Inoculation of Lacazia loboi into the subcutaneous tissue of the hamster cheek pouch.

The subcutaneous tissue of the hamster cheek pouch, a site of immunologic privilege, has been used to investigate the potential infectivity of different types of parasites. It has been demonstrated that the implantation of fragments of lesions induced by the fungus Lacazia loboi, the etiologic agent of Jorge Lobo's disease, into the subcutaneous tissue of the hamster cheek pouch resulted in parasite multiplication and dissemination to satellite lymph nodes16. Here we describe the evolution of lesions induced by the inoculation of the isolated fungus into this immunologically privileged site. The morphology of the inflammatory response and fungal viability and proliferation were evaluated. Inoculation of the fungus into the cheek pouch induced histiocytic granulomas with rare lymphocytes. Although fungal cells were detected for a period of up to 180 days in these lesions, the fungi lost viability after the first day of inoculation. In contrast, when the parasite was inoculated into the footpad, non-organized histiocytic lesions were observed. Langhan's giant cells, lymphocytes and fungal particles were observed in these lesions. Fungal viability was observed up to 60 days after inoculation and non-viable parasites were present in the persistent lesions up to 180 days post-inoculation. These data indicate that the subcutaneous tissue of the hamster cheek pouch is not a suitable site for the proliferation of Lacazia loboi when the fungus isolated from human tissues is tested.

Inoculation of BALB/c mice with Lacazia loboi.

In a previous study, the authors inoculated Swiss mice with Lacazia loboi (L. loboi) and succeeded in maintaining a granulomatous infiltrate and viable fungal cells up to one year and six months after inoculation. Considering the experimental work on paracoccidioidomycosis, 0.03 ml of a fungal suspension obtained from a biopsy of a Jorge Lobo's Disease patient were inoculated into both hind foot pads of 32 six week-old BALB/c mice of both sexes. The animals were sacrificed 1, 4, 7 and 10 months post inoculation. The suspension contained 1.3 x 10(6) fungi/ml and presented 38% viability. Seven months after inoculation, most of the animals presented profuse infiltrates consisting of isolated histiocytes, foreign body and Langhans' giant cells and a large number of fungi, most of them viable. Emergence of macroscopic lesions was observed during the 8th month. Based on fungal count, viability index before and after inoculation, presence of macroscopic lesions and histopathological findings similar to the findings in humans, the authors believe that BALB/c mice may be a good experimental model to study Jorge Lobo's Disease, mainly regarding therapeutic evaluation.

Jorge Lobo's disease: experimental inoculation in Swiss mice.

Sixty-four isogenic Swiss mice were intradermically inoculated in both hind foot pads. The inocula, consisting of fungal suspensions from biopsies obtained from Jorge Lobo's Disease patients, had the total number of fungi and the viability index determined using a Neubauer chamber and the fluorescein diacetate-ethidium bromide technique (FD-EB), respectively. The animals were sacrificed at times ranging from ten days to eighteen months after inoculation. The cellular infiltrate, mainly consisting of macrophages containing fungi, increased progressively up to end of the study; however, no macroscopic alterations were observed in the inoculated feet. After nine months, small numbers of Langhans' giant cells started to appear in the infiltrate. A considerable number of fungi was observed at the end of the experimental period, but only a few were viable when stained by the FD-EB technique. This fact suggests that there is a multiplication of fungal cells, which are destroyed by the macrophages but remain in the tissue for a long time due perhaps to the difficulties in their elimination. These findings led us to conclude that in spite of the maintenance of the infection in these animals, Swiss mice cannot be considered an ideal model to study Jorge Lobo's Disease. However, the authors call attention to the possibility of other mouse strains being more susceptible to Paracoccidioides loboi.

[An estimation of the prevalence of hanseniasis by research in non-specific demand for health services]. / Estimativa da prevalência da hanseníase pela investigação em demanda inespecífica de agências de saúde.

In view of the importance of knowing the prevalence and incidence rates of a disease to learn about its behavior and control at the collective level, a study was undertaken to determine the occurrence of hanseniasis among the clients of health agencies and to explore the use of this methodology for estimating the epidemiologic "iceberg" of the disease, i.e., the total number of cases including those that are not officially reported. The city of Taubaté in the Paraíba Valley, State of S. Paulo, Brazil, was chosen for the study. All clients aged 15 years or older were screened regardless of variables such as sex, age, social condition or marital status. The study was based on what is known about the populational distribution of the disease and the characteristics of health services. In view of the local peculiarities of operationalization (e.g., identity of the different clientele, availability of offices, hours of greatest flux), the sampling process used was simple randomization. The patients with active disease detected, 40 of the 10,013 persons examined, correspond to a prevalence of 3.99/1000, with a confidence interval (at the 5% level of reliability) of 3,365 to 4,625/1000, indicating that the minimum estimated increase of prevalence is of the order of 52% and the maximum estimated increase is of the order of 109%.(ABSTRACT TRUNCATED AT 250 WORDS)

[The Rubino reaction. Criterion for inactivation in lepromatous patients]. / Reação de Rubino. Critério de branqueamento para pacientes virchowianos.

Approximately 55% of active lepromatous patients respond positively to the Rubino reaction. With arrested cases this rate of positivity lowers considerably to about 15%. In an effort to associate this reaction with the presence of bacilli, a study of 796 cases was undertaken. The patients were divided into: a control group, active tuberculoid cases, arrested tuberculoid cases, active borderline cases, arrested borderline cases, active lepromatous cases, and arrested lepromatous cases. The patients were submitted to the following tests: Rubino reaction, presence of cryoglobulins, and VDRL and PCR positivity. By the results obtained we may conclude that: a) A positive Rubino reaction may be present in all the forms of leprosy studied, this reaction having an inverse relationship with the organism's resistance to "M. leprae". b) The Rubino reaction has specificity to leprosy. c) This reaction does not depend on the number of bacilli present in the host. d) A positive Rubino reaction is not related to the presence of cryoglobulins in the serum, nor to VDRL or PCR positivity, nor to the length of time the patient's disease has been arrested. The authors present these findings and suggest that this reaction be used as one of the criteria for determining cure.

[Developmental aspects of the Rubino reaction]. / Aspectos evolutivos de reação de Rubino.

The Rubino reaction was studied in 178 leprosy patients to correlate its positivity, clinical form, E.N.L., and time and activity of the disease. The patients studied were classified according to the criteria established by the 1953 Madrid Congress. The results show that the tuberculoid patients presented negative Rubino reactions regardless of any of the parameters studied. The borderline patients showed 9% positive Rubino reaction not related to any of the observed parameters studied. The lepromatous patients showed a significant difference of positivity to the Rubino reaction when grouped as to the disease's active or non-active presence. Active lepromatous patients showed 55% of positivity to the Rubino reaction whereas patients considered clinically inactive showed only 6% of positivity. The fact that the Rubino reaction is negative in the majority of patients who are clinically cured would indicate that this test could be included as part of the criteria used in determining which clinically cured lepromatous patients can discontinue medication without risking a relapse.

[Cellular immunity in leprosy patients]. / Estudo da imunidade celular em pacientes branqueados.

Forty eight clinically arrested lepromatous patients were submitted to several tests to evaluate cellular immunological competence (PPD, Trichophytin, DNCB, determination of the percentage of T lymphocytes in the peripheral blood, and early and late reactions to the lepromin). The results of the Trychophytin test and of the lymphocyte percentage test did not differ from those of the control group, whereas the results to the PPD test were more often positive for the arrested Virchovian cases. All the patients presented positivity to the DNCB test, and none of them showed positivity to the Fernandez reaction. As for the late reaction to the lepromin, all the patients were negative, although in histopathological testing four degrees of reactivity was observed ranging from focal, non specific inflammatory reaction to granulomatous histiocytic with an epithelioid outline. By means of these results the authors were able to confirm that: 1. There is no cross reactivity between the PPD and lepromin tests. 2. Sulfone therapy does not interfere in immunocellular results for the tests employed. 3. Lepromatous patients respond like the general population to the unspecific sensitivity tests utilized. 4. The finding of histiocytic reaction with epithelioid outline in some biopsies of Mitsuda reaction in lepromatous patients, suggests the presence of a sub-population within this group, probably Borderline patients that suffered during the infection's evolution downgrading of theirs clinic and histological characteristics.

[The teaching of leprosy in medical schools]. / O ensino da hansenologia nas faculdades.

All post graduated young doctors should be conscious of our major public health problems and should be able to diagnose and treat these endemic diseases. Hanseniasis is an infectious disease that has a special affinity by peripheric nerves and for that a high potential to produce disability and deformity leading to stigma and social out cast. There are 5,546,000 known patients on the world and the WHO estimates that the real figure is around 12 million in Brasil the registered number of patient is 259,000 but realistic estimations would increase it up to half a million. The disease spreads out all over Brasil but, in absolute figures the southeast region has the highest load. To cope with this problem one the basic actions is training and the teaching of hanseniasis in medical school is essential. As a systemic disease it should be present in all specialities involved leading to an increase of time devoted to the teaching of hanseniasis. This approach, which could be extended to other endemic diseases, will contribute to the eradication of many diseases that afflict our population. There is no reason for public health budget if there is no trained personal to use it.

[Myocardial disease, heart failure and reactional tuberculoid leprosy]. / Seção anátomo-clínica.

A 60-year-old woman was admitted with congestive heart failure, essential hypertension and abdominal distension. Her son reported that she appeared with red spots in the body and that she was under dapsone therapy. Seven months ago there was sudden increase of the skin lesions. In the 11th day after admission she underwent a stroke that progressed to decerebration and she expired on the fourth day. Autopsy confirmed CHF due to chronic myocarditis related to Chagas' disease. Aneurysm of the apical region of the left ventricular chamber was also observed leading to thrombosis and systemic embolism with brain and spleen hemorrhagic infarct. In the encephalous there was edema, uncus herniation and hemorrhagic infarct of the brain stem. The skin lesions were due to reactional tuberculoid hanseniasis (RHT) with focal lesions in axillary lymphnodes, nasopharyngeal mucosa and in the posterior tibial nerve. The pathogenesis of RHT is discussed as well as its differentiation with the BT group of Ridley and Jopling and its probably relationship with the secondary tuberculoid hanseniasis reported by Ridley. The focal lesions are also discussed with END to the involvement of a peripheral nerve trunk what is said to be uncommon in this form of Hansen's disease.

Oral lesions in borderline and reactional tuberculoid leprosy.

Thirty patients, fifteen with borderline and fifteen with reactional tuberculoid leprosy, were submitted to clinical and histopathologic studies of the buccal mucosa for detection of specific lesions. Five reactional tuberculoid and eight borderline patients presented specific conditions characterized by chronic granulomatous lesions with bacilli, chronic granulomatous lesions without bacilli, and nonspecific chronic inflammatory lesions with bacilli. The infiltrate had small extension, low bacterial levels and the mucosa, with the exception of one case, did not show ulceration. These results suggest that in the reactional tuberculoid and borderline patients the buccal mucosa is not an important source of bacilli elimination.

[Maintenance of armadillos in captivity and results of the inoculation of Mycobacterium leprae]. / Manutenção de tatus em cativeiro e resultados de inoculaçãso do Mycobacterium leprae.

The authors report their experiences in raising armadillos and the results obtained in inoculating them with M. leprae. They begin by reporting on the origin of the animals, how they were captured, and their efforts until they found the right kind of housing for the armadillos. They also tell of their attempts to develop the correct feeding procedures. The result is the feed used now in their laboratory: chicken feed mixed with bread, eggs, meat and milk. Along with this, they report on the way each animal is registered and the examination it undergoes upon admittance, namely, recording of its weight, rectal temperature, feces examination, hemogram and hemossedimentation. They also tell how animals are immobilized so as to take blood samples for the inoculations. Lastly, they present the results of the inoculations with M. leprae in three armadillos of the Euphractus sexticinctus species and 26 of the Dasypus novemcinctus species, with special reference to an armadillo of the D. novemcinctus species, which had systemic infection 15 months after being inoculated subcutaneously and intravenously. Bacilli were found in large quantities in various organs like the skin, lymph nodes, liver, kidneys and spleen. No bacilli were found in the myocardium, pancreas, thyroid and testicles. The authors are of the opinion that this is the first armadillo of the D. novemcinctus species to present experimental systemic infection with M. leprae in South America. Another armadillo, which has been inoculated with a suspension of 1.2 X 10(8) bacilli by the intracardiac route already showed nodules with bacilli on the abdominal wall 10 months after being inoculated. They believe that the results already obtained indicate that soon they will be able to provide material for institutions in Brazil as well as other countries that are interested in the study of M. leprae and in the preparation of the Mitsuda antigen which is so vital to the control of Hansen's disease.

A controlled trial to compare the therapeutic effects of dapsone in combination with daily or once-monthly rifampin in patients with lepromatous leprosy.

In this controlled trial in 35 patients with lepromatous leprosy the therapeutic effects of adding rifampin 450 mg daily (Regimen A) or 1200 mg once a month (Regimen B) to a standard dapsone regimen of 50 mg daily were practically identical. Moderate to marked clinical improvement was observed in 88% and 83% of the patients treated with Regimens A and B respectively. The average rates of decrease in the MI of the skin smears and nose-blow smears were similar. The average decreases in the BI of the skin smears were 0.7 and 0.6 in patients on the Regimen A and B respectively. Following 6 months' treatment with Regimens A and B the average decreases in the Logarithmic Bacterial Indexes of Biopsies were 4.7% and 7% respectively. The once-monthly rifampin schedule was well tolerated and did not lead to "flu" syndrome, anuria, oliguria, hemolytic anemia, thrombocytopenia, or anaphylactic shock. This trial revealed the satisfactory efficacy, good tolerability, and practicability of a supervised once-monthly 1200 mg single oral dose rifampin schedule as a component of combination regimes for the initial treatment of patients with lepromatous (LLs and LLp) leprosy.

Serial Mitsuda tests for identification of reactional tuberculoid and reactional borderline leprosy forms.

The authors studied the Mitsuda reaction in 37 leprosy patients (18 reactional tuberculoid, 19 reactional borderline cases) and compared the results with clinical findings, histopathology and bacilloscopy. Evaluation of the Mitsuda reaction was carried out on days 30, 60, 90 and 120. Most of the reactional tuberculoid patients showed a Mitsuda reaction of +3 in opposition to the reactional borderline patients who showed only +. Bacilloscopic analysis revealed that in 75% of the reactional tuberculoid cases there were rare or no bacilli; bacilli were present in 95% of the reactional borderline cases. The authors conclude that reactional tuberculoid cases have a greater ability to clear bacilli than reactional borderline cases, and that the Mitsuda reaction is a useful tool for the differentiation between these two types of leprosy.

Results of a surveillance system for adverse effects in leprosy's WHO/MDT.

The implementation of the World Health Organization's multidrug therapy (WHO/MDT) in Brazil began slowly and gradually in 1986, and in 1991 it was adopted officially by the Brazilian Ministry for Health. After 1991, during the intensive phase of WHO/MDT implementation, there was some concern about the number of cases of renal failure observed in several Brazilian states, including some fatalities. This was the motive behind the state of São Paulo's Health Department's decision to carry out a study that would evaluate not only the incidence rate of adverse effects of rifampin in relation to kidney function but also in relation to the use of WHO/MDT in general. Due to the existence in the state of São Paulo of health services with a program for the control of Hansen's disease and an organized and stratified system of epidemiological surveillance, it was possible to elaborate a subsystem for data collecting. During the period from July 1991 to December 1993, 20,667 patients were treated with WHO/MDT. Among this group there were 127 notifications considered as adverse effects, mainly: "flu"-like syndrome (54), acute renal failure (20), cutaneous reactions (15), toxic hepatitis (15), gastrointestinal complaints (8), hemolytic anemia (6), methemoglobinemia (4), thrombocytopenic purpura (2), hypotension (2) and disseminated intravascular coagulation (1). There was a predominance of adverse effects among multibacillary (MB) patients and the majority of the reactions occurred before the 6th dose; 82.7% of MB patients had had previous treatment with dapsone and rifampin and, due the fact that most severe reactions were related to rifampin, a booster mechanism could be an explanation for this occurrence. So far, there are seven published reports on renal failure in the world, and in Brazil only in the state of Søao Paulo there were 20 cases reported among 20,667 patients under WHO/MDT treatment, This striking difference deserves a better explanation, but in no way do these reports undermine the positive aspects of WHO/MDT. However, the authors believe that a world alert about its possible serious side effects is not only necessary but ethically required.

Experimental reproduction of the Jorge Lobo's disease in BAlb/c mice inoculated with Lacazia loboi obtained from a previously infected mouse.

Long-term maintenance of Lacazia loboi in the laboratory has not been reported. We report here the use BALB/c mice to maintain the Lacazia loboi for extended period of time. Eight to ten week-old mice were inoculated intradermally in both hind footpads with a fungal suspension from a macerated footpad obtained from an original mouse previously infected with the fungi and sacrificed 8 months after inoculation. The inoculated animals were sacrificed at different time intervals, footpads were excised, the right one was submitted to histopathological examination and the left one was macerated in sterile saline for fungal count and viability index determination. The inoculated animals presented the histopathological picture identical to the mice previously inoculated with material from human lesion. Granulomatous infiltrates with predominance of macrophages and giant cells were observed. The granulomas evolved progressively as observed in the different times of sacrifice. After 7 months of inoculation, macroscopic lesions were observed, and the number of fungi obtained from macerated footpads was higher than the number of inoculated fungi. The pattern of lesion development was similar to what was observed in animals infected with a fungal suspension obtained from a human lesion. Considering the histopathological findings, the clinical manifestations, and the finding of a higher number of fungi obtained than the inoculated into footpads of each mice, we believe the BALB/c mice strain is as an excellent way to maintain L. loboi in laboratory. Moreover, even after serial passages of the fungi, the granulomatous lesions are reproduced consistently in laboratory conditions.

No evidence of linkage between Mitsuda reaction and the NRAMP1 locus.

Thirty sib-pairs were ascertained through unrelated lepromatous probands. They consisted of 22 healthy individuals and 8 leprosy patients. The Mitsuda reactions of all sibs were evaluated both macroscopically and histologically, and high molecular weight genomic DNA was extracted from the white blood cells of all sib-pairs. Three DNA polymorphisms identified by polymerase chain reaction (274C/T, D543N, 1729 + 55del4) were used as chromosome markers at the NRAMP1 locus. Sib-pair comparisons did not disclose any sign of close linkage between the Mitsuda reaction and the genetic markers.

[Fernandez and Mitsuda reactions in patients with leprosy and their contacts]. / Estudo das reaçóes de Fernandez e Mitsuda em pacientes hansenianos e seus contatos.

Hanseniasis patients and contacts of Virchowian patients, totaling 467 persons have been submitted to Mitsuda's reaction. These individuals have been divided into five groups: group 1 compounded of 59 persons, relatives of Virchowian patients; group 2 - 171 persons, employees of the Hospital Lauro de Souza Lima working there for more than one year; group 3 - 127 hanseniasis patients in activity, Mitsuda negative; group 4 - 63 hanseniasis patients in activity, Mitsuda positive; group 5 - 47 Virchowian patients, inactive, Mitsuda negative. The analysis of the data showed that: a) hanseniasis patients, both Mitsuda positive and negative, do not present positivity to Fernandez's reaction; b) early reaction can be observed more frequently in individuals of group 1 than in those of group 2. Mitsuda's antigen causes the reversal of Fernandez's reaction in about 30% of indirect contacts; c) the percentage of negative Mitsuda's reactions (without induration) does not differ between groups 1 and 2, though indurations smaller than 5 mm have been as many as 10 times more frequent in individuals of group 1. A second application of the antigen in Mitsuda negative persons showed the reversal of the test in 100% of the tested subjects.