Leprae Bacilli Research Articles

Borderline Lepromatous Leprosy Masquerading as Granuloma Annulare: A Case Report

Background: Leprosy is a chronic granulomatous disease caused by Mycobacterium Leprae. Leprosy is a great imitator with its various atypical, unusual and varying clinical presentations which can be confused with many infectious and non-infectious diseases. Case Report: A 46-year-old female presented to the dermatology department with complaints of painful raised lesions over face, upper back, upper limbs, lower legs and feet for 12 months. Clinically suspected as a case of Granuloma annulare. Punch biopsy was taken which revealed epidermis with dermis showing nodules of histiocytes and lymphocytes with grenz zone at the dermoepidermal junction. Acid fast stain and Fite Faraco stain was performed which revealed the presence of leprae bacilli with bacteriological index of 4+. Conclusion: Detailed history taking and correlating clinical picture with morphology is required to diagnose atypical presentations of leprosy. Early diagnosis helps to provide appropriate treatment and thus prevent from devastating complications.

Mycobacterium leprae in Armadillo Tissues from Museum Collections, United States.

We examined armadillos from museum collections in the United States using molecular assays to detect leprosy-causing bacilli. We found Mycobacterium leprae bacilli in samples from the United States, Bolivia, and Paraguay; prevalence was 14.8% in nine-banded armadillos. US isolates belonged to subtype 3I-2, suggesting long-term circulation of this genotype.

Leprosy Epidemiology According to Leprosy Type in 13 Teaching Hospitals in Indonesia between 2018 and 2020

BACKGROUND: Leprosy or Hansen’s disease is an infectious disease caused by Mycobacterium leprae (M. leprae) bacilli which results in chronic infections in humans that affect the peripheral nerves, skin, and other organs such as the eyes, mucous membranes, bones, and testicles. Leprosy has a variety of clinical presentations, depending on the cell-mediated immunity of the host. Leprosy is classified by the World Health Organization (WHO) as paucibacillary type (PB) and multibacillary type (MB) depending on the number of lesions. This multicenter study aims to provide representation of leprosy types and its epidemiology in Indonesia. AIM: The purpose of this study is to find out the epidemiology according to the leprosy type in 13 teaching hospitals in Indonesia between 2018 and 2020. METHODS: This is a descriptive study with cross-sectional design. All patients with the diagnosis of PB or MB type of leprosy who underwent Multidrug Therapy (MDT) treatment between 2018 and 2020 were included in this study. Data collection was carried out by tracing medical records data in 13 teaching hospitals in Indonesia. RESULTS: A total of 2129 leprosy patients were included in this study. There were 232 cases (10.8%) of PB leprosy and 1897 cases (89.1%) of MB leprosy. The average age of the patients is 38.7 years (SD ± 16.2 years). Males are more affected by leprosy (n = 1,441; 67.7%) compared to female (n = 688; 32.3%). CONCLUSION: Leprosy remains as a national health problem. The increasing cases of MB leprosy in Indonesia are caused by several factors including the lack of knowledge about leprosy, the delay in diagnosis of leprosy, and the stigma of leprosy within the community; therefore, it becomes a challenge to eliminate leprosy in Indonesia.

Approach of Persisters Relapse in a Smear-negative Leprosy after Second MB-MDT Completion: An Arduous Case Report

Introduction: Many infectious disease intervention programs, including leprosy, are cautious about the establishment of relapse and drug resistance. This case report reports on a male patient who has had two relapses and had undergone twice MB-MDT treatment as well.
 Case Report: A 31 year-old male, came with the main complaint of new hypopigmented patches appeared on the body since 4 months ago, which arose from his second MB-MDT treatment. He started his first MB-MDT treatment in 2019 and second MD-MDT in 2020. Slit skin smear yielded negative results and histopathological examination showed no signs of leprosy. Serological anti-PGL-1 antibody tests yielded very high IgM (3.163) and normal IgG (2) levels. PCR amplification examination showed positive, but DNA sequencing targeting rpoB, folP, and gyrA genes showed no mutations. He was diagnosed with persisters relapse in a borderline-lepromatous leprosy and was treated with rifampicin 600 mg/day for 2 weeks; combination of minocycline 100 mg/ day, clarithromycin 500 mg/ day, and clofazimine 50 mg/ day for 6 months; and methylprednisolone 32 mg/ day for the neuritis. No new lesions appeared during current treatment and he is also showing gradual improvement of foot drop in his left leg.
 Discussion: The fact of a very high IgM levels after 2 courses of MB-MDT indicated that there is still bacteria that continued to induce antibody formation, thus supporting the persisters bacilli hypothesis in this case. Positive results of PCR amplification conclude that there is still M. leprae bacilli in skin tissues, which may be hidden in deeper tissues. We have to make a more proactive approach considering this was his second times of relapse, and knowing the fact that MB-MDT was consumed religiously in the two previous treatment.
 Conclusion: In an arduous case like this, proactive step must be taken. Whenever possible, the use of whole genome sequencing is strongly advised.
 Keywords: leprosy, persisters bacilli, relapse, drug resistance

The value of Real time PCR and High Resolution Ultrasound in Diagnosis of Suspected Pure Neural Leprosy

Background: Cases with pure neural leprosy (PNL) are difficult in their diagnosis and usually loss the early management opportunity. Objective: to evaluate Real time PCR and high resolution ultrasonography of peripheral nerves as diagnostic tools in PNL. Methodology The study included 25 suspected PNL and 25 healthy controls. All patients were subjected to ultrasonography cross-sectional area (CSA) measurement. Fine needle aspiration (FNA) was obtained, and followed by Ziehl-Neelsen (ZN) staining, and real time PCR. Results: ZN staining demonstrated M leprae bacilli in only 8 cases while their DNA was detected in 92 % cases (Sensitivity of both were 100%, and 44.4%, respectively). Only the left median and right and left posterior tibial nerves CSA measurements showed good performance in distinguishing patients of PNL. Median nerve cut off=15.5 mm2 , with a sensitivity of 72%), and right and left posterior tibial nerves CSA Cut Off were 12, 11.5 mm2 with sensitivity of 76-84%. Conclusions: ultrasonography of suspected nerves and real time PCR of nerve aspirates are simple accurate tests for diagnosis of PNL.

Functional biomarker signatures of circulating T-cells and its association with distinct clinical status of leprosy patients and their respective household contacts

BackgroundLeprosy is a chronic infectious disease classified into two subgroups for therapeutic purposes: paucibacillary (PB) and multibacillary (MB), closely related to the host immune responses. In this context it is noteworthy looking for immunological biomarkers applicable as complementary diagnostic tools as well as a laboratorial strategy to follow-up leprosy household contacts.MethodsThe cross-sectional study enrolled 49 participants, including 19 patients and 30 healthy controls. Peripheral blood mononuclear cells (PBMC) were isolated and incubated in the presence of Mycobacterium leprae bacilli. The cells were prepared for surface (CD4+ and CD8+) and intracytoplasmic cytokine staining (IFN-γ, IL-4 and IL-10). Multiple comparisons amongst groups were carried out by ANOVA, Kruskal–Wallis, Student T or Mann–Whitney test. Comparative analysis of categorical variables was performed by Chi-square. Functional biomarker signature analysis was conducted using the global median values for each biomarker index as the cut-off edge to identify the proportion of subjects with high biomarker levels.ResultsThe cytokine signature analysis demonstrated that leprosy patients presented a polyfunctional profile of T-cells subsets, with increased frequency of IFN-γ+ T-cell subsets along with IL-10+ and IL-4+ from CD4+ T-cells, as compared to health Controls (Venn diagram report). Moreover, statistical analysis was carried out using parametric or non-parametric variance analysis followed by pairwise multiple comparisons, according to the data normality distribution. L(PB) displayed a polyfunctional profile characterized by enhanced percentage of IFN-γ+, IL-10+ and IL-4+ produced by most T-cell subsets, as compared to L(MB) that presented a more restricted cytokine functional profile mediated by IL-10+ and IL-4+ T-cells with minor contribution of IFN-γ produced by CD4+ T-cells. Noteworthy was that HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB) that presented a cytokine profile limited to IL-10 and IL-4.ConclusionsOur data demonstrated that L(PB) displayed enhanced percentage of IFN-γ+, IL-10+ and IL-4+ as compared to L(MB) that presented functional profile mediated by IL-10+ and IL-4+ T-cells and HHC(MB) exhibited enhanced frequency of IFN-γ+ T-cells, contrasting with HHC(PB). Together, our findings provide additional immunological features associated with leprosy and household contacts. These data provide evidence that biomarkers of immune response can be useful complementary diagnostic/prognostic tools as well as insights that household contacts should be monitored to access putative subclinical infection.

Biotechnological and Immunological Platforms Based on PGL-I Carbohydrate-Like Peptide of Mycobacterium leprae for Antibodies Detection Among Leprosy Clinical Forms.

Phenolic glycolipid I (PGL-I) is an abundant antigen on the Mycobacterium leprae cell wall, commonly used for operational classification of leprosy patients. Our aim was to develop PGL-I mimotopes with similar characteristics and functions of the native antigen. We have used a random peptide phage display (PD) library for selections against the monoclonal antibody anti-PGL-I. After three selection cycles, six peptides were identified. All sequences were interspersed by a spacer generating a chimeric peptide (PGLI-M3) that was artificially synthesized. The highly reactive peptide was submitted to a reverse PD selection with a single-chain Fv (scFv) antibody fragment combinatorial library. The most reactive scFv was then validated by enzyme-linked immunosorbent assay (ELISA) against both native PGL-I and two derived synthetic (NDO and ND-O-HSA). We have further proved the scFv specificity by detecting M. leprae bacilli in leprosy lesions through immunohistochemistry. We then described its applicability in ELISA for all clinical forms and household contacts (HC). Afterward, we showed differential binding affinities of PGLI-M3 to sera (anti-PGL-I IgM) from all leprosy clinical forms through surface plasmon resonance (SPR). ELISA IgM detection showed 89.1% sensitivity and 100% specificity, considering all clinical forms. Positivity for anti-PGL-I IgM was twofold higher in both HC and patients with paucibacillary forms in hyperendemic regions than in endemic ones. The SPR immunosensor was able to differentiate clinical forms with 100% accuracy. This is the first time that a PGL-I mimotope has efficiently mimicked the carbohydrate group of the M. leprae antigen with successful immunoassay applications and may become a substitute for the native antigen.

Leprosy presenting as remitting seronegative symmetrical synovitis with pitting oedema syndrome \u2013 a case report

BackgroundLeprosy typically manifests with skin and peripheral nerve involvement. Musculoskeletal complaints are the third most common, and can be the sole presenting manifestation. They range from arthralgia/arthritis in reactional states to full mimics of systemic rheumatic diseases. Remitting Seronegative Symmetrical Synovitis with Pitting Oedema syndrome has only been described once in a patient with already diagnosed Leprosy.Case reportA 68-year-old male, from an endemic region of familial amyloid polyneuropathy, presented with an inaugural Remitting Seronegative Symmetrical Synovitis with Pitting Oedema like syndrome, more that 20 years after travelling to Leprosy endemic areas. Arthritis would resurface whenever oral prednisone was tapered, so methotrexate was started, controlling the complaints. Only one year later, after the appearance of peripheral neuropathy and skin lesions, it was possible to diagnose Leprosy, through the identification of Mycobacterium leprae bacilli in a peripheral nerve biopsy.ConclusionThis report is an example of the heterogeneity of manifestations of Leprosy, namely rheumatic, and the challenge of diagnosing it when typical complaints are absent. It is also a reminder that this disease should be considered whenever a patient with a combination of skin/neurologic/rheumatic complaints has travelled to endemic countries in the past.

Evaluation of 16S rRNA qPCR for detection of Mycobacterium leprae DNA in nasal secretion and skin biopsy samples from multibacillary and paucibacillary leprosy cases

Mycobacterium leprae bacilli are mainly transmitted by the dissemination of nasal aerosols from multibacillary (MB) patients to susceptible individuals through inhalation. The upper respiratory tract represents the main entry and exit routes of M. leprae. Therefore, this study aimed to evaluate the sensitivity and specificity of real-time quantitative polymerase chain reaction (qPCR) in detecting M. leprae in nasal secretion (NS) and skin biopsy (SB) samples from MB and paucibacillary (PB) cases. Fifty-four NS samples were obtained from leprosy patients at the Dona Libânia National Reference Centre for Sanitary Dermatology in Ceará, Brazil. Among them, 19 MB cases provided both NS and SB samples. Bacilloscopy index assays were conducted and qPCR amplification was performed using specific primers for M. leprae 16S rRNA gene, generating a 124-bp fragment. Primer specificity was verified by determining the amplicon melting temperature (Tm = 79.5 °C) and detection limit of qPCR was 20 fg of M. leprae DNA. Results were positive for 89.7 and 73.3% of NS samples from MB and PB cases, respectively. SB samples from MB patients were 100% positive. The number of bacilli detected in NS samples were 1.39 × 103–8.02 × 105, and in SB samples from MB patients were 1.87 × 103–1.50 × 106. Therefore, qPCR assays using SYBR Green targeting M. leprae 16S rRNA region can be employed in detecting M. leprae in nasal swabs from leprosy patients, validating this method for epidemiological studies aiming to identify healthy carriers among household contacts or within populations of an endemic area.

Deciphering the genetic control of gene expression following Mycobacterium leprae antigen stimulation.

Leprosy is a human infectious disease caused by Mycobacterium leprae. A strong host genetic contribution to leprosy susceptibility is well established. However, the modulation of the transcriptional response to infection and the mechanism(s) of disease control are poorly understood. To address this gap in knowledge of leprosy pathogenicity, we conducted a genome-wide search for expression quantitative trait loci (eQTL) that are associated with transcript variation before and after stimulation with M. leprae sonicate in whole blood cells. We show that M. leprae antigen stimulation mainly triggered the upregulation of immune related genes and that a substantial proportion of the differential gene expression is genetically controlled. Indeed, using stringent criteria, we identified 318 genes displaying cis-eQTL at an FDR of 0.01, including 66 genes displaying response-eQTL (reQTL), i.e. cis-eQTL that showed significant evidence for interaction with the M. leprae stimulus. Such reQTL correspond to regulatory variations that affect the interaction between human whole blood cells and M. leprae sonicate and, thus, likely between the human host and M. leprae bacilli. We found that reQTL were significantly enriched among binding sites of transcription factors that are activated in response to infection, and that they were enriched among single nucleotide polymorphisms (SNPs) associated with susceptibility to leprosy per se and Type-I Reaction, and seven of them have been targeted by recent positive selection. Our study suggested that natural selection shaped our genomic diversity to face pathogen exposure including M. leprae infection.

Interaction of ATP with a small heat shock protein from Mycobacterium leprae: effect on its structure and function.

Adenosine-5’-triphosphate (ATP) is an important phosphate metabolite abundantly found in Mycobacterium leprae bacilli. This pathogen does not derive ATP from its host but has its own mechanism for the generation of ATP. Interestingly, this molecule as well as several antigenic proteins act as bio-markers for the detection of leprosy. One such bio-marker is the 18 kDa antigen. This 18 kDa antigen is a small heat shock protein (HSP18) whose molecular chaperone function is believed to help in the growth and survival of the pathogen. But, no evidences of interaction of ATP with HSP18 and its effect on the structure and chaperone function of HSP18 are available in the literature. Here, we report for the first time evidences of “HSP18-ATP” interaction and its consequences on the structure and chaperone function of HSP18. TNP-ATP binding experiment and surface plasmon resonance measurement showed that HSP18 interacts with ATP with a sub-micromolar binding affinity. Comparative sequence alignment between M. leprae HSP18 and αB-crystallin identified the sequence 49KADSLDIDIE58 of HSP18 as the Walker-B ATP binding motif. Molecular docking studies revealed that β4-β8 groove/strands as an ATP interactive region in M. leprae HSP18. ATP perturbs the tertiary structure of HSP18 mildly and makes it less susceptible towards tryptic cleavage. ATP triggers exposure of additional hydrophobic patches at the surface of HSP18 and induces more stability against chemical and thermal denaturation. In vitro aggregation and thermal inactivation assays clearly revealed that ATP enhances the chaperone function of HSP18. Our studies also revealed that the alteration in the chaperone function of HSP18 is reversible and is independent of ATP hydrolysis. As the availability and binding of ATP to HSP18 regulates its chaperone function, this functional inflection may play an important role in the survival of M. leprae in hosts.

Oral manifestations of patients with leprosy: A disease, actually infectious but not always, still a stigma in society

Context Leprosy is a chronic granulomatous infectious disease process in humans primarily involving the skin, peripheral nerves, and nasal mucosa but capable of affecting any tissue or organ. The leprae bacilli were first observed by Hansen in 1868. Although this was the first bacterial human pathogen to be described, it continues to remain one of the least understood. Aims The aim of the study was to evaluate oral and facial manifestations in patients affected with leprosy. Materials and methods The present study was performed with an aim of reviewing oral and facial manifestations in patients suffering from leprosy. For this, 150 patients suffering from lepromatous and tuberculoid forms of leprosy were included in the study. The study was approved by the ethical committee appointed by SIBAR Institute of Dental Sciences, Guntur. Permission was also taken from asylum management and the district leprosy officer to conduct the study. All study samples were patients diagnosed with Hansen's disease by a leprologist using a bacteriological index. Results In the present study, oral and facial manifestations were observed more frequently in the lepromatous type of leprosy than in the tuberculoid form. Also, these manifestations were less pronounced in group I than in group II and were more in groups III and IV based on increased exposure to the disease process. Statistical analysis was performed using the c2 -test and P values were also found to be significant. Conclusion Present day diagnostic protocols for early detection and treatment are effective in controlling the disease and in preventing further progression with severe orofacial manifestations. A dentist should be able to recognize these manifestations and treat them under the supervision of a leprologist. Protective care must be taken in treating the disease, although it should not be held as a nightmare as was considered previously.

Therapeutic alternatives for the treatment of residual hansenoma in patients with cure criteria of leprosy

Hansenomas originate from the intense cutaneous infiltration by Mycobacterium leprae bacilli in Virchowian hansenic patients who tend to improve with treatme...

The challenge of producing skin test antigens with minimal resources suitable for human application against a neglected tropical disease; leprosy.

True incidence of leprosy and its impact on transmission will not be understood until a tool is available to measure pre-symptomatic infection. Diagnosis of leprosy disease is currently based on clinical symptoms, which on average take 3–10 years to manifest. The fact that incidence, as defined by new case detection, equates with prevalence, i.e., registered cases, suggests that the cycle of transmission has not been fully intercepted by implementation of multiple drug therapy. This is supported by a high incidence of childhood leprosy. Epidemiological screening for pre-symptomatic leprosy in large endemic populations is required to facilitate targeted chemoprophylactic interventions. Such a test must be sensitive, specific, simple to administer, cost-effective, and easy to interpret. The intradermal skin test method that measures cell-mediated immunity was explored as the best option. Prior knowledge on skin testing of healthy subjects and leprosy patients with whole or partially fractionated Mycobacterium leprae bacilli, such as Lepromin or the Rees' or Convit' antigens, has established an acceptable safety and potency profile of these antigens. These data, along with immunoreactivity data, laid the foundation for two new leprosy skin test antigens, MLSA-LAM (M. leprae soluble antigen devoid of mycobacterial lipoglycans, primarily lipoarabinomannan) and MLCwA (M. leprae cell wall antigens). In the absence of commercial interest, the challenge was to develop these antigens under current good manufacturing practices in an acceptable local pilot facility and submit an Investigational New Drug to the Food and Drug Administration to allow a first-in-human phase I clinical trial.

Resistance of M. leprae to Quinolones: A Question of Relativity?

Multidrug resistant leprosy, defined as resistance to rifampin, dapsone and fluoroquinolones (FQ), has been described in Mycobacterium leprae. However, the in vivo impact of fluoroquinolone resistance, mainly mediated by mutations in DNA gyrase (GyrA2GyrB2), has not been precisely assessed. Our objective was to measure the impact of a DNA gyrase mutation whose implication in fluoroquinolone resistance has been previously demonstrated through biochemical studies, on the in vivo activity of 3 fluoroquinolones: ofloxacin, moxifloxacin and garenoxacin.Methodology/Principal FindingsWe used the proportional bactericidal method. 210 four-week-old immunodeficient female Nude mice (NMRI-Foxn1nu/Foxn1nu) were inoculated in the left hind footpad with 0.03 ml of bacterial suspension containing 5×103, 5×102, 5×101, and 5×100 M. leprae AFB organisms of strain Hoshizuka-4 which is a multidrug resistant strain harboring a GyrA A91V substitution. An additional subgroup of 10 mice was inoculated with 5×10−1 bacilli in the untreated control group. The day after inoculation, subgroups of mice were treated with a single dose of ofloxacin, moxifloxacin, garenoxacin or clarithromycin at 150 mg/kg dosing. 12 months later mice were sacrificed and M. leprae bacilli were numbered in the footpad. The results from the untreated control group indicated that the infective inoculum contained 23% of viable M. leprae. The results from the moxifloxacin and garenoxacin groups indicated that a single dose of these drugs reduced the percentage of viable M. leprae by 90%, similarly to the reduction observed after a single dose of the positive control drug clarithromycin. Conversely, ofloxacin was less active than clarithromycin.Conclusion/SignificanceDNA gyrase mutation is not always synonymous of lack of in vivo fluoroquinolone activity in M. leprae. As for M. tuberculosis, in vivo studies allow to measure residual antibiotic activity in case of target mutations in M. leprae.

Development and pre-clinical assessment of a 73 kD chimeric fusion protein as a defined sub-unit vaccine for leprosy

Despite the advances toward the elimination of leprosy through widespread provision of multi-drug therapy to registered patients over the last 2 decades, new case detection rates have stabilized and leprosy remains endemic in a number of localized regions. A vaccine could overcome the inherent limitations of the drug treatment program by providing protection in individuals who are not already harboring the Mycobacterium leprae bacilli at the time of administration and effectively interrupt the transmission cycle over a wider timespan. In this report we present data validating the production of 73f, a chimeric fusion protein incorporating the M. leprae antigens ML2028, ML2346 and ML2044. The 73f protein was recognized by IgG in multibacillary (MB) leprosy patient sera and stimulated IFNγ production within whole blood assays of paucibacillary (PB) leprosy patient and healthy household contacts of MB patients (HHC). When formulated with a TLR4L-containing adjuvant (GLA-SE), 73f stimulated a strong and pluripotent Th1 response that inhibited M. leprae-induced inflammation in mice. We are using these data to develop new vaccine initiatives for the continued and long-term control of leprosy.

Sensitivity Of Fluorescent Microscopy In Detecting Mycobacterium Leprae In Tissue Sections

Detection of Mycobacterium leprae in tissue sections by Modified Fitefaraco method was compared with the sensitivity of fluorescence microscopy in tissue sections .AuramineRhodamine fluorescent stain as recommended by Kuper & May’s was used with minor alterations in deparaffinization to obtain optimal fluorescence. Out of 70 biopsies studied, 63 showed organisms by fluorescent method and 16 showed organisms by modified Fite -faraco method. We found that fluorescent stain was superior to modified Fitefaraco in detecting leprae bacilli in tissue sections particularly in paucibacillary cases where bacillary load is much low. The speed of observation and rapidity of finding the bacilli by fluorescent microscopy also reduced observer’s fatigue. Hence fluorescent microscopy can be used as a main tool to detect the bacilli in paucibacillary cases.

How to prevent immunological reactions in leprosy patients and interrupt transmission of Mycobacterium leprae to healthy subjects: Two hypotheses

The basis of World Health Organization strategy for leprosy elimination is that the only source and reservoir for infection are patients with the disease. It was assumed that multi drug therapy (MDT) would reduce transmission of Mycobacterium leprae, but there is no convincing evidence for this. Furthermore, even if MDT has been proved to be extremely effective against the infectious disease, a noticeable proportion of leprosy patients can suffer from immunologic hypersensitivity reactions which are now the most significant issue in the managements of the disease. In endemic areas it was found that: M. leprae survives outside human body; healthy individuals harbor M. leprae bacilli in nasal cavity and shed micro-organisms in environment; there is widespread subclinical transmission of M. leprae with transient infection of the nose resulting in the development of a mucosal immune response. This disparate clinical, epidemiologic, and microbiologic evidence leads to the first hypothesis: that antigenic load in local tissues, sufficient to trigger the immune response, comes from external supply of M. leprae organisms. The hypothiocyanite anion (OSCN-) is generated in vivo by the reaction of thiocyanate with hydrogen peroxide catalyzed by lactoperoxidase. OSCN- is an antimicrobial oxidizing agent that prevents growth of bacteria, fungi, and viruses. OSNC- exists in lower airway secretions and lung has never been reported to be affected by leprosy. There is a sufficient concentration of OSCN- in the saliva, and accordingly mouth is rarely affected by leprosy. By contrast, the concentration of this compound is low or nil in nasal and lacrimal secretions and leprosy very often affects nose and eyes. The second hypothesis is that OSCN- may also protect from leprosy. Recently a method of OSCN- production, not involving enzymatic steps or use of toxic heavy-metal salts, has been patented. Studies on the susceptibility of M. leprae to hypothiocyanite could be carried out and, in case of positive results, the substance might be used in order to sterilize the nasal cavity of healthy carriers and prevent transmission of M. leprae to healthy subjects and to leprosy patients in whom it may trigger an immune response.

High resolution shadowing of Mycobacterium leprae

Metal shadow casting techniques for transmission electron microscopic examination was used to determine the morphological characteristics of Mycobacterium leprae in untreated and treated patients. This technique is used to visualize bacterial surface structures by thermal evaporation of platinum alloys under moderate vacuum. This method gives a high contrast image at relatively low resolution and is useful for correlating micro-morphology quantitatively to early therapeutic effects of anti-leprosy drugs. Using these techniques in untreated cases, the surface structures of M. leprae were uniformly filled with relatively homogenous protoplasm surrounded by a cell wall. Most of the bacilli had thick cell walls with prominent banded and fibrous structures on the surface of the cell body. The cell wall was not detached in any of the solid bacilli in untreated cases. The bacilli varied in size and some of them were swollen in their mid-portion. Some bacilli were very short and completely filled with cytoplasm; therefore, these short bacilli were counted as solid bacilli in electron microscopic morphological index (EM-MI) determination. During treatment, mainly the cytoplasms of the bacilli were affected, and degeneration was observed. Ultrastructurally, the cytoplasm was shrunken and detached from the cell wall indicating mild degeneration. After moderate degeneration, the cytoplasm appeared fragmented. In advanced degeneration, all structures except the cell walls collapsed completely and no fibrous or band structures were visible on the surfaces of the cell walls. Therefore, these bacilli were counted as non-solid bacilli for EM-MI determination. This study shows that transmission electron shadowing gives more accurate counts than standard light microscopy of intact M. leprae bacilli in patient specimens.

Dendritic epidermal gamma/delta T cells (DETC) activated in vivo proliferate in vitro in response to Mycobacterium leprae antigens.

gamma/delta T-cell receptor (TCR)+ dendritic epidermal T cells (DETC) are part of a primitive defense system in the skin; they are capable of responding only to a limited number of antigens. The aim of the present study was to test whether DETC can proliferate in vitro in response to antigens of Mycobacterium leprae. DETC were obtained from CBA mouse ear skin by trypsinization and Histopaque gradient centrifugation. The resulting epidermal cell suspension contained up to 20% DETC, as analyzed by the fluorescence activated cell sorter (FACS) after staining with anti-Thy-1 or anti-gamma/delta TCR monoclonal antibodies (mAbs). The freshly isolated cells, or DETC cultured up to 4 weeks with interleukin-2 (IL-2), were exposed in vitro for up to 6 days to varying doses of the following M. leprae antigens: (1) integral (live) M. leprae bacilli; (2) Dharmendra antigen; and (3) PGL-1 (phenolic glycolipid of M. leprae). The DETC response was assessed by tritiated thymidine (3H-TdR) incorporation. The freshly isolated DETC, or DETC cultured up to 4 weeks with IL-2, did not respond significantly to any of the M. leprae antigens, although at the same time they were able to respond vigorously to concanavalin A (Con A), as positive control. If, however, DETC were isolated from skin, painted 7 days before with croton oil (10 microL/cm2 to cause irritant dermatitis, they were able to respond to all M. leprae antigens by a 3-4-fold incrase in the 3H-TdR uptake. The most effective stimulator was a 1 : 1 mixture of Dharmendra and PGL-1 (0. 01 microg/mL), which was as effective as 10-fold higher doses of either antigen alone. Cell counts confirmed that increased DNA synthesis was associated with cell proliferation. Experiments employing alpha/beta-TCR CBA murine spleen cells and epidermal cell suspension treated with anti-gamma/delta or antialpha/beta mAbs + C' proved that only the gamma/delta DETC were the responder cells to M. leprae antigens. The results suggest that activation of DETC in vivo may make them responsive to M. leprae antigens. A significant increase in the number of class II major histocompatibility complex (MHC) positive, nondendritic cells was observed in the croton oil-treated epidermis. We hypothesize that croson oil-induced upregulation of class II MHC expression, which endows epidermal cells with antigen-presenting capabilities, might be an important factor in vivo in delivering an immunogenic signal to resident DETC in the skin.