Presence of M. leprae in tissues in slit skin smear negative multibacillary (MB) patients after WHO-MBR

This study looked for M. leprae in the lymph node, nerve and skin of multibacillary (MB) leprosy patients who become slit skin smear negative after the completion of WHO-MBR. Twenty-five WHO-MBR-treated multibacillary leprosy patients were studied; borderline lepromatous (BL) leprosy (n = 11) and lepromatous (LL) leprosy (n = 14)). Fifteen patients had reaction (erythema nodosum leprosum 11, upgrading reaction 4) either at presentation or during therapy. All patients attained slit skin smear negativity after WHO-MBR (range 24-39 months. Sixteen (64%) patients with multibacillary leprosy showed fragmented bacilli in skin and nerve biopsy or lymph node aspirates after WHO-MBR. Lymph node aspirates alone revealed M. leprae in seven patients, followed by nerve in two and skin in one patient. Four cases showed M. leprae at all sites followed by nerve and skin or lymph node in one case each. A pretreatment bacteriological index (BI) of 4+ or more was significantly associated with the presence of M. leprae at the end of treatment. Also, significantly more lymph node aspirates contained M. leprae in comparison with nerve or skin biopsies. All seven cases in whom treatment was extended beyond 24 months showed M. leprae in tissues even after attaining slit smear negativity. In conclusion, M. leprae persist in tissues after 2 years of WHO-MBR and patients with an initial BI of 4+ or more need to be closely followed up after stopping MDT.     

Anti-Mycobacterium leprae monoclonal antibodies cross-react with human skin: an alternative explanation for the immune responses in leprosy.

A panel of 17 mouse monoclonal antibodies (MoAb) raised against Mycobacterium leprae (M. leprae) antigens was used to detect antigenic determinants in normal human skin. An indirect immunoperoxidase technique was used. Eight of the MoAb detected epidermal antigens similar to patterns well known for human sera. Five of these MoAb detected determinants in the dermis, too. These observations may indicate a certain degree of similarity between the antigenic determinants occurring in M. leprae and in the human host. We propose that such a similarity on the one hand may facilitate the survival of M. leprae in the human host when the antigens are not recognized as "non-self," a situation which seems to occur in lepromatous leprosy, when the patients' tissues are loaded with bacteria virtually without any immune response. On the other hand, M. leprae antigens which mimic host antigens may induce an auto-immune reaction against the host's own antigens, which could explain the immune reaction in tuberculoid leprosy and during a "reversal reaction" when M. leprae is not observed in the host tissues, but extensive granuloma formation occurs.     

Preliminary observations on experimental leprosy in tupaias (Tupaia belangeri yunalis)

The Tupaia belangeri yunalis (tree shrew) is one of the primitive primates. They were inoculated subcutaneously in the footpad or intravenously with Mycobacterium leprae from a patient with multibacillary leprosy. As controls, the footpads of CFW mice were inoculated with the same suspension of M. leprae. The results showed growth of acid-fast bacilli (AFB) in the footpads of locally inoculated CFW mice and in the footpads of both locally and intravenously inoculated tupaias. Whereas the numbers of AFB declined in the footpads of CFW mice after 12 months, they increased in the tupaia footpads, up to 2.44 x 19(9) AFB/g of tissue. The footpads of one tupaia were swollen, which on section revealed a granulomatous infiltration, including foamy and heavily infected macrophages. M. leprae were also seen in the branches of cutaneous nerves. Also AFB occurred in some viscera. Preliminary studies indicate that the AFB multiplying in tupaias are M. leprae.     

Experiences with Mycobacterium leprae soluble antigens in a leprosy endemic population

Rees and Convit antigens prepared from armadillo-derived Mycobacterium leprae were used for skin testing in two leprosy endemic villages to understand their use in the epidemiology of leprosy. In all, 2602 individuals comprising 202 patients with leprosy detected in a prevalence survey, 476 household contacts and 1924 persons residing in non-case households were tested with two antigens. There was a strong and positive correlation (r = 0.85) between reactions to the Rees and Convit antigens. The distribution of reactions was bimodal and considering reactions of 12 mm or more as 'positive', the positivity rate steeply increased with the increase in age. However, the distributions of reactions to these antigens in patients with leprosy, their household contacts and persons living in non-case households were very similar. These results indicate that Rees and Convit antigens are not useful in the identification of M. leprae infection or in the confirmation of leprosy diagnosis in a leprosy endemic population with a high prevalence of nonspecific sensitivity.     

The post-lepromin scar and its significance in the control of HD

In the past little attention has been paid to the post-lepromin scar (PLS) and its use in controlling Hansen's disease (HD), particularly in the prognosis, classification and measurement of CMI response. The immuno-information of the Mitsuda reaction is thought to be informative only in the extreme range of 10 + mm or in its absence. Previous studies have shown that the range of PLS formation increases proportionally to the degree of lepromin positivity. PLS positive HD patients have a stable form of the disease with good prognosis. Those unable to form a PLS have a marked tendency to downgrade towards the lepromatous form of HD. PLS formation appears to indicate a CMI response to M. leprae implying immunity. It is thought that there exists a correlation between the PLS and the lymphocyte transformation test (LTT), both reaching their optimum measurement three months after the M. leprae injection, either with lepromin or M. leprae suspension used for the anti-HD vaccine. It is proposed to study the use of the PLS in HD control programs on a trial basis with the objective of its general introduction as part of the management of HD control. Considerable improvements in the prognosis, classification and application of treatment can be expected from such a measure. The discovery of the armadilo as a source of M. leprae by Kirchheimer and Storrs facilitates the availability of lepromin A and its purified version, lepromin Ap. The relevant studies have shown that a 40 M/bact/ml lepromin A suspension should be used for the application of lepromin in control programs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Observations on the cultivation of M. leprae and M. tuberculosis in medium 'V' and 'V 1'.

Skin scrapings from five different active sites were collected from 14 leprosy patients and inoculated into medium V. Skin scrapings from three leprosy patients were inoculated into medium V 1. All the cultures were incubated at 8-10 degrees C. M. tuberculosis H37Rv, pretreatment isolates and streptomycin resistant strains were inoculated into medium V, with and without antibiotics, and incubated at 8-10 degrees C as well as 37 degrees C. Smears were made from the M. leprae and M. tuberculosis cultures at 0 hours and at different time points. The number of bacilli in the smears were counted. There was no increase in the number of M. leprae or M. tuberculosis in any of the cultures.     

Are viable Mycobacterium leprae present in lepromatous patients after completion of 12 months' and 24 months' multi-drug therapy?

A study was carried out to determine whether or not viable bacilli persist in MB patients treated with 12-month and 24-month multidrug therapy (MDT). In the first group, 60 untreated lepromatous patients who had an initial average bacterial index (BI) of 3+ or more were enrolled. At the completion of 12 months of MDT, skin biopsies were obtained and M. leprae concentrate was inoculated into the footpads of five thymectomized and irradiated (T900r) mice. Rees technique was used for the mouse footpad (MFP) experiment. Harvesting was done it the 6th, 9th and 12th months. Out of the 60 biopsies inoculated into mouse footpads to check the viability of bacilli, 2 skin biopsies (3.3%) showed significant growth and 10 (16%) showed equivocal growth. 27 patients also had nerve biopsies tested for growth in MFP studies. None of the inoculated nerve biopsies showed significant multiplication in the MFP experiments. However, 4 biopsies (14%) showed equivocal growth. In the second group, 20 patients had skin biopsies and 10 had nerve biopsies done at the end of 24 doses of MDT in order to test the viability of bacilli; none of the skin or nerve biopsies from these patients showed any growth. This study showed that M. leprae present in the tissues after 24 doses of MDT are not viable and the drug schedule of 24 doses is adequate to treat leprosy patients, irrespective of their BI. However, a small (3.3%) percentage of the patients with a high BI harbour viable bacteria in the skin after 12 doses of treatment. Since a large majority of the patients (38 patients) who had a high initial BI responded well to the treatment, it is important to find out the reason for the lack of response in two patients. One of the reasons may be the presence of drug-resistant strains. It is important to follow up on these patients for a longer duration to ascertain whether or not they would relapse.     

Effect of treatment on immune responsiveness in lepromatous leprosy patients

This study was performed in order to analyse whether the immune unresponsiveness to Mycobacterium leprae, largely seen in lepromatous patients, persisted after discharge from treatment. Lymphoproliferation and skin tests were performed using two mycobacterial antigens (M. leprae and BCG) in three groups of lepromatous patients grouped by treatment status. Forty-seven per cent of the lepromatous patients tested acquired reactivity to M. leprae after long-term treatment.     

Utility of beige mouse in leprosy research.

Dissemination of M. leprae to visceral organs is seen by four months onwards only in beige (C57BL/6/bgj) but not BALB/c mice following intravenous or intraperitoneal infections. Inoculation of the beige mouse derived M. leprae showed all the characteristics of M. leprae, including growth pattern in the foot-pads of BALB/c mice. M. leprae inoculated into foot-pads of beige mice multiplied faster than those in the foot-pads of BALB/c mice. The possibility of using beige mouse in chemotherapeutic studies in leprosy is discussed.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.     

The influence of topical steroid application on tuberculin skin reactions in healthy persons

Thirty-seven healthy persons were studied in order to evaluate the influence of topical steroid application on tuberculin skin reactions. Four areas measuring 4 cm in diameter were each treated with 50 micrograms of hydrocortisone cream 1%, or 50% micrograms of halcinonide (Halog) cream 0.1%, or 50 micrograms of unguentum cetacei simplex (cold cream), or not treated. The creams were applied once daily for 3 days before and one day after a tuberculin skin test. After 24 and 48 hours the area of induration were measured. We observed that application of unguentum cetacei simplex increased the size of the induration at the 24-hour reading, but not after 48 hours. Hydrocortisone cream 1% gave the same effect, whereas halcinonide cream (Halog) 0.1% caused ischaemia of the skin and reduced the induration of the skin test after 24 hours, but not after 48 hours. In 12 persons we found that simple rubbing of the skin with halcinonide cream base did not affect the size of the tuberculin skin reaction. In the present study we found that even very potent local steroid application on intact skin could only delay the development of tuberculin skin reactions, but could not diminish their size.     

Persistence of Mycobacterium leprae in the peripheral nerve as compared to the skin of multidrug-treated leprosy patients.

Skin and nerve biopsies obtained from 18 multibacillary (MB) and 16 paucibacillary (PB) cases of leprosy who had been fully treated by the WHO regimen were assessed for bacterial load using different staining techniques. In addition skin and nerve homogenates of 10 MB cases were tested for 'persistor' Mycobacterium leprae using immunosuppressed mice. While significant amounts of integral bacilli and BCG cross-reactive antigen of M. leprae were detected both in skin and nerve tissues of all the MB cases (100%), 56% of skin and 62% of nerve biopsies of PB cases also showed the presence of BCG cross-reactive antigen. Detection of 'persistor' M. leprae in 2/10 skin biopsies (20%) and 3/10 nerve biopsies (30%) of MB cases was thought to be unexpectedly high after 2 years of MDT.     

First lesion in experimental armadillo leprosy

Eighteen armadillos were infected intravenously with 10(8) M. leprae and 10 intracutaneously with 10(7) M. leprae. Among those which developed disseminated disease, a nodule at the site of inoculation was the first lesion noticed in 14 of the 16 infected intravenously and 4 of the 4 infected intradermally. It is possible that in human leprosy the first sign of infection is localized proliferation of M. leprae at the site of entry, and even nodule formation in lepromatous patients. It may be important to search for asymptomatic swelling or keloid-like lesions in skin or in nasal mucosa while screening a population for early leprosy.     

Role of immune complexes in alteration of lymphocyte subpopulation numbers and functions in experimental leprosy

Swiss albino mice (normal as well as thymectomised and irradiated were inoculated into the footpads with Mycobacterium leprae and divided into two main phases of study. Phase I comprised of animals not given preformed immune complexes (IC). Uninfected controls were however included. Phase II consisted of animals given in vitro prepared IC at zero day period (OdIC), three month period (3mIC) or six month period (6mIC) to both uninfected and infected groups. Splenic lymphocytes were isolated to quantify T and B cells and their responses to M. leprae antigen and four different mitogens. Significant decrease in T cell counts and blast transformation was seen in the M. leprae infected animals which were also administered with immune complexes. Immunosuppression by IC was therefore seen to be enhanced in the presence of M. leprae infection.     

Studies on lepromin and soluble antigens of M.leprae: their classification standardization and use.

Before the discovery of armadillo as a susceptible animal the source of M.leprae was limited and hence the use of lepromin was not common in the field. In recent times, the soluble antigens of armadillo-derived M.leprae have been used extensively in the field. Although the results of the study show that these antigens do not differentiate always a susceptible form from the resistant form, they are able to segregate the polar forms of leprosy. In a given field situation the criteria for diagnosis is so stressed that leprosy is overdiagnosed and within one year of follow up nearly half the number of cases are noted as not leprosy. Hence, in such situations lepromin reaction would be definitely a poor correlate with the type of leprosy. However, in hospital based studies the lepromin reaction has always been and would remain useful in confirming the classification (Sengupta et al 1984). Lepromins and M.leprae soluble antigens have gone through extensive standardization procedures. As these antigens contain mostly common mycobacterial antigens along with the M.leprae-specific antigens, these antigens are unable to specifically diagnose M.leprae infection. After purification of M.leprae from infected armadillo tissue, it was expected that the soluble antigen of M.leprae would probably be as useful as tuberculin. However, this was not found to be true in case of lepromin. Specificity for M.leprae has been noted in the epitopes (antigenic sites) on cross reacting molecules (12 kd, 18 kd, 28 kd, 35 kd, 36 kd) of mycobacteria (Ivanyi et al 1983; Watson 1989). These specific epitopes, if synthesized, could be of use as skin test antigens for determining M.leprae infection.(ABSTRACT TRUNCATED AT 250 WORDS)     

Leprosy: a review of laboratory and therapeutic aspects - Part 2

Leprosy is a chronic infectious condition caused by Mycobacterium leprae(M. leprae). It is endemic in many regions of the world and a public health problem in Brazil. Additionally, it presents a wide spectrum of clinical manifestations, which are dependent on the interaction between M. leprae and host, and are related to the degree of immunity to the bacillus. The diagnosis of this disease is a clinical one. However, in some situations laboratory exams are necessary to confirm the diagnosis of leprosy or classify its clinical form. This article aims to update dermatologists on leprosy, through a review of complementary laboratory techniques that can be employed for the diagnosis of leprosy, including Mitsuda intradermal reaction, skin smear microscopy, histopathology, serology, immunohistochemistry, polymerase chain reaction, imaging tests, electromyography, and blood tests. It also aims to explain standard multidrug therapy regimens, the treatment of reactions and resistant cases, immunotherapy with bacillus Calmette-Guérin (BCG) vaccine and chemoprophylaxis.     

Absence of mycobactin in Mycobacterium leprae; probably a microbe dependent microorganism implications

Ferric mycobactins were prepared from Mycobacterium phlei. Mycobacterium avium--intracellulare A and H, isolated respectively from armadillo and human leprosy specimens. Attempts were made to extract mycobactin from host grown M. leprae cells. The crude ferric mycobactin extracts were tested for growth supporting effect on the mycobactin dependent M. paratuberculosis strain ATCC 19698. Mycobactins prepared from M. phlei and the two M. avium--intracellulare strains had growth promoting effect on M. paratuberculosis. The same test organism did not grow in media supplemented with the extract prepared from M. leprae. Results indicate the absence of mycobactin from host grown M. leprae. Since M. leprae cells contain cytochrome c and since mycobactin is essential to growth of all mycobacteria, M. leprae might be considered as a microbe dependent microbe. It is proposed that secondary mycobacteria present in M. leprae infected humans and armadillos might provide mycobactin for in vivo multiplication of M. leprae.     

Severe local skin reactions to interferon beta-1b in multiple sclerosis-improvement by deep subcutaneous injection

Severe local skin reactions to subcutaneous injection of interferon beta-1b in multiple sclerosis are rare, and only 12 cases of severe skin reaction due to interferon beta-1b have been reported to date. We report two cases of severe skin reactions in multiple sclerosis patients following the injection of subcutaneous interferon beta-1b. In case 1, after five years of treatment, a painful indurated erythematous lesion appeared at the injection site on the left buttock. On histological analysis, the lesion showed septal and lobular panniculitis with lymphocytic infiltration. In case 2, cutaneous ulceration was surrounded by painful induration, which developed at the injection site on the right thigh after four years of treatment. The lesions resolved rapidly after discontinuation of interferon beta-1b treatment in both cases. Here, we review cases of similar lesions caused by interferon beta-1b reported in the literature, and discuss the characteristics, mechanism, treatment, and prevention of such lesions.     

ADCC function in Mycobacterium leprae inoculated normal and immunosuppressed mice

Normal and immunosuppressed mice were inoculated with Mycobacterium leprae obtained from untreated lepromatous patients. Besides monitoring the AFB counts in the footpads at 3,6 and 9 months post inoculation, antibody dependent cellular cytotoxicity (ADCC) function was studied. The ADCC function seen to be largely unaltered in the M. leprae infected animals, comparable to the observation made in human leprosy.