines against tuberculosis should also include dominant M. leprae antigens to ensure they provide cross-protection against leprosy.
1- Martin E, et al (2001) DNA encoding a single mycobacterial antigen protects against leprosy infection. Vaccine 19;1391-6
OI 13
IN SITU EXPRESSION PATTERN OF IFN-γ, 1L-4 AND M. leprae ANTIGENS ACROSS SPECTRUM OF LEPROSY REFLECT DISEASE ACTIVITY RELATED TO REACTIONS
C.E. Verhagen; A. Moens; Chi Han; I.C. van den Bos; R Fleury; W.R. Faber; B. Naafs; P.K. Das
Recent studies on lesional skin derived T cell clones (TCC) from leprosy patients experiencing reversal reaction (RR) showed a polarised shift of M. leprae responsive TCC to Type-1 like phenotypes with predominant production of IFN-γ/TFN-α and low production of IL-4/IL-5/IL-13. With this background knowledge, we assessed the presence of IFN-γ and IL-4 (both protein and mRNA) in lesional skin biopsies of untreated leprosy patients during RR and erythema nodosum leprosum (ENL) reactions. The in situcytokines were identified on frozen biopsies by both immunohistoehemical staining and in situ hybridisation and RT-PCR methods. On the other hand in situ presence of m.leprae antigens was identified in both frozen and parafin embeded biopsies by specific monoclonal antibodies to phenolic glycolipid-1 (PGL-1) lipoarabinomanan (LAM) by immunohistoehemical single and double stainings. We found that both IFN-γ and IL-4 protein and mRNA were opresent in varying amounts in the lesions of untreated paucibacillary (PB) and multibacillary (MB) patients irrespective of their spectral status. No significant differences were seen regarding the in toto presence of these cytokines in individual lesions although their presence varied indifferent granulomas within one lesion. However in lesions with RR and ENL higher levels of IFN-γ and IL-4 were seen although in ENL IL-4 was relatively higher although not significant. These data of in situ expression of T cell cytokines appear to indicate the ongoing disease activity as is the case in patients with reactions. The in situ presence of PGL-1 and LAM with the macrophages in lesions of MB patients decreased dramatically with the treatment. However, in some patients, presence of these antigens persisted in lesions of MB as well as in PB patients, but with differing staining pattern. Such dynamics in the expression pattern of PGL-1 and LAM, in leprosy lesions appeared to he associated with reactions. Our studies suggest that the evaluation of m situ expression pattern of IFN-γ, IL-4 and M. leprae antigens can be regarded as important differential diagnostic critérium for recognising leprosy lesions and may have predictive value for recognising reactions during the evalution of the disease
OI 14
INTERLEUKIN-10 PROMOTER SINGLE NUCLEOTIDE POLYMORPHISMS: MARKERS FOR DISEASE SUSCEPTIBILITY AND DISEASE SEVERITY IN BRAZILIAN PATIENTS
Moraes M.O.1; Santos A.R.1; Schonkeren J.J.M.2; Vanderborght PR.1; Coutinho D.1; Sales A.M.1; Ottenhoff T.H.M.3; Sampaio E.P.1; Huizinga T.W.J.2; Sarno E.N.1
1. Leprosy Laboratory. Department of Tropical Medicine, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, RJ, Brazil.
2. Department of Rheumatology.
3. Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands.
Single nucleotide polymorphisms at positions -3575, -2849, and -2763 on the promoter region of the inter-leukin-10 gene are arranged to form haplotypes that affect levels of IL-10 production. In this study we have determined IL-10 genotype and haplotype frequencies in leprosy patients compared to controls, and analyzed their occurrence with particular forms of leprosy (multi-vs. paucibacillary as severe and mild forms, respectively). No significant differences was observed in genotypes comparing patients to controls, but in -2849 SNPs genotypes were different in multi-bacillary (MB) when compared to paucibacillary (PB) patients (P=0.04). The observation of haplotypes suggested that -3575T/-2849A/-2763C is associated with the occurrence of the disease (P=0.038) irrespective of the further clinical outcome. Besides, AGC haplotype was diminished in patients group as compared to controls (P=0.001). The comparison among patient groups demonstrated that the rarely found IL-10 haplotype AAA was strongly associated to the development of the severe (P=0.003) form of leprosy and TGA haplotype was more frequent in paucibacillary group. The data suggest that distal IL-10 promoter haplotypes could be used as genetic markers that predict susceptibility as well as disease severity in leprosy.
OI 15
LATERAL FLOW ASSAY FOR CLASSIFICATION OF LEPROSY PATIENTS AND IDENTIFICATION OF HIGH-RISK CONTACTS
S. Buhrer-Sekula; H.L. Smits; G.C. Gusscnhoven; J. van Leeuwen; P.R. Klatser; L. Oskam
KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands.
The interruption of leprosy transmission is one of the main challenges for leprosy control programs since no consistent evidence exists that it has been significantly reduced after introduction of MDT. Sources of infection are particularly patients with high loads of bacteria and infected persons in which the clinical signs have not yet become apparent. Detection of antibodies to PGL-I of M. leprae to identify those cases may be a tool for the interruption of transmission. An operational applicability of serology within the leprosy control services requires a simple test system.
We have developed a lateral flow assay for the detection of antibodies to PGL-I which takes only 10 minutes to perform. We have compared its performance with that of ELISA. An agreement of 89.7% was observed between ELISA and the lateral flow assay when tested on 620 serum samples; the agreement beyond chance (Kappa value) was 0.76. No significant difference was found between the lateral flow assay and ELISA when seropositivity rates obtained in groups of leprosy patients, household contacts and controls were compared. Storage of the only reagents required, the lateral flow test and the running buffer, for up to a month at high temperatures, does not influence the results of the assay.
The lateral flow assay is a fast and easy-to-perform method for the detection of IgM antibodies to PGL-I of M. leprae; it does not require any special equipment and the highly stable reagents make the test robust and suitable for use in tropical countries.
OI 16
LEPROSY TRANSMISSION AND MUCOSAL IMMUNE RESPONSE
Dr. R. S. Jadhav; Miss A. Fernando; Miss V.S. Shinde; Ravindra R. Kamble; Mrs. S.P Madhale; Dr. V.K. Edward; Dr. J.R. Rao; Prof. W.C.S. Smith on behalf of MILEP-2 Study Group*
Stanley Browne Research Laboratories, Richardson Leprosy Hospital, Miraj, Maharashtra-416410. Tel. No Off: 0233-211213 Fax: 0233-211708 E-mail: sblabtlm@vsnl.com
Decrease in the prevalence rate of leprosy has not reflected in the incidence of new cases. Very little is known about the transmission of leprosy. Though household contacts of multi-bacillary cases are at high risk of developing disease, majority of the new cases have no history of household contact. A study was undertaken to look at the transmission and the development of mucosal immunity. Subjects (3035) from three villages were examined and followed twice at six monthly intervals. Polymerase Chain Reaction (PCR) was used to detect presence of M. leprae DNA on the nasal mucosa and mucosal immunity was tested by measuring the salivary M. leprae reactive IgA antibodies (sML-IgA) using ELISA. More than 60% of the subjects from all the three villages showed sML-IgA. This response was seen in all the age groups. The response between BCG vaccinated and non-vaccinated individuals did not show any difference. Overall PCR positivity (presence of M. leprae on nasal mucosal) was found to be 1.65% (42 out of 2552), 4.5% (56 out of 1252) and 1.9% (25 out of 1308) in the initial screening. 1st follow-up and 2nd follow-up respectively. Most of the positive subjects in the follow-up were negative in either the previous or subsequent follow-up suggesting transient nature of the PCR positivity. Presence of M. leprae reactive antibodies in the majority of population suggested a possible widespread exposure to M. leprae. This mucosal immune response could be of protective importance as most of the subjects showing presence of nasal M. leprae had these antibodies. Shorter intervals between the follow-ups may shed more light on role of mucosal immunity and late of M. leprae in the nasal passage.
OI 17
M. leprae INDUCES NF-B NUCLEAR TRANSLOCATION IN PBMC FROM LEPROSY PATIENTS
Hernandez M.O.; Lopes U.G.*; Redner P.*; Sarno E.N.; Sampaio E.P.
Leprosy Laboratory, Oswaldo Cruz Foundation -FIOCRUZ.
*Molecular Parasitology Laboratory -Carlos Chagas Filho Institute UFRJ. Rio de Janeiro - Brazil.
NF-B is a transcription factor involved in the regulation of many inflammatory genes, including TNF and IL-1. It has also been suggested to be involved in the regulation of apoptosis. M. leprae is known to induce TNF production by PBMC from leprosy patients in vitro. Recent data from our laboratory showed apoptosis to occur in a dose-dependent manner and that TNF seems to be a mediator of this process. In order to investigate whether NF-B is activated in response to M. leprae, nuclear proteins from stimulated PBMC were analyzed by EMSA. Initial results demonstrate that M. leprae, when added at 1 and l0g/ml, induces nuclear translocation of this transcription factor. To identify NF-B subunits activated by M. leprae, samples were assayed by super-shift. Subunits p65 and p50 were detected, while p52. c-rel and Rel-B were not. These results indicate that NF-B is activated in PBMC by M. leprae. Moreover, its role in the induction of TNF synthesis and apoptosis of TNF synthesis and apoptosis is under further investigation.
OI 18
M. leprae-SPECIFIC, HLA CLASS II-RESTRICTED KILLING OF HUMAN SCHWANN CELLS BY CD4+ TH1 CELLS: A NOVEL IMMUNOPATHOGEN1C MECHANISM OF NERVE DAMAGE IN LEPROSY.
H.T. Spielings; T. de Boer; Brigitte Wieles; L.B. Adams; E. Marani; T.H.M. Ottenhoff
Department of Imnuinohematology and Blood Transfusion and Department of Neurophysiology. Leiden University Medical Center, The Netherlands, National Hansen's Disease Programs Laboratory. Louisiana State University School of Veterinary Medicine. Baton Rouge. USA.
Peripheral nerve damage is a major complication of reversal (or type-1) reactions in leprosy. The pathogenesis of nerve damage remains largely unresolved, but detailed in situanalyses suggest that type-1 T cells play an important role. Mycobacterium leprae is known to have a remarkable tropism for Schwann cells of the peripheral nerve. Reversal reactions in leprosy are often accompanied by severe and irreversible nerve destruction and are associated with increased cellular immune reactivity against M. leprae. Thus, a likely immunopathogenic mechanism of Schwann cell and nerve damage in leprosy is that infected Schwann cells process and present Ags of M. leprae to Ag-specilic. inflammatory type-1 T cells and that these T cells subsequently damage and lyse infected Schwann cells. Thus far it has been difficult to study this directly because of the inability to grow large numbers of human Schwann cells. We now have established long-term human Schwann cell cultures from sural nerves and show that human Schwann cells express MHC class I and II. ICAM-1, and CD80 surface molecules involved in Ag presentation. Human Schwann cells process and present M. leprae, as well as recombinant proteins and peptides to MHC class II-restricted CD4( + ) T cells, and are efficiently killed by these activated T cells. These findings elucidate a novel mechanism that is likely involved in the immunopathogenesis of nerve damage in leprosy.
OI 19
MATRIX METALLOPROTEINASE (MMP) mRNA EXPRESSION IN THE SKIN LESION OF LEPROSY PATIENTS
Teles RMB; Samo E.N.; Geraldo N.T.; Moura D.F.; Vasconcelos B.S.; Shattock R.; Sampaio E.P.
Leprosy laboratory, FIOCRUZ. Rio dc Janeiro- Brazil-Infectious Disease' Department - St. George' Hospital. London, England
Introduction: MMPs are a zinc-dependent proteases family that collectively are able to degrade most extracellular matrix components. In general, MMPs are not constitutively expressed by the cell in vivo, but their expression can be modulated by cytokines such as TFN-á.
Objective: To determine the pattern of expression of MMPs in reactions in leprosy patients who showed enhanced TFN-á production.
Methods: Skin biopsies of 20 leprosy patients and 2 controls were collected and total RNA was extracted. RT-PCR to MMP-2, MMP-9 and TFN-á was performed, and the amplified products analyzed through electrophoresis in agarose gel.
Results: The dermis of all (n= 15) reactional patients (RR and ENL) were positive to TFN-á and MMP-9 mRNA, and 84% (n= 12) to MMP-2 mRNA. In the dermis of the 5 unreactional patients, 60% (n= 3) were positive to MMP-2, MMP-9 and TFN-á mRNA. The 2 health controls are positive to TFN-á mRNA, but negative to MMP mRNA. In 50% of the patients MMP mRNA expression decreased during the treatment. In the epidermis of leprosy patients the TNFá mRNA was detected in all patients and MMP-2 and MMP-9 in 50% (n=3) and 16% (n= 1), respectively. TNFa and MMP mRNA was not observed in the epidermis of the unreactional patients. Preliminary experiments with Real Time PCR confirm the above data.
Conclusion: The MMP mRNA expression was detected only together with TFN-á expression both in the dermis and epidermis. It is likely that these enzymes play a role in inflammatory reaction in leprosy.
OI 20
MHC AND IMMUNE REPONSE ACROSS THE LEPROSY SPECTRUM.
Meenakshi Singh1; D.K. Mitra1; K. Katoch2; S. Ghei2; U. Sengupta2; N.K. Mehra1
1. Department of Transplant Immunology & Immunogenetics, All India Institute of Medical Sciences.
2. Central JALMA Institute of Leprosy. India.
We earlier showed that HLA Class II genes modulate immune response following infection with M. leprae with preferential interaction among mycobacterial epitopes and particular Class II motif characterized by 'Arg' rich pocket 4 residues of the DRB1 molecule. It has been observed that cytokine polarized clinical states of leprosy correlate with the representation of phenolypable tine T cell subsets. We found that the functionally polarized subsets of CD4 memory T cells are identifiable on the basis of expression of CD11a, CD45RA and CD62L, viz those that primarily produce IL- 4 (MT2, CD45 RA- CD62 L + CD 11adim) or primarily y-Ifn (MT1, CD45 RA- CD62 L - CD 11abright). The frequency and representation of phenotypically definable memory T cell subsets (MT1 and MT2) was defined in all patients. We observed an overrepresentation of MT1 cells among BT subjects (Median, 3) compared to those with BL leprosy (Median, 15). Conversely MT, cells were over represented in BL patients (Median, 23) as opposed to BT subjects (Median, 3). Although median value of MT( representation among BL subjects was significantly lower than BT subjects, certain BL/LL patients had comparable higher frequency of MT1 cells as observed in BT cohorts. These results suggest a possible heterogeneity of immune response against M. leprae in these patients. Careful analysis of the data with regards to the bacillary load of BL patients revealed that MT1 cells were increased in only those BL/LL patients who had become bacillary negative following therapy. All BL/LL patients with a bacillary index of 2 or 4 showed lower frequency of MT cells. Our data indicates immune dynamics in leprosy and provides evidence that functional immune response as measured by cytokine producing memory T cells is dictated by the antigen load and presentation of relevant peptides of M. leprae by the host MHC.
OI 21
NEURAL PATHOLOGY DURING TREATMENT AND RFT
D. Porichha1; A. Mukherjee2; Ramu3
1. CGHS Medical Center, Parliament House Annexe, New Delhi, India. Pin. 110001. India.
2. Institute of Pathology, Safdarjung Hospital Campus, New Delhi. India, Pin 110023.
3. Sacred Heart Leprosy Center, Kubhakonam, Tamilnadu. India.
Histological findings of 24 nerves, 15 developing pain and tenderness during treatment and 9 after RFT are presented in this study. Eight cases showed macrophage type and 15 had epithelioid cell type of granulomas. All cases were active except 3 macrophage granulomas where regressive changes were evident. One case had fibrosis with lymphocytic infiltration. No morphological difference could be observed between nerves biopsied during treatment and during RFT. Histology of epitheliod cell granulomas had interesting and diverse manifestations such as severe type I reaction, caseation necrosis with leque-faction and calcification. Three macrophage granulomas showed regressive changes, two were in ENL, while one case showed histoid changes. All the cases Studied showed similar granulomatous response irrespective of whether the pain and swelling appeared timing treatment or during surveillance. Nerves are thought to be immunologically more protected structures but once the taction is triggered the special nature of neural tissue seems to make the reactional episodes more explosive. Ascertaing relapse in nerves is more ambiguous due to smouldering nature of neural pathology.
OI 22
QUANTITATIVE ESTIMATION OF SALIVARY IgA ANTIBODIES IN LEPROSY BY ELISA USING INDIGENOUS POOLED SALIVA AS STANDARD
Miss A.Fernando; Dr. R.S. Jadhav; Miss VS. Shinde; Ravindra R. Ramble; Mrs. S.P. Madhale; Dr. J.R. Rao; Dr. V.K. Edward; W.C.S. Smith on behalf of MILEP-2 Study Group*
Stanley Browne Research Laboratories, Richardson Leprosy Hospital, Miraj, Maharashtra-416410. Tel. No Off: 0233-211213 Fax: 0233-211708 E-mail: sblabtlm@vsnl.com
Background: M. leprae being the first organism to be discovered, yet a specific method for cultivation of M. leprae is still lacking. Leprosy is a unique disease; the disease is difficult to define due to the lack of gold standard for diagnosis. It is known that the primary lesions may be in the nasal mucosa. All mucosal sites are linked by lymphocyte re-circulation. The mucosal immune system is of importance in a putative protective response to infection.
Aim: To investigate M. leprae reactive antibodies in saliva as a marker of anti-M. leprae immunity using indigenous pooled saliva as standard.
Materials and Methods: Saliva samples were collected from subjects in leprosy endemic areas. ELISA studies were performed on all the saliva samples using whole a-irradiated M. leprae. Saliva showing high IgA concentration was pooled in appropriate quantities and a two-fold dilution of the pooled saliva was done to construct the standard curve. HRP conjugated antihuman IgA was added and the reaction was developed using o-phenylenediamine containing 0.05% hydrogen peroxide.
Results: Standard curves prepared using pooled saliva samples were used to determine the concentration of IgA in terms of arbitrary units (AU) and used to compare the antibody levels in different individuals. For most of the saliva samples tested, 1:04 dilution of saliva appeared to be the right dilution. The optimum concentration of M. leprae cells used for coating was 1x107 cells/ml and the secondary antibody was diluted 1: 40,000.
Conclusion: The pooled saliva used as a standard contributes to the uniformity in ELISA results. It was seen that this kind of quantitation was sufficiently a robust technique to give reproducible results.
OI 23
REAÇÃO DE MITSUDA EM PACIENTES PORTADORES DE HANSENÍASE NA FORMA TUBERCULÓIDE E DIMORFA DURANTE E APÓS O SURTO REACIONAL.
Joel C. Lastória; Vladimir de A. Opromolla; Raul N. Fleury; Alexandra P. Bononi; Karina G. Pedroso; Carlos R. Padovani
Depto de Dermatologia - F. M. de Botucatu- UNESP
A hanseníase, de acordo com o sistema de classificação adotado pelo VI Congresso Internacional de Leprologia, 1953, apresenta duas formas polares, clínica e imunologicamente distintas: o tipo virchoviano e o tuberculóide. Apresenta ainda dois grupos instáveis, o indeterminado e o dimorfo. Em todas elas, exceto na indeterminada, a evolução crônica pode ser interrompida por surtos agudos, denominados de tuberculóide reacional (TR), dimorfo reacional (DR) e, quando na virchoviana, de eritema nodoso. A resistência à Hanseníase pode ser avaliada através da Reação de Mitsuda e, neste sentido, diferentes autores relatam que esta pode estar aumentada, diminuída ou inalterada em pacientes Tuberculóides e Dimorfos durante os surtos reacionais, sem contudo realizarem trabalhos específicos sobre o tema. Com esse objetivo, nesta apresentação, avaliou-se o comportamento clínico da reação de Mitsuda em 43 pacientes das formas Tuberculóide e Dimorfo, durante e após o surto reacional, e observou-se que há predominância da manutenção dos resultados da Reação de Mitsuda - tanto quando a avaliação foi feita separadamente, nos grupos TR e DR, quanto quando conjuntamente. Ademais, as alterações, quando ocorreram, foram predominantemente no sentido de aumento após o surto, ou seja, é possível que, na realidade, durante o surto reacional, alguns indivíduos possain apresentar uma diminuição de sua resistência específica ao M. leprae
OI 24
RT-PCR ANALYSIS OF PATHOGEN AND HOST EXPRESSION IN LEPROSY PATIENTS
Shabaana, A.K.1; N.P. Shankernarayan2; D.C. Hoessli3; K. Dharmalingam1.
1. School of Biotechnology, Madurai Kamaraj University, Madurai, India.
2. VHS, Leprosy Project, Shakthi Nagar. India.
3. University of Geneva Medical School. Geneva, Switzerland.
Molecular analysis of nucleic acids in Paraffin-embedded infected tissues (PET) using RT-PCR is an effective approach to analyse gene expression of both host and pathogen genes. Punch biopsies obtained from leprosy patients across the leprosy spectrum (n=46) were fixed in formaldehyde and embedded in wax. RNA extraction conditions were optimized and first strand cDNA synthesis was carried using oligo dT primers. Semi-quantitative PGR was carried out using human cytokine specific primers as well as M. leprae specific primers.
Analysis of the expression of regulatory cytokines, TGE- and IL-10 in relation to Thl/Th2 cytokines in different disease states confirm and extend the earlier observations that the LL condition is a mixed type of response having both Thl/Th2 cytokine production. Also, TGF- mRNA was found to be up- regulated at the LL end of the spectrum and could be responsible for the absence of any effect of TNF- and IFN-γ in borderline and lepromatous conditions. Finally the reactional conditions, reversal and ENL show similar cytokine profiles.
Till date there is no documentation of the gene expression of M. leprae in infected host tissue. The same RNA extracted for cytokine analysis was also used for analysis of M. leprae specific genes 18kDa heat shock protein, 35kDa Major Membrane Protein I and two genes belonging to the Mce operon (Mammalian cell entry) namely mcelA and IprK. Amplification of these genes by RT-PCR from leprosy biopsies confirmed the expression of these genes in vivo. All amplified products have been cloned and sequenced to confirm the reliability of the system.
OI 25
SCREENING NEW LEPROSY ANTIGENS FOR POTENTIAL AS LEPROSY SKIN TESTS
Murdo Macdonald; Niraj Shrestha; Parmeshwor Amatya; Rakesh Manandhar; Kees Franken; Tom Ottenhoff; Warwick Britton; Paul Roche
Mycobacterial Research Laboratory. Anandaban Leprosy Hospital, PO Box 151. Kathmandu, NEPAL. E-mail: anandaban@mail.com.np
There remains a requirement for leprosy-specific tests to detect leprosy exposure in communities with high levels of tuberculosis. We have previously demonstrated that levels of the cytokine interferon-gamma (IFN-γ produced in a simple overnight whole blood culture with leprosy antigens are increased in healthy contacts of leprosy patients.
Aim: To investigate the efficacy of three M. leprae antigens (35kD antigen, 45kD antigen, and the M. leprae homologue of ESAT-6 (ESAT-6 ML)) as potential new skin test antigens.
Methods: Whole blood was collected from almost 300 Nepali leprosy patients. TB patients, leprosy household contacts and unexposed subjects and used in overnight whole blood assays. Cells were stimulated with the above named antigens, and IFN-γ was measured in supernatants. The resultant cytokine levels in these stimulated short-term cultures were compared with longer (5-day) culture and with T-cell proliferation.
Results: Very high IFN-γ response levels were observed in leprosy health workers compared with healthy control subjects. Both 24 hour and 5-day cultures gave similar results. In both cases cytokine levels observed were highest in response to M. leprae 35kD antigen, followed by ESAT-6 and 45kD.
Conclusion: These data indicate the potential of these three relatively leprosy-specific antigens for use as leprosy skin tests in the future.
OI 26
SCREENING OF NEW Mycobacterium leprae ANTIGENS AS CANDIDATES FOR THE DEVELOPMENT OF TESTS FOR THE EARLY DIAGNOSIS OF LEPROSY
Monica C.B.S. Lima 1, 2, 3*; Márcia V.B.S. Martins2*; John S. Spencer1; Maria A.M. Marques1; Heejin Kim1; Bruce C. Gregory1; Nadia C. Duppre1; José A. C. Nery1; Geraldo M.B. Pereira2,3; Euzenir N. Sarno2; Patrick J. Brennan1; Maria C.V. Pessolani2
1. Department of Microbiology, Colorado State University, CO. USA.
2. Leprosy Laboratory, Oswaldo Cruz Institute. Oswaldo Cruz Foundation2.
3. Laboratory of Immunopathology, School of Medical Sciences, State University of Rio de Janeiro. RJ, Brazil.
*These authors contributed equally to this work.
The greatest needs from leprosy research are definitive diagnostic antigens to help understand transmission and allow early detection of disease. In order to further investigate the in vitro potential of new M. leprae antigens for in vivo skin tests or related in vitro tests, PBMC from leprosy patients, contacts of leprosy multibacillary patients, operational contacts, TB patients, and exposed or non-exposed healthy controls from leprosy endemic and non-endemic areas were stimulated with antigens from a collection of 26 M. leprae antigens, including crude and fractionated subcellular fractions of M. leprae and recombinant antigens. Cell-mediated responses were measured through IFN-γ secretion using ELISA, and some T-cell activation parameters (such as CLA and CD69 expression) were estimated by flow cytometry. Initial results obtained with these different groups of subjects indicate that sonic fractions/antigens are good inducers of IFN-γ production, and of CLA and CD69 expression, in leprosy patients, but not in TB patients. Responses were lower in lepromatous leprosy patients. These preliminary results suggested that some of our M. leprae antigens hold promise as specific diagnostic tools for leprosy. (Research supported by NIAID, NIH).
OI 27
STUDY OF EXPERIMENTAL LEPROSY IN INTERLEUKIN-12 DEFICIENT MICE
Linda B. Adams; Nashone A. Soileau; Marilyn A. Dietrich; David M. Scollard; James L. Krahenbuhl
National Hansen's Disease Programs Laboratory Research Branch at Louisiana State University. Baton Rouge, LA
IL-12, a key regulatory cytokine of the immune system, induces the production of IFN-γ by T cells and NK cells and promotes the development of a Th1 type cell mediated immune response. To study its role in experimental leprosy, Mycobacterium leprae infection was evaluated in IL-12 knockout (KO) mice. Wild type control mice (C57B1/6) and IL-12 KO mice were infected in both hind foot pads with 6 × 103 viable M. leprae and bacterial growth, cell profiles, histology, and gene expression were monitored for over twelve months. In wild type mice, growth of the bacilli in the foot pads peaked on the order of 105 at six months post infection. In contrast, growth of M. leprae was enhanced in IL-12 KO mice, reaching 105 by three months post infection (P<0.01) and continuing to multiply to reach 106 by 12 months post infection (P<0.01). Histopathologically, control mice exhibited mild lymphocytic and histiocytic infiltrates at 12 months post infection. IL-12 KO mice also developed a mild inflammation with equal numbers of lymphocytes, macrophages and epithelioid cells. Lymph node cells from the draining popliteal lymph nodes were examined throughout infection for lymphocyte differentiation and activation surface markers. Cells bearing the CD44high CD45RBlow markers (activation/memory phenotype) constituted only 15.57 ± 3.93% of the CD4+ cells in the lymph nodes of wild type mice at three months post infection. This cell population increased to 29.14 ± 4.17% by six months and to 36.26 ± 10.49% by twelve months. A similar profile was observed in the lymphocytes of IL-12 KO mice. In summary, IL-12 KO mice exhibited a decreased ability to control M. leprae KO mice, yet did so without the massive granulomatous infiltration observed in those mice.
OI 28
T CELL RESPONSES TO PEPTIDES FROM M. leprae 10 KDA PROTEIN IN THAI LEPROSY PATIENTS, HEALTHY CONTACTS AND NON-CONTACTS
B. Chua-lntra; S. Srisungngam; S. Wattanapokayakit; K. Mahotarn; J.Ivanyi
Leprosy Division, Department of Communicable Disease Control. Ministry of Public Health, Non-thaburi, Thailand.
Detection of M. leprae infected individuals using aT cell based assay is hampered by the lack of suitably specific test reagents. Thus the objective of this study was to identify M. leprae-specific immunogenic peptides from M. leprae 10 kDa protein. Although this protein has a homologue in M. tuberculosis, it was previously found to contain M. leprae-specific epitopes located within amino acid residues 24-39 as tested by murine T cell hybridoma and T cell clone. In this study, we analyzed the proliferative responses of peripheral blood mononuclear cells (PBMCs) to three synthetic peptides from 10 kDa protein of M. leprae among 73 paucibacillary (PB) and 124 multibacillary (MB) leprosy patients, 57 healthy household contacts and 20 non-contacts. These 18-mer peptides were located in 3 areas between residues 11-28. 22-39 and 55-72 containing 3-4 residues distinct between M. leprae and M. tuberculosis. Surprisingly, the result showed that frequencies of responders to all peptides were similar among the PB and MB patient groups. The most immunogenic peptide was p55-72, recognized by 34% and 48 % of PB and MB leprosy patients, respectively. M. leprae-specific P22-39 was recognized by 23-27% of patients but only 7% of healthy contacts which might be useful for discriminating between disease and sub-clinical infection. All peptides were recognized by non-contacts with significantly lower frequencies than the patient groups, suggesting that they were likely to be M. leprae-specific. Determination of the exact species-specificity would require further evaluation using T cell lines or clones. Combination of these peptides may increase the sensitivity of a prospective diagnostic test reagent above that, observed for the individual peptides.
OI 29
T CELL SUBSETS EXPRESSING NEURAL CELL ADHESION MOLECULE: ASSOCIATION WITH ANTIGEN INDEPENDENT, MHC UNRESTRICTED T CELL CYTOTOXICITY IN LEPROSY PATHOLOGY
Eric Spierings; Tjitske de Boer; Tamara Dekker; Birhane Kaleab; Ben Naafs; William R. Faber; Pranab K. Das; Brigitte Wieles; Tom H.M. Ottenhoff
Department of Immunohematology and Blood Transfusion and Department of Dermatology, Leiden University Medical Center, The Netherlands, Department of Dermatology. Amsterdam Medical Center, The Netherlands
We have investigated the role of Neural Cell Adhesion Molecule (NCAM or CD56) in the killing of Schwann cells and other NCAM positive targets by an NCAM expressing human T cell subset, isolated from leprosy patients. Involvement of NCAM expressing T cells in leprosy pathology was suggested by the observations that NCAM expressing T cells could be isolated from inflamed neural tissue. Furthermore, antigenic stimulation of these cells with Mycobacterium leprae increased both the number of NCAM+ T cells and their cytolytic activity against NCAM+ target cells. The cytolytic activity of NCAM+ T cells was antigen independent and could be attributed to the CD8+ T cell subpopulations. NCAM expression was not a stable but rather seemed an acquired characteristic, since it could be modulated in vitro on sorted, NCAM+ cell populations.
In addition, a longitudinal analysis of leprosy patients undergoing active erythema nodosum leprosum (ENL or type 2 leprosy reactions) showed that M. leprae stimulation increased NCAM expression on CD8+ peripheral T lymphocytes only at the time of active ENL. In line with these observations, stimulation with M. leprae increased antigen independent lysis of NCAM positive target cells in close association with the period of active ENL. At the same time, CD8+ NCAM+ T cells could be visualized in ENL skin lesions.
These results reveal a novel mechanism of antigen independent, T cell mediated tissue damage, which is likely to play a role in leprosy and possibly other peripheral neuropathies
OI 30
THE APPLICATION OF SEROLOGICAL TOOLS IN PATIENT MANAGEMENT AND LEPROSY CONTROL
Samira Bührer-Sékula; Linda Oskam
KIT Biomedical Research, Meibergdreef 39, 1105 AZ Amsterdam, The Netherlands
The detection of human antibodies to the Mycobacterium leprae cell wall component phenolic glyco-lipid I (PGL-I) and its semi-synthetic derivatives can be performed using ELISA, agglutination tests, dipsticks or lateral flow tests. The results from these tests used to answer a number of important questions that are of direct relevance to patient management and leprosy control.
Specifically, serological testing can be used to assist with the classification of leprosy patients into multi-bacillary (MB) and paucibacillary (PB) after clinical diagnosis, to detect patients who have an increased risk of relapsing after treatment and to identify contacts of leprosy patients that are in danger of developing leprosy in future.
This presentation will give a critical overview of the various techniques in use. their relative advantages and limitations and the way in which these tools can be used to assist in patient management and leprosy control.
OI 31
THE ROLE OF MANNOSE BINDING LECTIN AND INFLAMMATORY RESPONSE IN LEPROSY
N.T. Foss; M A.N. Ferreira; J.L. Andrade; E.M. Ranialho; T.L. Nunes
Faculty of Medicine of Ribeirão Preto of São Paulo University.
Mannose binding lectin (MBL) is a serum protein component of the innate immune system. MBL is able to enhance the phagocytosis of the pathogens by binding to the sugars on the microbial surface through an opsonin mechanism resulting in complement system activation. The deficiency of MBL is generally associated to susceptibility to infections. Leprosy is a chronic inflammatory and infective disease caused by the intracellular parasite Mycobacterium leprae which strongly bind to MBL. To evaluate the involvement of MBL on the evolution of the disease were selected 58 untreated leprosy patients, classified by Ridley & Jopling criteria (lepromatous leprosy-LL = 14, borderline lepromatous-BL = 8, borderline tuberculoid-BT = 11 and erythema nodosum leprosum-ENL = 25) and 10 healthy controls. Sera samples from the patients and controls were analyzed for determination of MBL, inflammatory cytokines (TNFα, IL6, IFN-γ), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and anti PGL1 antibody (APGL 1). It was observed that all ENL patients presented significantly higher levels of MBL (M = 2335.3 ng/ml) than LL (M = 301,3 ng/ml), BL (M = 428,2 ng/ml). BT (M = 486,1 ng/ml), or the normal control (M = 508,7 ng/ml). Additionally significantly elevated values of all the inflammatory parameters were found in ENL, when compared with the other forms of the disease and to controls. ALthough the bacilloscopic index (BI) of ENL and LL patients were similar (4.5 ±1.1 and 5.0 ± 1.5 respectively) the APGL1 levels (ENL 7.5; LL = 3.1), inflammatory cytokines (TNFα, IL6. IFN-γ, ESR and CRP levels were significantly higher in ENL than in LL, indicating that the reactional episode, type 2 reaction, could stimulate the liver cell to produce more MBL and induce the phagocitosis of the parasite, increasing the intracellular destruction. Thus, the data suggests that the protein MBL may act protecting the leprosy patients against the dissemination of the infection.
OI 32
THE ROLE OF TH1 AND TH2 CYTOKINES IN ACUTE LEPROSY NEURITIS
S. Khanolkar-Young; A. Coulthart; S. Suneetha; D.N.J. Lockwood
Department of Infectious and Tropical Diseases, London School of Hygiene & Tropical Medicine, Keppel Street, London, WCIE 7HT, United Kingdom.
BPRC, Hyderabad, India
Study: To assess cytokine production in nerves from patients with acute neuritis (defined as tenderness and/or loss of function within the last six months). The clinical samples skin and nerve biopsies from 57 patients with acute neuritis (BT = 30, BL = 18 and LL = 9) were collected.
Imnumohistochemistry was done on skin and nerve sections to detect the cytokine proteins IFN-γ, IL-6, IL-10, IL-12, IL-13, TFN-α, TGF-β and iNOS.
Results: Morphology: Granulomas were better defined and organised in nerve lesions. Cellular infiltration also more prominent in nerve. Th1 type cytokines (IFN-γ and IL-12) were present at high levels in skin and nerve. Nerves from LL patients had both low levels of IFN-γ and IL-12 and moderate levels of IL-6 and TGF-(3. Th2 type cytokines IL-6, IL-10 and IL-13 were present across the spectrum.
Comments: Nerve damage may occur through two mechanisms, a Th1 dependent mechanism in BT and BL patients and a Th2 dependent mechanism in LL patients.
OI 33
TNF PROMOTER GENOTYPE INFLUENCE TNF PRODUCTION IN LPS- BUT NOT Mycobacterium leprae- STIMULATED WHOLE BLOOD CELLS IN VITRO.
Moraes M.O.; Salgado J.; Abreu A.P.; Alves C.F.R.; Santos A.R.; Nery J.A.C.; Sampaio E.P.; Sarno E.N.
Leprosy laboratory, Tropical Medicine Department, IOC-FIOCRUZ
Single nucleotide polymorphisms (SNP) on TNF promoter are associated with the risk and progression of infectious and inflammatory diseases. Mutations at position -308 in TNFα (TNF2) promoter gene might affect levels of the cytokine production that are central in the outcome and the natural course of leprosy. The study was set out to investigate the contribution of TNF2 SNP in the cytokine mRNA expression and protein secretion in vitro. Paucibacillary leprosy patients were genotyped by PCR-RFLP for the presence of TNF2 allele (carriers = 13, non-carriers = 21). Whole blood cells from these patients were stimulated with LPS (1 ng/ml) and M. leprae ( 1 µg/ml). To mRNA expression analysis, semi-quantitative RT-PCR was performed after 3h stimulation. TNFα mRNA did not show any differences among the patients analyzed regardless the stimulus or the genotype of the patients. Nevertheless, LPS induced an increased in TNFα secretion in TNF2 carriers as compared to non-TNF2 carriers at 6h only (p<0,05). In M. leprae-stimulated cultures no significant differences were achieved. TNF2 allele influence the increased production of LPS-stimulated TNFα production was time dependent and restricted at the protein levels suggesting a post transcriptional regulatory role associated to the promoter polymorphism.
MICROBIOLOGY & MOLECULAR BIOLOGY
OM&BM 1
ACCUMULATION OF NORFLOXACIN AND DAPSONE IN M. smegmatis
K.Venkatesan; Nirmala Deo; A. Mathur*
Central JALMA Institute for Leprosy, Agra -282 001 (India); *Deceased
Quinolones are being increasingly used as second-line agents in the treatment of tuberculosis caused by multidrug-resistant strains. Dapsone is the main component of the MDT regimen for leprosy. At this juncture adequate knowledge of the transport of these chemotherapeutic agents will be of help in the development of new agents. A preliminary study has been conducted at this Institute on the accumulation of norfloxacin and dapsone using modified fluorescence methods. By employing exogenous norfloxacin concentration of 10 ug/ml, a steady state concentration (SSC) of 100 ng of norfloxacin/mg cells, by dry weight was obtained for M. smegmatis. Adequate care was taken to nullify the errors due to drug adsorption to the cell surface and to maximise the desorption using several standardised washings in the buffer. The addition of either dinitrophenol (2.0 mM) or CCCP (150 uM) 10 minutes before or or after addition of norfloxacin did not affect drug accumulation suggesting an absence of energy involvement in its transport process. In a parallel study on dapsone accumulation, the drug accumulated to a level of 78-106 ng/mg cells of M.smegmatis (by dry weight) during 15-60 minutes of incubation at an exogenous concentration of 10 ug/ml. Further studies using other agents like ofloxacin, rifampicin and clofazimine and several other mycobacteria are being planned. The experimental conditions for each mycobacterial strain and each antimicrobial agent are to be suitably standardised in order to get useful information. The method, once standardised, will be applied to drug resistant strains so as to evaluate the role of efflux pump in the emergence of drug resistance.
OM&BM 2
AN IN VITRO MODEL FOR STUDYING THE EFFECTS OF M. leprae ON SCHWANN CELL/NEURON INTERACTIONS
Deanna Hagge1; David Seollard2; Greg McCormick2; Diana L Williams2
1. Biological Sciences Dept. Louisiana State University, Baton Rouge, LA.
2. Laboratory Research Branch. National Hansen's Disease Programs at LSU-SVM. Skip Bertman Dr. Baton Rouge. LA, USA.
Globally, millions of leprosy patients suffer from irreversible nerve damage, resulting in disabilities or blindness as a consequence of infection with Mycobacterium leprae, an obligate intracellular pathogen. The mechanisms of nerve damage have not been fully elucidated due to a lack of a well-developed in vitro model which maintains the viability of M. leprae and closely mimics disease conditions. Therefore, an in vitro model was developed using freshly harvested nude mouse-derived M. leprae, rat Schwann cells and Schwann cell/neuron co-cultures incubated at 33º C, a conductive temperature for M. leprae viability. At 33º C, Schwann cells and mitogen-expanded Schwann cells appeared to be morphologically similar, express similar levels of Schwann cell markers and function in a comparable manner when seeded onto cultured neurons as those cells maintained at 37ºC. M. leprae within Schwann cells retained 56 % of their original viability for at least 3 weeks post infection at 33ºC compared to only 3 % at 37º C. Infected cells exhibited morphological changes, gene expression alterations 33º C, but were capable of interacting with and myelinating neurons. Infected myelinated co-cultures maintained myelin sheath architecture and were morphological comparable to non-infected cultures at both temperatures. In conclusion, an improved model for studying the effects of M. leprae on Schwann cells has been described. Preliminary results using this model indicate that M. leprae, under the conditions specified, do not appear to have detrimental effects on Schwann cell functional capabilities in the peripheral nerve and suggest that the majority of the neuropathy observed in leprosy is most likely due to an aggressive immune response to infection within the nerve.
OM&BM3
CARBOHYDRATE METABOLISM IN LEPROSY PATIENTS
V.P. Tsemba; V.Z. Naumov; N.G. Urlyapova
Leprosy Research Institute, Astrakhan, Russian Federation.
Leprosy infection is often accompanied by metabolic disturbances, and carbohydrate metabolism is no exception. To a large extent, parameters of carbohydrate metabolism depend on sex and age of the patients and reflect a metabolic state at the moment of taking samples. A state of carbohydrate metabolism was estimated in 150 leprosy cases, including 117 (78%) patients with multibacillary (MB) and 33 patients (22%) with paucibacillary (PB) forms of leprosy. Among the patients (of 45 to 93 years old) there were 75 males and 75 females. Carbohydrate metabolism was assessed by integral index of glycemia, i.e. glycolized hemoglobin (HbAlc) defined for the past 3 months by means of colorimeter method. Upper normal limit of HbAlc was 7,5%. In leprosy patients with normal carbohydrate metabolism level of HbAlc did not depend on either sex or age. Disturbances in carbohydrate metabolism were found out in 37 patients (25% of all studied), out of them there were 30 MB (81%) and 7 PB (19%) patients. Ratio of the patients with and without metabolic disturbances was approximately 1:3 in MB and 1:4 in PB leprosy. Diabetes II type was found out in 13 MB-patients (11%) and in 2 patients with PB leprosy (6%). Prevalence of diabetes in patients under observation was 10%. In one case diabetes preceded the development of PB-leprosy. and in other cases sugar disease occurs against the background of leprosy process. Latent disturbances of carbohydrate metabolism were detected in 15% of cases observed. Thus, the data obtained showed a higher prevalence of carbohydrate metabolic disturbances in leprosy patients (to 11%) as compared with that in general population, necessitating further investigations to elucidate their possible causes and mechanisms.
OM&BM 4
COMPARISON OF PCR MEDIATED AMPLIFICATION OF DNA AND THE CLASSICAL METHODS FOR THE DETECTION OF Mycobacterium leprae IN LEPROSY PATIENTS AND CONTACTS
Torres P.1, Gomez J.R.1, Gimeno V.1, Camarena J.J.2; Nogueira J.M.2; Navarro J.C.2; Olmos A.2
1. Sanatorio San Francisco de Borja, Fontilles (Spain).
2. Servicio de Microbiología, Hospital Dr.Peset. Universidad de Valencia (Spain).
Traditional staining and microscopic examination techniques for the detection of Mycobacterium leprae and Polymerase chain reaction (PCR), DNA amplification of a 531 -bp fragment of the Mycobacterium leprae specific pra gene were compared on different clinical specimens on 60 leprosy patients attending the Sanatorium of Fontilles and divided for the purpose of the study in. multibacillary patients (MB) with positive Bacteriological Index (BI), 30 MB patients with negative BI and, 10 paucibacillary (PB) together with 4 non-leprosy patients as controls.
The results in the multibacillary BI positive group show a good correlation between practically all methods and specimens, most techniques detecting 100% of the cases.
The results in the MB negative group reveal that a combination of test (humoral response to D-BSA, together with PCR biopsy and PCR post biopsy swab) are the most sensitive in some cases of this group for monitoring leprosy patients who have completed chemotherapy. In the paucibacillary group no level of positivity was detected by conventional or PCR methods.
The prevalence of antibodies to Mycobacterium leprae antigens in serum was measured together with the presence of Mycobacterium leprae DNA in the nose and lepromin status in a group of 43 contacts of leprosy patients. Two individuals were found to form a potential high risk group.
OM&BM 5
DETECTION OF ANTILEPROTIC DRUG (S) INDUCED DNA DAMAGE IN HUMAN PERIPHERAL BLOOD LYMPHOCYTES BY THE ALKALINE SINGLE CELL GEL ELECTROPHORESIS
P. Rajaguru1; K. Kalaiselvi1; M. Palanivel1; G. Ramu2
1. Department of Environmental Science, PSG College of Arts & Science, Coimbatore 641 014, India.
2. Leprologist, GKNM Hospital Quarters, Coimbatore, India.
Multidrug treatment (MDT) is the WHO recommended method of treatment for leprosy. In MDT, dapsone and rifampicine are effective chemotherapy followed by other frontline drugs like clofazimine, and ofloxacin. These drugs are reported to induce cytogenetic damage in different test systems. Our previous studies indicated higher incidence of DNA strand breaks, chromosomal aberration, and micronucleus frequency in the peripheral blood lymphocytes of leprosy patients treated with MDT. Therefore, to clarify the possible role of components of MDT in inducing DNA damage in leprosy patients, in this study, the induction of DNA damage by antileprotic drugs (dapsone, rifampicin, clofazimine, minocycline and ofloxacin) and subsequent repair was investigated by the alkaline comet assay in human blood lymphocytes. Lymphocytes isolated from leprosy patients and healthy individuals were treated with increasing concentrations of antileprotic drug(s) for varying duration of exposure and subjected for the comet assay. Metabolic aclivation/inactivation of the drugs was studied by incorporating rodent liver microsomal activation system (S9-mix). DNA damage data in lymphocytes of leprosy patients were compared with that of health individuals.
OM&BM 6
DETECTION OF Mycobacterium leprae NASAL CARRIERS BY POLYMERASE CHAIN REACTION IN SINGLE LESION LEPROSY PATIENTS
Silva, M.H.M.; Castro, F.; Visconde, A.M.; Sousa, A.L.O.M.; Rebello, P.F.B.; Gomes, M.K.; Nararashi, K.; Sacchetim, S.C.; Costa. M.B.; Stefani, M.M.A.; Marlelli, C.M.T.; Gillis, TP
IPTSP/UFG, Rua Delenda Rezende s/n Setor Universitário, CEP 74605-050 Brazil: National Hansen's Disease Programs/USA
Objective: Detect M. leprae DNA by PCR in nasal swabs among Brazilian single skin lesion paucibacillary leprosy patients (SSL-PB).
Methods: 259 newly detected SSL-PB leprosy patients, negative baciloscopy, were recruited in 3 endemic regions. 155 nasal swabs and 134 skin biopsies were collected before ROM therapy, snap-frozen and stored (liquid nitrogen) for M. leprae DNA detection by PCR. After DNA extraction each specimen was amplified, undiluted and at 1:5, using pairs of primers for a 360 bp M. leprae specific fragment and products detected by slot blot hybridization using digoxygenin-labeled 212bp DNA probe. Specimens were coded and tested blinded to patient's characteristics at IPTSP/Brazil in collaboration with National Hansen's Disease Programs.
Results: In nasal swabs, M. leprae DNA was detected in 9.7% (15/155) of SSL-PB. Higher positivity (14.1%) was found among specimens from patients living in the North region compared with samples from Southeast and Central Brazil, compatible with endemic levels. No association was found between patient's characteristics or presence of household leprosy contact with PCR positivity. There was no agreement between positivity of M. leprae DNA PCR in skin biopsies and nasal swabs (Kappa=0.07).
Conclusion: Detection of M. leprae DNA in nasal swabs from SSL-PB patients may reflect exposure in endemic areas without agreement with bacilli detection in skin biopsy by PCR. TDR/WHO grant 981007
OM&BM 7
DISTINGUISHING VARIANTS OF M. leprae LABORATORY STRAINS.
Richard Truman, Ph.D.; Thomas P. Gillis, Ph.D.
Laboratory Research Branch, Division of National Hansen's Disease Programs, HRSA, Baron Rouge, La. 70894, USA
Genotyping has practical application in outbreak investigations and variant classification of cultured strains. Though remarkably little variability has been noted among M. leprae, in recent times a few loci for allelic diversity have been identified. These include mainly small insertion sequences and tandem repeating elements. At least one of these, the TTC triplet occurring in the putative sugar transporter pseudo-gene, has been found to occur at variable copy numbers in different clinical isolates. To better understand the suitability of this and other VNTR markers in differentiating variant strains of M. leprae, we examined a battery of 12 M. leprae isolates derived from leprosy patients in different regions of the United States, Brazil, Mexico, and the Philippines, as well as from wild nine-banded armadillos and the Sooty Mangaby Monkey. The stability of the TTC VNTR was compared among the individual isolates as well as to those from bacilli obtained on subsequent passage in nude mice and armadillos. Copy numbers for the TTC repeat ranged from 10-15 among the isolates tested. No regional clustering was noted and all of the U.S. isolates showed a variable number of repeats. Strains derived from wild animals were not identical. Greatest variability in TTC was seen over long term passage with the Thai-53 strain, which has been maintained continuously in nude mice for many years. Thai-53 TTC copy number varied markedly over 8 passage intervals. However, the TTC VNTR genotype of most individual strains remained relatively constant for isolates passaged outside man for fewer than 12 generations. In addition, the TTC VNTR genotype of these strains tended to remain constant when passaged through an alternate animal host, the experimentally infected nine-banded armadillo. Even though the TTC VNTR occurs in a non-coding region of the M. leprae chromosome, its apparent stability among most short term passaged isolates suggests that it has utility for differentiating laboratory strains of M. leprae, and may be useful in assessing drift amongst isolates carried in long term culture.
OM&BM 8
EVALUATION OF TFN-α AND IE-10 SINGLE NUCLEOTIDE POLYMORPHISM (SNPS) AMONG HIV/M. leprae CO-INFECTED AND LEPROSY PATIENTS
Galhardo, M.C.C.; Vanderborght, P.R.; Nery, J.A.C.; Silva-Filho, V.F.; Sarno, E.N.; Sampaio. E.P.; Santos, A.R.
Oswaldo Cruz Foundation, Evandro Chagas Hospital, Leprosy Sector. Av. Brasil, 4365 Manguinhos Rio de Janeiro - Brasil CEP: 21045-900
The effective immune response against pathogens depends on an interaction of different cells and molecules from which pro and anti-inflammatory cytokines like TFN-α and IL-10 have a fundamental role. Thus, up or down regulation of these genes can influence clinical manifestations and outcome of several diseases including aids and leprosy. Recently, several SNPs have been described in cytokine genes and associated with gene expression and a number of diseases. Although the mutant -308A TFN-α allele have been associated with protection in leprosy, no polimorphic TNF alleles was associated to outcome in HIV infection. The aim of this study was to evaluate the possible association of promoter SNPs on TFN-α (-238, -308) and IL-10 (-819, -1082) positions with the outcome in HIV/ M. leprae co-infected compared with leprosy patients. Twenty one co-infected patients classified as multi (10) and paucibacillary (12) MB/PB leprosy were evaluated besides a group of 300 leprosy patients (210 MB and 90 PB). The results indicated that for the TFN-α polymorphisms the frequency of -238A was higher in the co-infected group compared with leprosy patients (p=0,04) corroborating with previous studies in which this allele was associated with the more severe MB forms of leprosy. For the IL-10 polymorphisms only the -819T allele showed an increased frequency in co-infected patients (p = 0,01). Frequencies of-308 and -1082 did not show difference between groups. However, horizontal analysis of the co-infected group shows the higher frequency of -1082A (related to the down regulation of IL-10 gene) linked with a low frequency of -308A (related to the up regulation of TFN-α gene) suggesting a combination of genetic factors probably associated with susceptibility for the co-infection HIV/M. lepraeng laboratory strains of M. leprae, and may be useful in assessing drift amongst isolates carried in long term culture.
OM&BM 9
FURTHER STUDIES ON M. leprae - PERIPHERAL NERVE PROTEIN INTERACTION AND THE ROLE OF THE 25 kDa GLYCOPROTEIN MYELIN P0.
Lavanya M Suneetha; Venkat Rami Reddy; Meher Vani; Deena Vardhini; David Scollard1; Jaun Archelos2; Sujai Sineedia
LEPRA India - Blue Peter Research Centre, Cherlapally, Hyderabad - 501301.
1. National Hansen's Disease Centre. Baton Rouge, USA.
2. Department of Neurology. University of Graz, Austria.
The invasion of Schwann cells and axons by M.leprae results in demyelination and axonal degeneration leading to motor, sensory and autonomic nerve damage and disfigurement which is the hallmark of leprosy. Other workers shown that tissue proteins such as fibronectin, β integrin, laminin-2 and αdystroglycan are involved in M. leprae - target tissue binding. Our earlier biochemical studies have revealed that a 25 kDa glycoprotein of the peripheral nerve has an affinity for M. leprae and is involved in binding. This glycoprotein is a major phosphorylated protein of the human peripheral nerve. Its molecular weight, carbohydrate content and phosphorylatable nature are similar to myelin P0.
The present study is an immunological confirmation that this protein is the myelin P0. The 25 kDa phosphorylated protein was confirmed as myelin P by the following experiments - dot blot assays, immuno-precipitation, western blot and by immuno-hislo-chemistry using monoclonal antibodies to P0 and the HNK-I epitope.
Since myelin P0 is a peripheral nerve specific protein, it could be one of the key target molecule for M. leprae binding/internalisation and may also explain the neural prediliction of M. leprae
OM&BM 10
GENE EXPRESSION IN Mycobacterium leprae
Diana L. Williams1; Richard Truman2; Thomas Gillis1
1. Molecular Biology Research.
2. Microbiology Depts, Laboratory Research Branch, National Hansen's Disease Programs, at LSU-SVM, Skip Bertman Dr, Baton Rouge, LA, USA
The genome of M. leprae has been completely sequenced and annotated. 1604 open reading frames and 1104 pseudogenes have been identified, however, the minimum gene set required for growth and survival (transcriptome) has not been defined. We have developed a protocol for M. leprae RNA purification and obtained RNA from two strains of M. leprae (T-53 and 4089). The expression of approximately 5% of the potential transcriptome was analyzed using RT-PCR. cDNA was produced using 1ug of RNA from each strain, random hexamers and reverse-transcription (RT). Gene transcripts were amplified from cDNA using PCR with primer sets flanking several potentially functional families. The cDNA from both strains was amplified and results demonstrated that genes encoding several enzymes including those involved with, folic acid synthesis, iron utilization, co-factor biosynthesis, gluconeogenesis, degradation of phosphorous compounds, degradation of DNA, detoxification, synthesis of mycolic acids, modification and maturation of ribosomes, synthesis of RNA, glycolysis, glyoxylate bypass, and genes containing secretion motifs or encoding stress proteins, and several genes with unknown functions were transcribed in both strains. These data have provided the first insight into the transcriptome of M. leprae. However, not all genes were expressed in both strains. Comparative analysis of gene expression theses strains will be discussed in greater detail. It is anticipated that this analysis along with cDNA array analysis will help to identify a larger set of functional genes in M. leprae which will potentially help us to understand the minimal requirements for growth and replication of this pathogen. This information may lead to the identification of new drug targets, skin test antigens and to identify factors that allow this pathogen to evade the immune system and destroy peripheral nerves.
OM&BM 11
GENETIC SUSCEPTIBILITY TO ERYTHEMA NODOSUM LEPROSUM (ENL) IN LEPROSY
Murdo Macdonald; Niraj Shrestha; Ruby Sidiqui; Paul Roche; Gilla Kaplan
Mycobacterial Research Laboratory. Anandaban Leprosy Hospital. PO Box 151. Kathmandu, NEPAL. Email: anadaban@mail.com.np
Erythema nodosum leprosum (ENL) is a distressing complication, experienced by up to 40% of lepromatous leprosy patients, which is characterized by severe systemic symptoms, including fever, painful cutaneous lesions, and neuritis, which often result in permanent nerve damage. The determinants and mechanisms underlying the onset of reactional states, progressive nerve damage and the regulation of immunity in these patients are not well understood.
Aim: To investigate the role of genetic factors in leprosy patients in their propensity for developing ENL.
Methods: We have recruited over 950 Nepali individuals, including both leprosy patients and their first-degree relatives. DNA was obtained from blood samples taken from each of these participants, and the SSO technique used to estimate the prevalence of polymorphisms in a number of candidate genetic loci: specifically, HLA-DR, TNE-á and Vitamin D receptor genes.
Results: Our results indicate that while the genetic loci under investigation may play a role in a patients' susceptibility to ENL, other factors may also have an effect. We will present data with regard to our analyses of the incidences of polymorphisms at these loci in each of the groups studied.
Conclusions: We have applied a rapid technique to determine the prevalence of specific genetic polymorphisms among leprosy patients and their first-degree relatives. In addition, the establishment of a large databank of DNA from patients susceptible to KNL will be an important resource for future studies.
OM&BM 12
GENOMIC DIVERSITY IN RPOT GENE OE Mycobacterium leprae AND GEOGRAPHIC DISTRIBUTION IN LATIN AMERICA
Masanori Matsuoka1; Yoshiko Kashiwabara1; Pedro Legua2; Carlos Wiens3; Mary Fafutis4
1. Leprosy Research Center, National Institute of Infectious Diseases, Tokyo, Japan.
2. University Peruana Cayetano Heradia, Lima, Peru
3. Hospital Mennonita Asuncion. Paraguay.
4. Universidad de Gaudalajara. Jalisco. Mexico
In the study to establish the genotyping of Mycobacterium leprae, two genotypes of the rpoT gene were detected among isolates. Some of them showed the rpoT gene with 4 copies of 6 base tandem repeats and other isolates harbored 3 copies of 6 base tandem repeats in the gene. Most striking finding was the apparent dominant distribution of the 6bp 4 tandem repeat genotype of M. leprae in the main island of Japan and Korea. In contrast, almost all isolates from other regions in the world revealed 3-copy type. It is clear 6bp 4 tandem repeat genotype spread in Japan in concordance with the migration of the people from Korea to Japan. Biased distribution of each genotype in the world led us to imagine the spread of the leprosy concordant with the migration of Mongoloids to Latin American countries as revealed for other microorganisms. Geographic distribution of different rpoT genotypes of M. leprae isolated in Paraguay. Peru and Mexico was investigated in connection with human prehistoric migration. All M. leprae genotype of rpoT gene isolated in Paraguay and Peru showed three tandem repeats of 6bp. On the contrary, isolates from Mexico showed the 6bp 4 tandem repeat genotype. It seems that the M. leprae distributed in Mexico was carried by the movement of Mongoloid but the bacilli in two South American countries is originated in another source.
OM&BM 13
Mycobacterium leprae DNA DETECTION BY POLYMERASE CHAIN REACTION FOR EARLY LEPROSY
Sousa. A.L.O.M.; Castro, F.; Silva, M.H.; Rebello, P.F.B.; Gomes, M.K.; Nararashi, K.; Sacchetim, S.C.; Visconde, A.M.; Costa, M.B.; Martelli, C.M.T.; Stefani, M.M.A.; Gillis.T.P.
IPTSP/UFG, Rua Delenda Rezende s/n Setor Universitário. CEP 74605-050 Brazil; National Hansen's Disease Programs/USA
Objective: To detect M. leprae DNA by PCR in skin biopsies among Brazilian single skin lesion paucibacillary leprosy patients (SSL-PB) prior to one dose ROM therapy.
Methods: 259 newly detected SSL-PB leprosy patients, negative baciloscopy, were recruited in 3 endemic regions from 97/98 and followed-up for 3 years. Before drug intake, 4 mm punch skin biopsies were collected for conventional histopathology. In a subgroup of 134 patients, half of the skin biopsy was snap-frozen and stored (liquid nitrogen) for M. leprae DNA detection by PCR. After DNA extraction (phenol/chloroform/ isoamyl alcohol) each specimen was amplified, undiluted and at 1:5, using pairs of primers for a 360 bp M. leprae specific fragment. Products were detected by slot blot hybridization using digoxygenin-labeled 212bp DNA probe. Specimens were tested blinded to patient's characteristics at IPTSP/Brazil in partnership with National Hansen's Disease Programs.
Results: 43.3% (95%CI 34.8-52.1) of M. leprae DNA positivity was detected among SSL-PB, representing an increase of 37.3% (50/134) bacilli detection when compared to the rare bacilli found in histopathology readings (12/134). There was an increased positivity trend with age (p<0.01). Patients with skin lesion on the face, Mitsuda negative (<5mm), anti PGLI negative were independently associated with positivity.
Conclusion: M. leprae DNA by PCR was a valuable tool for diagnosis confirmation among early paucibacillary leprosy patients and to explore prediction factors of disease progression.
TDK/WHO grant 98100
OM&BM 14
NASAL PRESENCE OF Mycobacterium leprae AND MUCOSAL IMMUNITY IN HOUSEHOLD CONTACTS OF LEPROSY PATIENTS
Mrs. S. P. Madhalc; Dr. R.S. Jadhav; Miss A. Fernando; Miss V.S. Shinde; Ravindra R Ramble; Dr. V. K. Edward; Dr. J.R. Rao; Prof. W.C.S. Smith on behalf of MILEP-2 Study Group*
Stanley Browne Research Laboratories. Richardson Leprosy Hospital, Miraj. Maharashtra-416410. Tel. No Off: 0233-211213 Fax: 0233-211708 E-mail: sblabtlm@vsnl.com
Transmission of leprosy in the household contacts (HC), as reflected in new case detection rate does not appear to be affected significantly in the post-MDT era. Incidence rates have been reported 8-10 times higher in the HC than the general population. Major route of transmission of M. leprae is thought to be mainly through the respiratory system with nose as the site of initial infection. The aim of the study was to see the mucosal immunity and exposure to M. leprae in HCs of patients and non-contacts (NC) to understand transmission. The principal methods employed for this were the polymerase chain reaction (PGR) to detect small quantities of M. leprae DNA and measurement of mucosal immunity by ELISA. 201 subjects out of 3035 were identified as HCs. Saliva samples and nasal swab were collected from subjects to carry out this study. Overall analysis of all the samples shows that the percentage of PCR posi-tivity is almost same in HC (2.3%) and NC (2.5%). Similarly in both groups 68% of the subjects show mucosal immune response. Both the groups show similar pattern of exposure to M. leprae with PCR positivity peak seen in monsoon. Amongst the household contacts, females show higher PCR positivity (3%) than males (1.5%). The difference in the PCR positivity in noncontacts in males (2.2%) and females (2.8%) is relatively small. Exposure to M. leprae is likely to be followed by immunity in most individuals, which is consistent with wide spread transmission of M. leprae producing transient nasal carriage and the development of a mucosal immune response, which may be protective.
OM&BM 15
PATTERN & SIGNIFICANCE OF PARASITAZATION OF ENDOTHELIAL CELLS IN LEPROSY: MORPHOLOGICAL & INVITRO STUDIES.
Sujai Suneetha; Lavanya Suneetha; David Scollard1
LEPRA India - Blue Peter Research Centre, Cherla-pally. Hyderabad-501 301. India
1. National Hansen's Disease Centre. Baton Rouge, USA.
Previous studies have implicated the role of the endothelial cell in the dissemination of leprosy. In this paper we present a detailed morphological study of 17 skin biopsies (4 BT, 3 BL & 10 LL) in which acid fast bacilli were found in the endothelial cells and relate it to other morphological features in the biopsies.
Among the 17 biopsies in whom bacilli were present in the endothelial cells: bacilli were also prevent in the nerves in 13 biopsies, in the macrophage in 16; smooth muscle in 10 and in the sub epidermal zone in 2 biopsies. Bacilli were present also in the walls of the blood vessels in 5 biopsies and in the lumen in 1 biopsy. Interestingly there was 1 biopsy in a BT patient in which bacilli were present only in the endothelial cells and absent elsewhere in the section.
In vitro studies on M. leprae-endothelial cell interaction were carried out using immortalized endothelial cell lines. The short term cultured endothelial cells were isolated and phosphorylated with gamma P32 ATP. M. leprae binding studies were carried out on nitrocellulose blot. Preliminary experiments suggest that there is a phosphorylated glycoprotein receptor (55 kDa) on the endothelial cells that interacts and binds to M. leprae.
These morphological and in vitro studies suggest that M. leprae has an affinity for endothelial cells which it parasitizes. The organism is then probably released into the blood stream resulting in its dissemination to distant sites of prediliction in the body
OM&BM 16
PERSISTERS IN LEPROSY AFTER MULTIDRUG TREATMENT IN MB PATIENTS
U.D.Gupta; K. Katoch; H.B. Singh; M. Natrajan; VM. Katoch
Central JALMA Institute for Leprosy (ICMR), Taj-ganj, Agra, India
With the Multi-Drug Treatment (MDT) of leprosy, the results have been satisfactory all over the world. However, the presence of drug sensitive viable organisms is well recognized in MB leprosy. These persisting bacilli have special significance clue to their relapse potential. This study has been initiated to gain an overview of this problem and follow the trends in multibacillary cases treated with MDT. In this study, biopsies for Mouse Foot Pad (MFP) have been obtained from MB patients treated with (i) standard MDT +Minocycline + Ofloxacin for 12 months, (ii). Standard MB MDT after 12,24 and 36 months Bacilli harvested from the biopsies were inoculated in to mouse foot pad and estimation of bacillary ATP levels by bioluminescence assay as per established methods. Available results indicate that despite reduction in viability after MDT, viable persisters are detected even beyond one and 2 years of treatment. There has not been much change in the trends over the last 5-10 years. It would be important to carry out such surveillance in larger number of MB cases to know the trends and the resultant relapses.
OM&BM 17
PROTECTION OF MICK AGAINST Mycobacterium leprae INFECTION BY A DNA VACCINE ENCODING M. lepraE ANTIGEN 85A AND MUTANT MURINE IL-12
M. Ngamying; R Sawanpanyalert; J. Nikasri; R. Butraporn; S-N. Cho; P.J. Brennan; L. Levy
National Institute of Health. Department of Medical Science. Ministry of Public Health. Nonthabnri. Thailand.
Female BALB/c were administered one of three DNA vaccines: M. leprae DNA: Ag85A; DNA: AgSSA + wild-type murine DNA: IL-12w; and DNA: Ag85A + mutant murine DNA: IL-I2m. Expression of Ag85A by the preparation of DNA: AgS5A had been confirmed by specific stimulation of I FN by murine spleen cells before it was employed in this experiment. Control mice were administered saline or the empty vector; live BCG served as a positive control. The mice were injected into the posterior tibial muscles with 200 g/dose/mouse of one of the preparations on four occasions four weeks apart, except for BCG, only two doses of which were injected. Four weeks after the last dose, the mice were challenged with 5000 M. leprae into a hind foot pad, and the organisms were harvested approximately live months later. The results of the harvests are summarized in the table. As shown by the control group, the results of the harvests demonstrate that the inoculum employed included only a small proportion of viable organisms. BCG appears to have conferred modest protection. Only the mixture of the DNAs encoding Ag85A and IL-I2m conferred protection, whereas the mixture of the DNAs encoding Ag85A and IL-12w appears to have enhanced the infection.
OM&BM 18
SCHWANN CELL GENE EXPRESSION PROFILE IS MODULATED BY Mycobacterium leprae
Tempone, A.J.1; Silva, T.P.1; Rossle, S.2; Lopes, U.G.2; Brennan, P.J.3; Sarno, E.N.; Pessolani, M.C.V.1
1. Instituto Oswaldo Cruz - Laboratorio de Hanseniase - Fiocruz.
2. Instituto de Biofisica Carlos Chagas Filho - UFRJ.
3. Dept. Microbiology Colorado State University Fort Collins. CO.
The primary effects of Mycobacterium leprae invasion on the physiology and metabolism of Schwann cells, and to whar extent these effects might be related to the progressive, irreversible degenerative nerve damage observed in leprosy, are poorly understood. In this study, we have applied differential display PGR and DNA microarray techniques to identify genes selectively expressed or repressed in Schwann cells in response to M. leprae infection. Schwann cell lineage ST-8814 was cultured and incubated with M. leprae isolated from armadillo and from human biopsies between 1 and 24 hours. Complementary DNA synthesized from RNA isolated from these cultures was used lor differential display RT-PCR reactions and hybridizations against oligonucleotide chips. Currently bands identified in polyacrilamide gel electrophoresis as differentially expressed have been cloned and sequenced for subsequent northern blot and real time PCR confirmation. Images of microarray hybridizations have been acquired using the Gen Pix software. The cluster analysis has been performed using the Tree View software. Preliminary results indicate that M. leprae is able to alter the gene expression profile of in vitro cultured Schwann cell.
NIH, WHO/TDR. sponsored this work.
OM&BM 19
SIMPLIFIED REVERSE TRANSCRIPTION POLYMERASE CHAIN REACTION FOR DETECTION OF Mycobacterium leprae IN SKIN SPECIMENS
B. Phetsuksiri1; J. Rudeeaneksin1; P. Supapkul1; S. Wachapong1; K. Mahotarn1; P.J. Brennan2
1. Sasakawa Research Building, Rajprachasamasai Institute, Leprosy Division, Department of Communicable Disease Control, Ministry of Public Health, Nonthaburi. Thailand.
2. Colorado State University, Fort Collins. Colorado, USA.
Diagnosis of leprosy based on detection of Mycobacterium leprae RNA remains a complicated process. To simplify the detection procedure, a one-step RNA extraction and reverse transcription polymerase chain reaction (RT-PCR) was established and evaluated for its potential in rapid detection of leprosy patients. The assay relies on the extraction of M. leprae RNA, and single-tube reactions of reverse transcription, followed by PCR amplification. Using M. leprae-specific primers targeting 171-bp fragment of the M. leprae 16s rRNA gene, the RT-PCR designed for convenience, and reproducibility resulted in detectable M. leprae in both slit skin smears and skin biopsies. The assay was specific for M. leprae in comparison with results obtained from Mycobacterium tuberculosis and Mycobacterium smegmatis. The use of digoxigenin-label DNA enhanced the positive signal of the amplified RT-PCR product. The method could detect less than 10 CPU of mycobacteria in analyzed samples indicating the sensitivity of the test. In the initial application, diagnostic results were obtained from 24 leprosy patients. Of these, 20 were multibacillary (MB) and 17/20 patients were positive for 16s rRNA of M. leprae in skin specimens. The assay particularly useful since slit skin smears negative in staining for acid fast bacilli were positive by RT-PCR. The method has also been evaluated for its potential to help monitor bacterial clearance in leprosy patients during chemotherapeutic treatment. We propose that this form of RT-PCR gives values in term of its simplicity and sensitivity to identify M. leprae in skin specimens especially when acid-fast bacilli are not discernable. The usefulness of RT-PCR in detection of viable leprosy bacilli needs to be extensively explored.
OM&BM 20
SINGLE NUCLEOTIDE POLYMORPHISMS (SNPS) OF TFN-α AND IL-10 GENES AND SUSCEPTIBILITY TO LEPROSY AMONG HOUSEHOLD CONTACTS.
Santos. A. R.; Moraes, M. O.; Vanderborght, P. R.; Matos, H.J.; Silva-Filho, V. F.; Vasconcellos, S. E. G.; Maniero, V.C.; Sampaio, E.P.; Sarno, E.N.
Oswaldo Cruz Foundation, Leprosy Sector. Av. Brasil, 4365 Manguinhos Rio de Janeiro - Brasil cep: 21045-900.
The interindividual variations in the host response to a certain pathogen are one of the most important variables for the determination of susceptibility and severity of the disease, which is the result of environmental effects against the background of genetic factors. Thus, identification of such factors, which are somehow associated to a higher or lower susceptibility, is of fundamental importance for the prediction of development or establishment of the disease. The aim of this study was to evaluate the possible association of the SNPs at positions -238 and -308 of the TFN-α and -819, -1082 and -2849 of the IL-10 genes among household contacts of leprosy patients.
Two hundred and sixty seven household contacts were enrolled in this study from which 67 became patients and 200 remained as healthy contacts. The results showed no statistic difference on the distribution of carriers and non-carriers of the -238a allele among sick and healthy contacts. For the -308 position, the number of carriers was significantly higher among sick in comparison to healthy contacts (p < 0,01). Moreover, when analyzed through the clinical spectrum of leprosy, all the -238A carriers developed multibacillary (MB) forms of the disease whereas 73,3% of the -308A carriers developed the paucibacillary (PB) forms. Regarding the IL-10 SNPs, the allelic frequency of the -819T was significantly higher in the healthy and -1082A in sick contacts (p <0,01 for both). Analysis according to the clinical forms revealed an increased frequency of the -819T carriers in the PB forms when compared to the MB (p < 0,01).
The present data suggest that SNPs of cytokine genes could be used to screen contacts of leprosy patients as a prognostic marker of diseases susceptibility and severity.
OM&BM 21
SITE OF ENTRY: AN IMPORTANT FACTOR IN THE GROWTH AND DISSEMINATION OF M. leprae IN MICE.
Gigi J Ebenezer; Sheela Daniel; Shantha Arumugam; Charles K. Job
Schieffelin Leprosy Research and Training Center, Karigiri. Vellore District, Taniilnadu. India - 632106.
Sixty eight thymectimized and irradiated mice were randomized and 36 inoculated intra-dermally in the flank and 36 in the footpad. In each of these two groups four different concentrations of M. leprae inoculation were used namely 107 in 0.1 ml, l06 in 0.1 ml, 105 in 0.1 ml and 104 in 0.1 ml. Mice were sacrificed at the 6th, 8th, 12th and 15th month and growth of M. leprae at the site of inoculation was estimated. Internal organs were subjected to histo-pathological examination.
The 104 in 0.1 ml inoculum did not promote growth in mice injected in the Hank but growth was seen in all mice that were inoculated in the foot-pad. In all other groups there was growth of M. leprae but it was quantitatively more in the foot-pad inoculated animals than in the flank inoculated ones. Further, growth in the foot-pad inoculated mice was associated with disseminated of M. leprae to the internal organs while such dissemination was not seen in flank injected mice.
We conclude that in mice, entry of M. leprae through a relatively cooler entry point (foot pad) allows better growth of M. leprae locally, needs smaller doses of inoculum to promote growth and allows dissemination of the organism to the internal organs. The site of entry of M. leprae and the dose may have a role in determining whether the person will be infected or not.
OM&BM 22
STUDIES ON NASAL TRANSMISSION BY M. leprae SPECIFIC GENE AMPLIFICATION
H.B.Singh; V.M. Katoch; M. Natrajan; K. Katoch; Raj Kamal; V.D. Sharma; D.S. Chauhan; R. Das; K. Srivastava; P. Gupta
Central JALMA Institute lor Leprosy (ICMR). Taj Ganj, Agra. India
Nose has been considered as an important portal of exit and entry in leprosy. Due to continued high incidence rates in leprosy, there is a great need to understand the sources and spread of M. leprae. This study has been carried out to study the nasal positivity on in leprosy cases by using M. leprae specific PCR. Nasal scrapings were collected from leprosy cases across the spectrum. These were from untreated as well patients treated with standard MDT for varying duration. These scrapings were suspended in IE buffer, decontaminated and DNA was extracted by a physiochemical procedure already established at the laboratory. Gene amplification was carried out by using a system targeting 36 kD gene (Hartskeerl et al 1989). Amplicons were analysed by gel electrophoresis and southern blot hybridization. PGR positivity was been analysed in relation to type of disease and duration of treatment. Positive results were observed in a section of PB cases (classified according to current WHO criteria) and most of MB cases. This positivity persisted for varying periods after treatment. The relevance of these findings will be discussed keeping in view the potential application of this approach in studying the transmission of leprosy.
OM&BM 23
STUDIES ON STRAIN VARIATION BY M. leprae USING TTC REPEATS
V. M. Katoch; Mallika Lavania; H.B. Singh; M. Natrajan; K. Katoch; Raj Kamal; V.D. Sharma; D.S. Chauhan; R. Das; K. Srivastava; P. Gupta
Central JALMA Institute for Leprosy (ICMR). Taj Ganj, Agra. India
There is a great need to develop molecular markers for eliciting the strain variation among M. leprae for understanding the dynamics of transmission of leprosy. This study has been carried out to study the strain variation in leprosy cases by using TTC repeats as markers. Biopsies were collected from leprosy cases across the spectrum. These biopsies were homogenized and DNA was extracted by a physiochemical procedure already established at the laboratory. TTC regions were amplified by using the primers and procedure described by Shin et. al. (2000). Amplicons were analysed by gel eletrophoresis. The polymorphism observed in the size of amplicons has been analysed in relation to geographical distribution, type of disease and possible sources. The relevance of these findings will be discussed in context of potential application in the molecular epidemiology of leprosy. Such techniques become very important due to persistent high incidence rates seen in our populations.
OM&BM 24
SUBCLINICAL TRANSMISSION OE M. leprae: OCCURENCE OE NASAL PGR POSITIVITY AND MUCOSAL IMMUNITY - ANALYSIS IN SCHOOLCHILDREN
Miss A.Fernando; Dr. R.S. Jadhav. Miss V.S. Shinde; Ravindra R. Kamble; Mrs. S.P. Madhale; Dr. J.R. Rao; Dr. V.K. Edward; Prof. W.C.S. Smith on behalf of MILEP-2 Study Group*
Stanley Browne Research Laboratories. Richardson Leprosy Hospital. Miraj, Maharashtra-416410. Tel. No Off: 0233-211213 Fax: 0233-211708 E-mail: sblabtlm@vsnl.com
Background: The transmission of leprosy is less well understood. Infection from sub-clinical sources could play an important role than from active clinically apparent cases. Most of the individuals in high leprosy endemic areas have immunological evidence to M. leprae. Thus the first exposure, probably exposure in childhood is important and also, is the related mucosal immune response to characterize the immune status of the individual.
Aim: To define the means by which M. leprae is transmitted and the development of immunity in school children in a population in which multidrug therapy had been used for more than 10 years.
Materials and Methods: Three villages in South Maharashtra, where leprosy is endemic were selected. These villages were comparable in size, socio-economic status and prevalence of leprosy. The principal methods employed in this study were the PCR to detect small quantities of M. leprae DNA, and measurement of mucosal immunity by assay of salivary IgA.
Results: 633 school children (267 of the total population) were analysed for the presence of M. leprae DNA and mucosal immunity. Analysis of the data show that the incidence of nasal PCR positivity (PCR+) in school children and rest of the population (ROP) is same (2.7%) whereas IgA positivity is 61% and 70% respectively. PCR+ percentages in school children and ROP in monsoons is 3% and 4% respectively as compared to 1.6% (school children) and 0.7% (ROP) in the summer months. In the group (5-9 years) the PCR+ percentage in household contacts is higher (7.7%) than 10-14 years group (1.3%). A significant difference in PCR+ percentage is observed in males (1.9%) and females (3.7%) in 5-9 years group.
Conclusion: Results suggest though there is no obvious differences in between the groups, the exposure and mucosal immunity to M. leprae is affected by seasons and shows marked variation in males and females
OM&BM 25
THE Mycobacterium leprae HLP PROTEIN: A PUTATIVE ADHESIN THAT BINDS MULTIPLE EXTRACELLULAR MATRIX COMPONENTS.
Lima. C.S.1,2,*; Marques, M.A.M.1,*; Sarno, E.N.2; Brennan, P.J.1; Pessolani, M.C.V.2
1. Dept. of Microbiology. Colorado State University, Fort Collins. CO. U.S.A.
2. Leprosy laboratory, Oswaldo Cruz Institute, FIOCRUZ, Rio de Janeiro, Brazil;
*These authors contributed equally to this work.
Recent reports have identified a 21 kDa histone-like protein (H1p) as a laminin-binding protein of the Mycobacterium leprae cell wall (Shimoji et al, Proc. Natl. Acad. Sci 96: 9857-9862. 1999; Marques et al. Microbes & Infection 2: 1407-1417. 2000). The C-terminal domain of H1p (also known as ML-LBP21) contains Ala/Lys-rich repeated motifs, which are also found in the heparin-binding hemaglutinin (HBHA), a major adhesin of M. tuberculosis. These repeated sequences constitute the heparin-binding site of HBHA, suggesting that M. leprae H1p might also interact with glycosaminoglycans (GAG). In this study, we have further characterized the interaction of H1p with laminin-2 and other extracellular matrix components. To map the functional binding sites of H1p, truncated recombinant fragments corresponding to the N-terminal (rH1p-N) and the C-terminal (rH1p-C) domains of the protein were produced by a PCR cloning strategy. The capacity of recombinant H1p and truncated proteins to interact with extracellular matrix components was investigated using a solid phase-based assay. In these assays, soluble laminin-l and -2 were able to bind in a dose-dependent manner to rH1p and rH1p-C, but not to rH1p-N, rH1p and rH1p-C were also able to bind heparin and collagen I, III and IV, but not fibronectin. These observations suggest that the Ala/Lys-rich sequences present in the C-terminal half of M. leprae H1p constitute the binding sites to extracellular matrix proteins. The capacity of H1p to interact with other extracellular matrix components expands the potential role of H1p as adhesin in mycobacterial pathogenesis. Currently, in vitro adherence assays are under way to evaluate the role of H1p, collagen and GAG in the interaction of M. leprae with Schwann cells and epithelial cells.
This work was upported by FAPERJ, WHO/TDR and NIAID, NIH.
OM&BM 26
THE USE OE POLYMERASE CHAIN REACTION (PCR) IN LEPROSY RESEARCH AND CONTROL
Linda Oskam; Evi Beukelaar; Julia Teerling
KIT Biomedical Research. Meibergdreef 39. 1105 AZ Amsterdam, The Netherlands
The PCR is a sensitive and specific technique, that allows the detection of minute amounts of DNA in a matter of hours. Since the development of the first PCR assay for the detection of Mycobacterium leprae more than 10 years ago. the technique has been used on a whole range of samples, varying from biopsy material and nose swabs from patients and contacts to dust samples from the environment.
The PCR has been used to investigate a variety of matters of clinical and epidemiological importance. We have now a better insight into the spread of leprosy in the society, because PCR made it possible to show that the presence of the bacterium on the nasal mucosa is widespread in the population. Also, PCR and another amplification technique, NASBA, have been used to monitor the presence of M. leprae DNA and RNA during and after treatment.
This presentation will give a critical overview of the possibilities, applications and achievements of molecular amplification techniques and the way in which they have influenced and will influence leprosy research and control.
OM&BM 27
THREE-COLOR 1MMUNOFLUORESCENT STAINING TO IDENTIFY M. leprae WITHIN ENDOTHELIUM OF HUMAN PERIPHERAL NERVE.
Shi, Ling; McCormick, G; Scollard, P.M.
Laboratory Research Branch, National Hansen's Disease Programs at LSU. Baton Rouge, LA. 70803. USA.
Studies in an animal model have suggested that M. leprae enter peripheral nerves by colonizing epineurial blood vessels and lymphatics, gaining access to the endoneurial compartment by passing through the vascular endothelium. To evaluate this possibility in human lesions, where excision and dissection of major nerve trunks is not possible, we have developed a method to assess endothelial involvement of cutaneous nerves in skin biopsies.
Archived, paraffin-embedded skin biopsies from HD patients were selected based on lepromatous classification (LL or BL) and presence of at least one large cutaneous nerve. Schwann cells were identified using rabbit anti-S-100, biotin-goat anti-rabbit, and strepta-vidin-Alexa-Flour-350; bacilli were identified using guinea pig anti-M. leprae and FITC-goat anti-guinea pig; endothelium was identified using rhodamine-Ulex earopaeus -1 (UFA-1). Examined under appropriate filters, this allowed positive identification of nerve (blue), M. leprae (green), and endothelium (red). Images were captured by digital photography and superimposed using Adobe Photoshop software.
Preliminary results from 5 biopsies indicate that the endothelium is infected in 29% of blood vessels associated with nerves, and 32% of blood vessels not associated with nerves. At this time, the sample is too small for differential analysis of infection of vessels at different levels of the dermis. The method appears to offer a sensitive means of positive identification of these and other structures that may be involved in vascular endothelial infection of nerves in HD.
OM&BM 28
UNIQUE METABOLIC PROPERTIES OF Mycobacterium leprae
K. Prahhakaran; E.B. Harris; B. Randhawa
OWL Hansen's Disease Center
5111 Hickory Ridge Boulevard, Baton Rouge. LA 70817. U.S.A.
The sequencing of the genomes of several microorganisms, including Mycobacterium leprae and Mycobacterium tuberculosis has been reported in recent years. M. tuberculosis contains a full complement of genes needed for survival and independent growth. On the other hand, M. leprae is deficient in genes coding for many biosynthetic enzymes, that makes the organism incapable of independent growth and survival, contradicting the claim that M. leprae is a competent bacterium. The finding explains the obligate intracellular parasitism of the organism and failure of attempts for over a century to culture the bacterium in chemically defined media. In addition, M. leprae was found to possess unique genes, not found in M. tuberculosis. These genes code for enzymes characteristic of the Hansen bacterium. We have discovered a unique enzyme activity, o-diphenoloxidase, in M. leprae. The enzyme is not present in M. tuberculosis or any other mycobacteria, including M. lepraemurium recovered from infected mouse tissues. It acts on phenolic substrates like 3.4-dihydroxyphenylalanine and related compounds, converting them to quinones. No rational explanation has been available for the unusual affinity of M. leprae for the Schwann cells of peripheral nerves, and for the hypopigmentation of skin lesions. Both Schwann cells, and melanocytes of the skin contain tyrosine hydroxylase that generates 3,4-dihydroxyphenylalanine (dopa), metabolized by the bacteria. Tyrosine hydroxylase occurs in the adrenal medulla that synthesizes dopa, epinephrine and norepinephrine from tyrosine. We found that adrenal medulla is a preferred site for early multiplication of M. leprae. In tuberculoid HD (Hansen's Disease) where the bacteria are restricted to specific areas of the skin, there is hypo-pigmentation of skin lesions. Melanocytes continually generate trace amounts of dopa, which is converted to melanin pigment. M. leprae diverts the substrate for its own metabolism, which prevents pigment formation. In melanocyte cultures, granules of melanin can be observed. When we added live M. leprae to such cultures, pigment production was suppressed. In lepromatous condition where the bacteria are distributed diffusely, only hypo-pigmented mottling results. The quinones generated by the bacteria can undergo reversible oxidation-reduction, helping in the utilization of other metabolites by the bacilli. Mycobacteria in general, can synthesize their own ATP. M. leprae, on the other hand, possesses a mechanism for the active transport of ATP from the surrounding milieu. β-Lactamase is a constitutive enzyme in mycobacteria, including M. tuberculosis. But M. leprae unexposed to β-lactam antibiotics showed no β-lactamase; bacteria recovered from experimentally infected armadillos treated with Bicillin (penicillin G benzathine), to control secondary infections, contained active β-lactamase. The enzyme activity persisted when these bacteria were used as inoula to infect other armadillos, which received no bicillin treatment subsequently. Once the enzyme is induced, it is not lost when the inducing agent is withdrawn: the phenomenon is referred to as derepression. A potent β-lactam-β-lactamase inhibitor combination. UNASYN, was bactericidal to M. leprae and M. tuberculosis, even resistant to other drugs. The compound could serve as an effective alternative drug for treating HD patients.
OM&BM 29
USE OF PCR IN THE RAPID DIAGNOSIS OF RI-FAMPICIN RESISTANCE IN LEPROSY
Murdo Maedonald; Niraj Shrestha; Andrea Thomas; Paul Roche; Nadine Honore; Stewart Cole.
Mycobacterial Research Laboratory. Anandaban Leprosy Hospital, PO Box 151. Kathmandu, NEPAL. E-mail: anandaban@mail.com.np
As rifampicin is the major bactericidal drug used in MDT therapy of leprosy, it is essential that resistance trends be monitored. The established method of assessing drug resistance, using culture in the mouse footpad, has recently been augmented by the development of a rapid PCR detection method.
Aim: To test for defined mutations in the M. leprae RNA polymerase a chain gene (rpoB), and to correlate these with drug resistance in the mouse footpad system.
Methods: A novel PCR based technique was used to examine bacteria obtained from skin biopsies from MDT defaulters or non-responders, and from samples which had previously been passaged in the mouse footpad. M. leprae DNA was extracted from these and a set of oligonucleotide probes immobilized on a nylon membrane used to probe for mutations associated with rifampicin resistance. The test combined positive and negative controls and used chemiluminesence for detection.
Results: A number of samples were found to have the rifampicin resistant genotype in the PCR assay. We will present data on all of these M. leprae strains genotyped for rifampicin resistance and tested at full (10mg/kg) and half (5mg/kg) doses in mouse footpad cultures.
Conclusions: While the rapidity of PCR based methods is a major advantage over MFP, the validation of genotype methods of detecting drug resistance in leprosy is critical for their wider use in monitoring this important problem
OM&BM 30
VIABILITY OF M. leprae IN LEPROMATOUS PATIENTS AFTER COMPLETION OF 12 MONTHS OF MULTI-DRUG THERAPY.
Gigi J Ebenezer; Thomson Sugumaran; Sheela Daniel; Geetha S. Rao; S. Arunthathi; P.S.S. Sunder Rao; Charles K. Job
Schieffelin Leprosy Research and Training Center, Karigiri. Vellore District, Tamil Nadu, lndia-632106
The Seventh WHO expert committee had recommended shortening the duration of multi-drug therapy (MDT) to 12 months from 24 months for multi-bacillary (MB) patients. We carried out a study to determine whether viable bacilli can persist in the body of treated MB patients after 12 months of MDT. 34 untreated lepromatous patients who had an initial average bacterial index (BI) of 3+ or more were enrolled in the study. At the end of 12 months of MDT, skin biopsies were obtained from a site, which displayed the maximum number of bacilli on skin smear examination. An M.leprae concentrate was prepared from each of the biopsies and inoculated into the footpads of five thymectomized and irradiated (T900r) mice. The preparation of innoculum, method of inoculation, harvesting and counting of M.leprae from the footpad tissue was done using the method described by Rees. Harvesting was done at 6th, 9th and 12th month. Skin histopathological examination was also done on 32 patients on completion of 12 doses of MDT. In nine (26%) out the 34 biopsies M.leprae continue to exist in the footpads of T900r mice. These nine patients had an initial average BI of 4+ or more at the time of starting MDT. Histopathologically, resolving granulomatous lesions were found only in eleven (34%) of the 32 skin biopsies at 12 months. Skin smears at the completion of 12 months of MDT showed a fall of one log BI or more in only 18 (56%) patients. This study demonstrates that at the completion of 12 doses of MDT a considerable proportion of MB patients with initially high average BI, harbor bacilli. It is possible that these are dead bacilli, not yet absorbed by the tissue. Long-term follow up of these patients will reveal whether these bacilli are alive or not. It may be necessary to maintain these mice for longer periods to study the behavior of persisting bacilli.
OPERATIONAL ASPECTS OF ELIMINATION
OOAl
ACTIVITIES OF THE TASK FORCE IN THE ACCELERATION OF THE ELIMINATION OF LEPROSY IN BRAZIL
Vera Andrade1; Tadiana Maria Alves Moreira2; Gerson Fernando Mendes Pereira3; Marcos Virmond;4 Artur Custodio de Souza5
1. WHO.
2. Secretary of Health of Rio de Janeiro State.
3. Ministry of Health.
4. Institute Lauro de Souza Lima Gil Soares - PAHO.
5. Movement for the reintegration of leprosy affected persons (MORHAN).
The strategy to encourage municipal health secretaries to be committed to the elimination of leprosy, by increasing coverage of MDT services, is a conjoint initiative of the National Council of Municipal Health Secretaries (CONASEMS) and WHO with support from the Technical Area of Sanitary Dermatology of the Ministry of Health, MORAHN and PAHO. To establish such strategy CONASEMS has created in 1998 the Task Force for Accelerating the Elimination of Leprosy (GT/HANSEN/ CONASEMS), which aim is to identify practical solutions at the local level within the available structure and resources of the basic heath system. At the methodological level it is stressed the need to strengthen the participation of various social and institutional partners, involving mainly the municipal managers and the community. At the political level, after including the issue of elimination in the agenda of local managers, it was created adequate condition to increase the coverage for diagnosis and treatment of leprosy with the additional outcome of a political profit to the local manger due to the success of eliminating leprosy from his municipality. In august 2001 the project has covered 52% of the municipalities through the country (2898 municipalities in 14 states), out of them 38% are priority municipalities for the MoH, mainly in the north and northeast region. In Tocantins. Piaui and Rio de Janeiro the process of decentralization is in its stage of consolidation. To support the deconcentration of diagnose and treatment the following material has been distributed: 25.000 booklets, 25.000 posters on signs and symptoms, 2 million leaflets on signs and symptoms in simple language to the community, leaders of the Children's Pastoral from 3379 municipalities and their families and educational videos for 5600 dioceses. It was sent to all municipal managers (5559), trough CONASEMS, technical information, a video with two vignettes and the film produced by the Global Alliance (WHO). Nowadays, it is difficult to identify in Brazil a municipal health secretary that is not aware of the strategy for elimination of leprosy. No doubt, this Strategy, by its content and quality, represents an innovative and effective contribution towards elimination of leprosy and. in addition, citizenship.
Financial support was provided by the Brazilian Ministry of Health. CONASEMS, WHO and Novarlas Foundation for Sustainable Development.
OOA 2
ANALYSIS ON THE DETECTION OF NEW LEPROSY CASES BEFORE. DURING AND AFTER THE YEAR OF LEPROSY ELIMINATION CAMPAIGNS
Shen Jianping; Li Wenzhong; Yu Meiwen; Yang Jun; Zhou Longchao; Wang Rongmao; Hu Lufang; Mou Hongjiang; Ye Fuchang; He Xinguo; Pan Liangde
In order to analyze the impact on the situation of ease finding after Leprosy Elimination Campaigns, the data of newly detected leprosy cases in the leprosy high endemic area have been collected before, during and after the year of carrying out Leprosy Elimination Campaigns. The result showed that the number of new leprosy cases detected during the year of leprosy elimination campaigns was significantly high. The number of newly detected cases after the year of Leprosy Elimination Campaigns was similar to that of detected before the year of carrying out Leprosy Elimination Campaigns in counties with persisting case finding activities. But the number of newly detected cases after the year of Leprosy Elimination Campaigns significantly decreased in counties without active ease finding activities. The average distance from the house of leprosy cases detected during Leprosy Elimination Campaigns to the leprosy control unit at the count town is 62.8 kilometer which is farther more than that of other leprosy cases detected before and after the year of Leprosy Elimination Campaigns. The average disease delay-time of leprosy cases detected after the year of LEC shortened. The results also showed that carrying out Leprosy Elimination Campaigns will have no the significant impact on the trend of cases finding within a short time in local areas. But it may improve some indicators of leprosy patients and so promote the leprosy control in local areas.
OOA 3
AS AÇÕES DO PROGRAMA DE ELIMINAÇÃO DA HANSENÍASE NO MUNICÍPIO DE FERNANDÓPOLIS - ESTADO DE SÃO PAULO/ BR.
Gaggini, M.C.R.; Gomes, A.A.L; Mencaroni; D.A.; Pansani. A.A.; Pinto Neto. J.M.
Escola de Enfermagem de Ribeirão Prelo/ USP. Av. Bandeirante. 3900. Campus Universitário - Ribeirão Preto - CEP 14040-902. São Paulo/ Brasil. CADIP Av. Brasília, 756 - Vila Regina. Fernandópolis -CEP: 15600-000-São Paulo/Brasil.
O município de Fernandópolis situado a noroeste do estado de São Paulo, a 553 Km da capital do estado, constitui-se em um pólo regional com 60.521 habitantes. Configura-se como referência na área da saúde para uma micro-região de treze municípios. Adotou dentro do processo de municipalização da saúde a Gestão Plena de Atenção Básica Ampliada. Como problema de Saúde Pública destaca-se a endemia hansênica, objeto de vários estudos. O objetivo desse estudo é descrever como ele está se organizando para cumprir as metas de eliminação dessa endemia que até 2001 estavam sob responsabilidade do estado. Mantém altos coeficientes de prevalência a mais de trinta anos. sendo considerado atualmente hiperendêmico com 10.25 casos/ 10 mil habitantes. Apesar de possuir onze unidades básicas de saúde concentra as ações de tratamento e seguimento dos doentes e contatos em uma unidade de saúde específica para o atendimento de doenças infecto-contagiosas e parasitárias, ficando sob responsabilidade das demais portas de entradas do SUS a suspeição diagnostica. Assim, acreditamos que a centralização de algumas ações poderá melhorar alguns indicadores operacionais. No entanto, os grandes desafios permanecem: o diagnóstico precoce; a descentralização c ou desconcentração das ações para todas as Unidades de saúde e atingir a meta da eliminação até 2005
OOA 4
BRIDGING EFFECTS OF INTEGRATION: COULD INTEGRATION OF LEPROSY HAVE UNINTENDED IMPACTS?
Nimal D. Kasturiaralchi
Consultant Novartis Foundation and Director. Medical Education Unit. Faculty of Medicine, University of Peradeniya, Peradeniya 20400, Sri Lanka
In many former colonial countries there still exists a sharp demarcation between preventive and curative sectors of healthcare. The bureaucracies in most health services are divided along these lines with little or no interaction between them. One of the reasons for the continuation of this division seems to be that the two sides have evolved to be relatively independent of each other in carrying out their routine duties. However, with the introduction of new health policies such as the integration of leprosy services, new perspectives are unfolding which provide practical guidance to bring the curative and preventive sectors closer.
This paper discusses the influence of integration on the general health system of Sri Lanka based on empirical evidence. It could serve as an eye opener for individuals trying to bring together existing health services to facilitate the provision of better and more cost effective healthcare.
OOA 5
CHARACTERISTICS AND TREATMENT OUTCOME IN LEPROSY PATIENTS DIAGNOSED DURING ACTIVE AND PASSIVE CASE-FINDING ACTIVITIES
C. Phaff; J. van den Brock; Y. Stuip
Netherlands Leprosy Relief (NLR), P.O.Box 95005. 1090 HA Amsterdam, The Netherlands
Objective: To assess whether the case-finding method is a determinant for diagnostic characteristics and treatment outcome of newly diagnosed leprosy patients in northern Mozambique.
Methodology: A retrospective cohort study about the differences between entrance characteristics and treatment outcome in self-reporting patients and active case-finding during a Leprosy Elimination Campaign in 1999 in northern Mozambique.
Results: As a consequence of LEC activities three times more patients were found compared to a comparable period one years earlier. More young (<15 years) PB cases were diagnosed during LEC activities with -surprisingly - equal percentage of disability grades. No gender imbalance was found in diagnosed LEC patients contrary to self-reporting patient groups.
Comparing active case finding in 1999 with the passive group of 1998 and 1999 showed a slight but statiscally significant better treatment result for the passive group. The classification of leprosy (in favour of PB) and age (in favour of older age groups) were also determinants for favourable treatment outcomes.
Finally, the type of health worker proved a major determinant of a favourable treatment outcome. Limited trained volunteers had a significant better result of treatment compared to trained nurses.
Conclusions: LEC proved to be a useful addition to the national Leprosy and Tuberculosis program in Northern Mozambique. As a result, many new cases were diagnosed and put on treatment, and their treatment results were comparable to those of self-reporting patients.
The type of health worker appeared to be a major determinant of a favourable treatment outcome. Limited trained volunteers have a significant better result of treatment compared to trained nurses, regardless of detection method.
OOA 6
COMMUNITY INVOLVEMENT FOR LEPROSY ELIMINATION
P.R. Manglani; B.L. Sharma; S. Postma
Delay in achieving elimination in an area enables the programme managers to analysis the factors responsible. The reasons elicited were enlisted. They are lack of Community involvement and support, stigma attached to the disease and passive role of service recipients.
The process of community involvement was given higher priority through service based action programs like; Care & concern Camps, Skin Disease Diagnosis Treatment & Education Camps. Dastak i.e. knocking the doors to knockout leprosy, introduction of festivity in Leprosy Elimination etc. This has lead to creation of concern, demystification and destigmatisation of disease and openness for early diagnosis and treatment. This has also helped for Zeroing distances between the patients, people around and providers.
OOA 7
CO-OPERATION BETWEEN NATIONAL AND INTERNATIONAL N.G.O.'S IN THE FIGHT OF LEPROSY -YEMEN EXPERIENCE
Dr. Abdul Rahim Al-Samie
NLEP. Office of the National Leprosy Control Program. P.O.Box.No.55722 -TAIZ. Republic of Yemen. Tel: 967-4-242306/7/9 and 967-792976 (Mobile) Fax: 967-4-242308.
Leprosy in Yemen is considered as a public problem more than a health problem. Before 1964, leprosy patients were subjected to an obligatory isolation in unsanitary houses outside the main cities.
Between 1973 and 1982. some leprosy patients were given medical care by dapsone monotherapy. Though MDT was officially adopted in Yemen in 1983. there were no real leprosy control activities due to lack of support till an agreement between Ministry of Public Health (MOPH) - Republic of Yemen and German Leprosy Relief Association (GLRA) - Wurzburg - Germany was signed in 1989.
In 1992, a local non-government organization called Yemen Leprosy Elimination Society (YELEP) was formulated. This Society together with GLRA further strengthened our lighting against leprosy in Yemen. With the support of GLRA. YELEP and other national and international non-government organizations the prevalence of leprosy was brought down from 0.70 per 10,000 populations in 1992 to 0.32 per 10.000 population in 1999
OOA8
DEVELOP PARTNERSHIP, STRENGTHEN INTEGRATION, TRANSFER SKILLS AND OWNER-SHIP TO HASTEN ELIMINATION
Mahmood K., Dr.
State Leprosy Officer. Tamil Nadu. India
The presentation evolves around Tamilnadu's success story. The PR was 118 / 10000 in 1983 when MDT was introduced, which was drastically reduced to just 31 / 10000 in 1991 when total coverage was reached. In October 2001 the PR was 3.7, indicating elimination a definite possibility.
Since maintaining a vertical structure with declining PR was not cost effective the programme was integrated with the Primary Health Care system in 1997.
Massive capacity building measures were undertaken to ensure that the PHC system provides better MDT services. This means, to suspect and refer cases for confirmation, treat, manage complications and refer, maintain simple information and reporting system and counseling to patient, family and community.
Integration has not reduced detection of new cases by routine methods. Instead, voluntary reporting has increased due to easy accessibility. The availability of the Female Health Worker has helped women in terms of coverage and accessibility to services.
In essence, integration ensures full participation of the PHC services in Leprosy Elimination. The deficiencies are addressed by regular capacity building measures to upgrade skills and equip the PHC system to accept responsibility and ownership of the programme to hasten Elimination.
The presentation records with appreciation the sacrifices made by all those involved in the Programme beginning with the Missionaries, various Partners and the Community. It is their contribution that has helped in greatly reducing the disease burden and the stigma attached with it.
We shall move forward in building a World without Leprosy with all our Partners
OOA9
EARLY LEPROSY CASE DETECTION BY VOLUNTEERS IN DIFFICULT AREAS IN THANE DISTRICT, INDIA
Prakash R. Dewarkar; M. Joy; B. Geeta; S. Vinaya; C. Kamlesh; A.A. Samy
ALERT-India; Association for Leprosy Education, Rehabilitation & Treatment - India. B-9 Mira Mansion, Sion (West), Mumbai - 400 022. India.
House to house leprosy case detection is very expensive and time consuming if we depend only on regular trained Para Medical staff. Given the fact that sufficient number of trained paramedical personnel not available one may have to seek alternate human resources for the primary task of leprosy case detection. ALERT was required to survey for identifying new cases in far Hung remote villages of Thane District that had become part of the Navi Mumbai Municipal Corporation limits in the recent years and is also part of ALERT's leprosy control project area. There was an urgent need to ascertain the leprosy situation in 40 villages newly added.
As qualified persons were not available, particularly because numbers were not adequate to complete the survey within a short period of 5 to 6 months ALERT decided to engage volunteers and give them intensive training to identify cases of suspected leprosy.
These volunteers made house-to house visits and examined 1.29,383 persons in 40 villages. Volunteers suspected 332 'leprosy cases'. Of these, the doctors and trained paramedical workers confirmed as high as 54 per cent as leprosy cases. A further 10% were kept under observation. This study indicates that a significant number of new cases 14/10,000 has been detected with less expenditure and in a short duration by utilizing the services of adequately trained volunteers in difficult areas too.
OOA10
FIRST STEP TOWARDS INTEGRATION: DEVELOPING A BLUEPRINT
Nimai D. Kasturiaratchi; Sunil Settinayake; Penny Grewal
University of Peradeniya (Sri Lanka), Anti-Leprosy Campaign (Sri Lanka), Novartis Foundation for Sustainable Development (Switzerland)
Planning and implementing the structural changes to integrate leprosy in the general health services is a challenging task as established procedures, responsibilities and relationships, both institutional and personal have to be altered. A blueprint which clearly articulates the vision of how the integrated system should function is critical to guide the implementation process. In Sri Lanka the blueprint was drawn up in a highly participative manner involving intensive discussions with various categories of health care staff both at peripheral and central levels over a three month period.
The blue print clearly outlines the new procedures, roles and responsibilities as well as monitoring mechanisms based on a careful understanding of the tunctioning of the general health services, likely problem areas and pragmatic ways to deal with them. Various technical details had to be worked out including, a distribution system for MDT, simplifying records and the reporting system, monitoring procedures at the local level, and role definitions for the most important partners involved. This process culminated in a goal oriented project planning workshop at which the detailed plan for the integration process was developed, which was then presented to the National Steering Committee and WHO for approval.
This paper describes the process adopted, the components of the blueprint, proposals For action and how it was used as a springboard for action. The blueprint also serves as a source of institutional memory and a shared reference document for the different players to be involved in the process
OOA 11
FOCAL SURVEY FOR INTENSIFIED CASE DETECTION - A COMPARATIVE STUDY CONDUCTED AT ENDEMIC AND NON ENDEMIC STATES IN INDIA
Thomas Abraham; T. Jayaraj Devadas; M.V. Ramana; Shibu George
German Leprosy Relief Association-India
No.4, Gajapathy Street, Shenoy Nagar, Chennai-600 030
This is an intensive case detection activity implemented in selected pockets of certain endemic and non-endemic states in India, where the ease detection is low due to various reasons. The survey team consist of 20 Paramedical workers (PMW's), 2 Non Medical Supervisors (NMS) and a Medical Doctor. 10 PMW's, 1 NMS and the Doctor are from out side the state. The duration of survey is one week, covering a population of 7000 - 10000. In most of the places the focal survey team could detect 2-3 fold more new cases than the normal case finding activity. It was also observed that, whether it is an endemic or non-endemic state the case detection was almost same. The results of this survey helped the management to decide the future strategy of leprosy work in the area. It was also reported that after the focal survey there was an increase in new case detection in the respective places
OOA 12
IMPLEMENTATION OF MODIFIED LECS WITH INTEGRATED APPROACH IN THE STATE OF ORISSA- INDIA HELPED IN REDUCTION OF NCDR
P.K.B. Patnaik
Assistance State Leprosy Officer, OrissaDirectorale of Health Services, Government of Orissa, Bhubaneswar, Orissalndia751001
Orissa is one of the constituent states situated along the east coast of India with a population of 36.7 million. Leprosy was highly endemic in the State with PR 121/ 10000 in 1983. In 1998 though PR had come down to 9.6/10000 but NCDR with 21.7/10000 was posing main hindrance in leprosy elimination within targeted period. Successive 3 rounds of Modified LECS in the State with regular intervals have helped in bringing down NCDR to 7.8 and now goal of elimination looks real possibility in next 3 years. MLECs are well planned, short time, intensive, integrated community approach for detection of all undetected cases of leprosy in a community where disease is highly prevalent and dealt by vertical infrastructure. MLEC-I was implemented in Orissa in 1998 resulted in detection of 62844 cases in 28961085 populations with NCDR 21.7. MLEC-II was implemented in 2000, resulted in detection of 27197 cases in 27715988 popl. With NCDR 9.8. MLEC-III was implemented in 2001 with detection of 12326 cases in 15802564 popl. with NCDR 7.8/ 10000. Fall in detection rate in 3 and 1/2 years was 64%. This fall would have not been achieved even in another 10 years of MDT implementation with routine manner through vertical infrastructure. 3 rounds of MLEC in Orissa not only helped in detection of large number of undetected cases within shortest possible time but also helped integration of leprosy control activity with General Health care System and at village level with integrated child and women development scheme, in reduction of average duration of case presentation from more than 2 years to only 6 months, improvement in drug compliance from 78% to 99.6% and voluntary reporting of cases from 50% to 79.6% and have created new hope for elimination of leprosy in a highly endemic State of Orissa.
OOA 13
INTEGRATING LEPROSY CONTROL INTO GENERAL HEALTH SERVICE IN A WAR SITUATION: THE LEVEL AFTER FIVE YEARS IN EASTERN CONGO
Denis Byamungu; Osahon Ogbeiwi
Provincial Leprosy Control Programme. Bukavu, South Kivu Province DR Congo
Although plagued by insecurity and inaccessibility due to two consecutive wars, South Kivu Provincial Leprosy Control Programme, DR Congo since 1995 started a process to integrate leprosy into general health facilities. General health workers (GHW) were trained, and a network of district TB/Leprosy supervisors provided, as regularly as possible, drugs, logistics and supervision to general facilities having patients. A questionnaire survey of 9 of the 14 districts in the province assessed the level of integration after 5 years. Structural integration was assessed by the proportion of health facilities with MDT and functional integration, by the proportion of health facilities where general health workers (GHW) are involved in leprosy activities. 37.5% of facilities had MDT and 73% had a trained nurse. GHW were involved in screening in 59% of facilities but diagnosed in only 36%. For drug dispensing and POD, they were involved in 78% and 26% respectively. Their degree of involvement put health facilities into four grades of functional integration: 1. Fully functional, fully integrated: tasks performed entirely by GHW. 2. Semi-functional: jointly performed with supervisors, 3. Semi-integrated (structured but not functional): leprosy supervisors did these activities alone, and 4. Non-integrated: nobody performed these activities. 80% of facilities had some degree of integration. 70% of the facilities were fully integrated in dispensing MDT and keeping records; 31% were semi-functional and 49% semi-integrated in diagnosis of leprosy.
The leprosy prevalence at the districts directly correlated with the levels of structural integration, dispensing MDT and case finding. The presence of a trained nurse significantly related to performance of case finding and records keeping, but it was irrelevant to suspecting leprosy, dispensing MDT or doing POD. Structural integration is thus low in South Kivu and the gap between % of facilities with MDT and % with a trained nurse suggests a delay in actually integrating leprosy after training. This could be a direct result of movements of trained nurses because of the war. Functional integration is higher in MDT activities and low in case finding and POD, where obviously more skills are required.
OOA 14
INTEGRATION OF LEPROSY SERVICES AND THE CONCERNS ABOUT QUALITY OF CARE. THE EXPERIENCE OF JIGAWA STATE OF NIGERIA.
Dr. Kefas Samosn
Netherlands Leprosy Relief, Office of the representative in Nigeria. Rm 3, Yelwa Club. Bukuru Nigeria.
Since the inception of the Nigerian National Tuberculosis and Leprosy Control Programme (NTBLCP), integration into the Primary Health Care system (PHC) has been a major objective. Jigawa State, located in northern Nigeria, is currently one of the most leprosy endemic states in the country and in the fore front of the integration initiative. During a Leprosy Elimination Campaign (LEC) organised by Jigawa in 1999, 304 PHC workers from various health units, and 368 volunteers were trained in basic leprosy, all of whom participated in case finding and initiating MDT treatment. Consequently there was rapid expansion of MDT services from 75 clinics prior to the LEC to 264 in 2000. Since then, leprosy patients are managed by the PHC workers, while specialised staff provides technical advice.
In order to assess the impact of die integration on the quality of leprosy services in Jigawa State, treatment records of 159 selected leprosy patients in Jigawa State were studied. 76 of these patients were detected and treated by the vertical staff between 1997 to 1998 (pre-integration), while 83 were detected and managed within the period 1999 onwards (post-integration).
The study found that quality of care for leprosy patients including assessment at diagnosis, monthly follow up, disability prevention and management ami treatment results were not significantly affected by the integration of the MDT services. It is therefore concluded leprosy care is not necessarily jeopardised by integrating into the GHS, instead could improve the accessibility of MDT services.
Details of findings to be discussed.
OOA 15
INTEGRATION OF LEPROSY WORK - AN EXPERIMENTATION
Sudhakar Bandyopadhyay
German Leprosy Relief Association-India. 23 Market Street. Kolkata-700087. India
It was expected that the leprosy services would be integrated with the general health services in the area with reduced prevalence rate. The introduction of MDT has resulted in a sharp decline in prevalence rate by over 907c with a drastic reduction of visible deformities among new cases. Districts where MDT has been implemented for 15 years have the NCDR of 20/10,.000 on an average. In a 5000-population area, the estimated active caseload will be 10 and this load is expected to be managed by a general health worker. Accordingly a 10-years retrospective study was conducted in an experimental zone of the Balarampur control unit of Gandhi Memorial Leprosy foundation in Purulia district of west Bengal. Total 41 General Health workers were involved in the programme from 1988 to 1997. It was observed that the contribution of the general health workers was substantial in relation to case-detection, patient persuasion, and inclusion of leprosy in health-talks and handling adverse situations. Total 440 persons were referred, 373 diagnosed as leprosy cases, 1029 patients were persuaded for regular drug intake and leprosy was discussed in 1204 health talks. Six social problems were handled. It was observed to be cost effective with better utilization of logistics and human resources. The integration processes should be supported with adequate training and supervision and monitoring system at least for next five years till the GHWs develop their own expertise. They should also gradually be exposed to and entrusted with the processes of rehabilitation and POD activities, which is utmost necessity in leprosy field
OOA 16
IS LEPROSY ERADICATION PROGRAM FEASIBLE IN VIETNAM?
Hong Hai Phan
Hospital of Dermato-Venereology, Hồ Ch� inh City. Vietnam
Since leprosy is no longer a serious national health problem in Vietnam, the decreasing workload in terms of leprosy disease prevalence has pushed the stakeholders to find the ways to sustain leprosy works. The target set for eradicating leprosy is to cut the transmission of leprosy, and the provincial managers have to focus on areas where leprosy is endemic, or leprosy pools.
LEC is continued where leprosy prevalence is still high or previously known to have had many leprosy cases. Recruitment of ex-leprosy patients as volunteers among community members can be a good idea: Health education materials should be distributed to the community to create people's awareness on leprosy, break down the silence and stigma surrounding leprosy and promote early reporting of leprosy patients. The use of community volunteers and ex-leprosy patients as health educators is considered. Education for school children is seen as the most effective approach to broadcast messages on leprosy to the people. A kind of quizgame called democratic picking flowers has been initiated and proved that it is very promising.
Besides the traditional training, the problem-based teaching and learning as well as field training are adopted. A new module of training called active education has been realized, setting a new style of training in Vietnam.
According to the WHO proposal, Vietnam begins to implement for a post-elimination surveillance system. A part of leprosy program is shifted to rehabilitation aspect, which is the rising concern of the people and local government for leprosy-affected-people. These efforts for improving the (ex-) leprosy patients lives should be mobilized nation-wide, to respond to the noble mission: working together for a world without leprosy.
OOA 17
LEPROSY ELIMINATION MONITORING (LEM) 2001, BANGLADESH
Jalal Uddin Ahmed; Safir Uddin Ahmed; S.K.S. Hossain; Sivaprakasam; L.R. Talukder
National Leprosy Elimination Programme. Directorate General of Health Services
Leprosy Control Institute & Hospital Compound. Mohakhali, Dhaka-1212. Bangladesh.
Leprosy Elimination Monitoring Exercise was carried out in Bangladesh between 21 January 15 February 2001. To validate data on prevalence, detection, integration and quality of MDT Services. The sample covered 1202 cases, records collected from 60 MDT centers (10%) of 20 randomly selected districts. Data collection was done by qualified Medical officers, especially trained for this activity using WHO recommended schedules.
Exercise was guided supervised and lead by WHO nominated Monitor along with one independent national Monitor nominated by the national Govt.
The actual data collection in the field was carried out between 30 January - 08 February 2001. The data consolidation and report preparation was done between 09-14 February 2001.
Over 5 years reduction in prevalence is observed. However this fall is not associated with expected change in clinical profile of new cases. As majority of the cases were detected by voluntary Reporting (40%) with long duration of delay (average 20 months) and grade 2 disability (10%). This pattern confirms further the need for intensive BCC in population and reorientation of staff on identification/detection of early Leprosy.
The summary of the main findings and observations of LEM will be presented
OOA 18
NEPALESE LESSONS ON COMMUNITY AWARENESS AND CAPACITY BUILDING OF HEALTH SERVICE DELIVERY SYSTEM TOWARDS LEPROSY ELIMINATION
Shushil Battarai M.A., M.P.H.; Suraj Chalise M.A., D.H.P. Ed; Mitha Ram Thapa B.A., NMSS
Leprosy Control Division. DHS/MOH (HMG/N). Kathmandu, Nepal
Nepal, a tropical country in South Asia has a history of high prevalence of Leprosy since last 150 years. Nepal has adopted the WHO resolution to eliminate leprosy by 2005 from the world and by the end of 2003 from the country.
Leprosy elimination activities were escalated from 1996 onwards with intensive Leprosy Elimination Campaign (LEC) during 1999 and 2001. Nepal is actively engaged in Information Education and Communication methods and mediums. The country is building up the capacity of health service delivery system / basic level health care workers to provide diagnostic and treatment services up to the Village Development Committee level, which is lowest. It is found that strong political commitment followed by an efficient programme support on intensive IEC component helps achieve this time bound vision.
This paper discusses the objectives, strategies, activities and major outcomes of elimination efforts. Paper includes secondary information (data) gathered from LCD/ DHS/MOH and presented with the help of simple statistical tools.
The study revealed the fact that prevalence rate has gone up significantly alter planned LEC; such sustained campaign has been contributing to reach the un-reached population (hidden/undetected/cases of consequence) in the areas where the prevalence rate is more than 3/10, 000. The current trend shows that the MB proportion, child proportion and GII disability is declining considerably providing hopes of elimination within the stipulated time
OOA 19
PROBLEMS ASSOCIATED WITH INTEGRATION OF LEPROSY IN BIHAR STATE IN INDIA AND POSSIBLE SOLUTIONS
Raman. D.K.; Menezes, L
State Leprosy Officer, Patna, Bihar C/O. Damien Foundation India Trust, 27, Venugopal Avenue. Spurtank Road, Chennai 600 031 India. E-mail: damienin@vsnl.com
Bihar is the second largest populated state in the country comprising 11% of the population of India. It is highly endemic for leprosy. It has the highest number of cases of leprosy in India and it accounts for 28% of caseload of the country. Since introduction of MDT in 19% more than 800,000 cases have been treated and at present (Jan 2002) total of 105000 cases are registered for treatment with MDT. The phase II of NLEP has identified integration with General Health Care Services as one of the key strategies for easy accessibility of leprosy services for early detection and treatment. Integration in Bihar has been undertaken from July 2001. These are however a few problems being faced in applying integration in practice and therefore the emphasis is presently being given on functional integration down to Health Sub Center level. Integration of following aspects is being implemented viz. Provision of diagnosis facilities on all days at PHC. Addl. PHC and Government hospitals and drug delivery to patients. Some of the factors which contribute to the problem are deficiency of GH staff.(44%) and NLEP staff (56%) with a wastage of 3% every year, poverty, low literacy and low awareness level among the medial fraternity and community. The Government has taken various measures to tackle these problems. The details will be discussed.
OOA 20
PROJETO PRIORITÁRIO "TOLERÂNCIA ZERO: MATO GROSSO SEM HANSENÍASE"
Secretaria de Estado de Saúde de Mato Grosso
Mato Grosso é campeão brasileiro de prevalência em hanseníase com 20 casos por 10 mil habitantes. Por esta razão o Governo em parceria com a SES-MT, lançou o projeto "Tolerância Zero" que tem por objetivo a eliminação da doença como problema de saúde pública até o ano 2005. Atualmente são 2.913 casos notificados e 3.900 casos estimados. Como incentivo a detecção precoce a SES-MT proporciona um "bônus" para equipes e saúde da família e agentes comunitários. Para cada caso detectado, o agente irá receber RS 20 (vinte reais) e os integrantes da unidade de saúde RS 100 (cem reais) por paciente com alta por cura. Na primeira etapa que compreende 2001 a 2002, o projeto foi implantado em todos municípios do Estado, que foram divididos por ordem de prioridade em 4 categorias, tendo por parâmetro a prevalência e o número de habitantes. Cada município deve elaborar um plano de intervenção para implementar atividades em sua área de abrangência, considerando o perfil epidemiológico e operacional da região, identificando as áreas de maior risco da endemia para definição de ações a serem desencadeadas, compor equipe técnica com profissionais qualificados, desenvolver parcerias com demais entidades, ONGs e órgãos, além de criar agenda de treinamentos e acompanhamento da equipe. Os municípios que atingirem as metas estabelecidas receberão um incentivo de acordo com os níveis de prioridade que vão de R$ 30 mil a R$ 2 mil reais, visando a detecção de 95% dos casos estimados nos municípios e redução da prevalência de 30% ao ano. Este recurso deverá ser investido, pelos municípios, na implementação das atividades de promoção, prevenção c atenção básica de saúde.
OOA21
REACHING NLEP SERVICES TO THE DISTANT POOR TRIBAL POPULATION IN CHHATT1S-GARH
B.P. Mukherjee; B. Sharma.
DANLEP Chhattisgarh, Civil Lines. Raipur (Chhattisgarh). 491001 INDIA Ph. No. -+91-0771-423058 Fax-+91-0771-423057
DANIDA assistance in the programme of NLEP helped in the satisfactory coverage of under-privileged poor people living in the distant inaccessible tribal areas. The indicators also reflect that prevalence of leprosy have been reduced considerably in these areas since DANLEP extended services to cover wider areas in all the districts including tribal districts. As women population who in general gets neglected and deprived of many health facilities, in NLEP particularly coverage of women in the programme has been seen to be satisfactory. The trend of reduction is an indication of probability of elimination of leprosy by 2003.
OOA 22
REGIONALIZAÇÃO DAS AÇÕES DE ELIMINAÇÃO DE HANSENÍASE INTEGRADAS NA ATENÇÃO BÃSICA - BAHIA. PERNAMBUCO E PIAUÍ
2001 -2002
Lúcia Possídio1; Geania Rocha2; Tâmara Stélvia3; Vera Andrade4
1. 8a DIRES - Petrolina Pe.
"PIRES - Juazeiro Ba.
3. Secretaria de Saúde Pi.
4. GT/HANSEN/CONASEMS
Através da articulação com GT/HANSEN/ CONASEMS e principalmente a partir da vontade e adesão dos gestores estaduais e municipais da Bahia, Pernambuco e Piauí foi elaborado um plano na perspectiva da estratégia de aceleração proposta pelo referido grupo técnico, que prevê o aumento da oferta do diagnóstico e tratamento da hanseníase com a descentralização e desconcentração das ações, integrando-as nas atividades da atenção básica, passando pela implementação de uma rotina de atualização sustentável dos profissionais de saúde da rede, pela ampla divulgação sobre a universalização da cura da doença e pelo estabelecimento de parcerias com instituições e áreas afins, sociedade organizada e comunidade. A 8ª Regional de Saúde /Pe. expandiu o PCEH para 100% dos seus municípios, a cobertura anterior ao plano era 43%. Em relação aos serviços a cobertura passou de 11,4% para 82,3%.No Piauí as ações de eliminação foram descentralizadas para mais 50 municípios e o número de unidades em 53 municípios aumentou de 50 para 136. Na 15ª Regional de Saúde/ Ba, o PCEH estava implantado em 100% dos municípios. A cobertura dos serviços no período anterior ao plano, era de 15,7% que passou para 64,5%.O trabalho que vem sendo desenvolvido busca atingir o objetivo proposto de facilitar o acesso da população ao diagnostico e ao tratamento integrando as ações de eliminação nos serviços da atenção básica, detectando precocemente os casos reduzindo a morbidade e o aparecimento de casos com incapacidade física. Nessa perspectiva pode-se afirmar que o controle e eliminação da hanseníase como problema de saúde pública pode ser efetivado
OOA 23
RESISTANCE TO CHANGE; HIGH POINTS IN THE SRI LANKAN INTEGRATION PROCESS
Sunil Settinayake; Nimal D. Kasturiaratchi; Penny Grewal
Anti-Leprosy Campaign (Sri Lanka), University of Pcradeniya (Sri Lanka), Novartis Foundation (Switzerland)
The sheer scale of the process of converting a vertically structured leprosy service into a horizontally structured system, during decentralization inevitably involved a number of formidable challenges. The Anti-Leprosy Campaign had for decades been accustomed to working directly only with the 24 leprosy workers - with the general health services playing only a supportive role. Now it had to collaborate on a broader basis with provincial health directors, epidemiologists, dermatologists, pharmacists, and directors of numerous local hospitals, motivating them to provide the necessary services without the necessary "authority" to ensure that the services are provided. It became clear that considerable skills in the areas of team building and teamwork, conducting negotiations, and monitoring were needed.
Integrating leprosy services into the local health-care system has also involved a sometimes delicate exercise in sharing responsibility and adjusting to new roles. Natural resistance to these changes was observed both within and outside of the ALC.
This paper shows how the different levels of players perceived integration and how they reacted. The paper also proposes what should be done to sensitize stakeholders and what they ought to consider before launching integration.
OOA 24
ROLE OF COMBINED MONITORING AND UPDATING REGISTERS IN ELIMINATING LEPROSY IN AFRICA, GUINEA AND CAMEROON EXPERIENCES
Bide Landry; Tiendrebeogo Alexandre
WHO/AFRO, DDC/LEP, PO BOX 773 BE, Harare Zimbabwe
Leprosy Elimination Monitoring (LEM) is a process to collect data and build three groups of 22 indicators enabling to identify leprosy programme weaknesses and to propose measures for improving activities towards the elimination of leprosy. Updating Leprosy Registers (ULR) is an exercise to review leprosy information forms and examine leprosy patients under Multiple Drug Therapy (MDT) in view of obtaining the real prevalence according to the definition of a case of leprosy. These two activities can be combined and implemented by external monitors, leprosy programme national managers and district health teams. Combined LEM and ULR exercise is a strong tool for the reduction of leprosy prevalence and improvement of leprosy programme activities. The Regional Office of WHO for Africa initiated 2 combined LEM/ULR in Guinea and Cameroon, respectively in November-December 2000 and August 2001. These combined exercises permitted to reduce the prevalence of leprosy of 50% in Guinea and 38% in Cameroon. Recycling old cases of leprosy, late or false diagnosis, over-treatment of MB patients, mismanagement of MDT blister packs were main problems identified with LEM indicators. Recommendations were proposed to solve those problems. They consisted mainly the integration of MDT services into general health services and routine ULR during supervisory visits to peripheral health facilities.
[Key words] Updating, Monitoring, and Elimination
OOA 25
SURVEY OF LEPROSY IN UNAPPROACHABLE AND UNCOVERED AREA
Shri U.H. Thakar; R. Ganapati; S.S. Naik; Pralibha Kathe
Hind Kushta Nivaran Sangh Hind Kushla Nivaran Sangh C/o Acworth Leprosy Hospital for Research. Rehabilitation and Education in Leprosy, Wadala. Society for Research. Rehabilitation Mumbai - 400 031 INDIA.
There is no doubt that due to the implementation of multidrug therapy the prevalence rate (PR) of leprosy has declined drastically. New case detection rate, however, has reduced only marginally or has remained static in certain areas, this may be due to the foci of infections in the society lurking in junap-proachable areas or due to mobility of the community members and spreading the disease.
a) The examination 3030 labourers of five different construction work places revealed six leprosy cases giving a PR of 20/10000.
b) The group of male fishermen (304 persons at Panvel) who remained 8 months on sea were examined in rainy season evealed four new cases (PR 131/1000) of which one was smear positive MB ease.
c) The examination of 3457 tribal population on hilly area of Pen yielded 11 leprosy cases (PR 32/10000) of which live were MB. Such pilot studies suggest that special surveys of selected population groups may have to be undertaken systematically, if the leprosy elimination target by the year 2005 AD is to be reached.
OOA 26
TECHNICAL SUPPORT TEAM (TST) FOR THE PROMOTION OF LEPROSY AND TUBERCULOSIS WORK IN THE DISTRICT - A CONCEPT PAPER
Thomas Abraham; T. Jayaraj Devadas
German Leprosy Relief Association-India. No.4. Gajapathy Street. Shcnoy Nagar, Chennai-600 030
The National Leprosy Elimination Programme (NLEP) and the National Tuberculosis Control Programme (NTP) are the two effective programmes taking care of the leprosy and tuberculosis problems in India respectively. The involvement of Non Governmental Organisations (NGO) in the National Leprosy and Tuberculosis programmes are well recognized. German Leprosy Relief Association (GLRA) and Swiss Emmaus Leprosy Relief work (ALES) are two international organisations engaged in leprosy and tuberculosis work in India. These two organisations already launched Technical Support Teams (TST) in 5 districts of Andhra Pradesh for providing support to the ongoing National Leprosy Elimination Programme (NLEP). This district based Technical Support Team consist of a well-experienced Medical Officer, trained both in leprosy and tuberculosis, a trained supervisor and a driver. The same team will be used for the support of the Revised National Tuberculosis Control Programme (RNTCP). by augmenting the system.
The concept of TST for leprosy and tuberculosis conveys the following:
Intensified leprosy elimination process and effective integration with General Health System.
Improved cure rate of sputum positive tuberculosis to more than 85%.
Through a well drawn out work plan with the government this concept will prove to be an effective strategy.
OOA 27
URBAN LEPROSY CONTROL IN ENDEMIC COUNTRIES - AN UNDER - RECOGNIZED PROBLEM
R. Ganapati; V.V. Pai; H.O. Bulchand
Bombay Leprosy Project. Sion-Chunabhatti. Mumbai-400 022. India
Since the recommendations of the International Leprosy Congress held in Bergen. Norway in 1973 focused world attention on the need for research in urban leprosy, several national and international meetings have stressed the exclusive importance of this aspect of leprosy management. It is however debatable whether the reduction in mean prevalence rate (PR) through MDT is due to an exclusive strategy adopted consciously in urban areas by the planners of leprosy control.
Perhaps we are driven to the necessity of bestowing attention on this subject as the incidence rate of leprosy is not coming clown clue to urban pockets, especially those in the slums, with hidden leprosy. Due to rapid industrialization and migration of population into the slums, especially of metropolitian cities of the endemic world, special challenges are posed to reach the target set for elimination of leprosy. The population in major cities in the Indian sub-continent is expected to increase by nearly 40% by 2015.
The slum population (about 6 million) in Bombay it-self reaches the dimension of the entire population of some countries in the developed world.
Though the lesser cities also face the problem in varying degrees, the exact magnitude is not known. The anomaly of using P.R to judge the effectiveness of leprosy elimination is classically exemplified by the unrealistic statistics relating to the megalopolis of Bombay, where the P.R is reported to be just 2 per 10.000 in the face of about 5000 new cases (10% skin smear +ve) being detected every year.
It is strongly urged that the recommendations already made by a series of Seminars. Workshops, Congresses etc., especially in the Indian subcontinent be implemented meticulously