Culturas de bacilos ácido-álcool resistentes isolados de hematófagos infectados em leprosos: evidências de se tratar do bacilo de Hansen

The A. summarises the history of his first culture of acidfast bacillus isolated directly from leprosy lesions (Sample José) and refers about two samples recovered from guinea pig and white rat inoculated with said culture. Then the A. completes his previous descriptions of four cultures of acidfast bacilli isolated by him from ticks (Amblyomma cajaennense and Boophilus microplus, two cultures from each species) infected experimentally in lepers. The A. having found specimens of two species of Triatomidae (Triatoma infestans and Panstrongylus megistus) naturally infected with HANSEN bacillus in huts habited by lepers in the State of Minas Gerais (Dec. 1942), started a series of experiments, using larvae and nymphs of T. infestans bred in laboratory at the Instituto Oswaldo Cruz, to infect in active cases of leprosy, in the city of Rio de Janeiro, could obtain two new samples of cultures of acid-fast bacilli (Ns. 6 and 7 of his set). In this papaer the A. studies the biological properties of said cultures, proving that Penicilin has not effect upon them, like other substances. The sulphuric and acetic acids were used to purify some of the cultures, with good results, the cultures becoming more rich and growing faster. Potassium hydroxide Sodium (10% solution) was also used with success to isolate and to purify the cultures, but it seems that it affects the bacilli in some way. In flud glycerinated media the majority of such cultures produce velum suitable for the preparation of antigens for skin tests and for therapeutical use. At last the A. says that he is becoming convinced that the HANSEN bacillus is in cause, especially after thee evidences of culturing the bacillus from one patient, in different opportunities.

Endocardites sépticas: (contribuição etiopatogênica)

In the present paper the A. A. reviewed the more fundamental problems in the ethiopathogenic process of bacterial endocardits and report the bacte¬riological observations done by them in 20 cases of the disecase observed at the Section of Pathological Anatomy and Bacteriology of the Hospital S. Francisco de Assis in charge of the Instituto Oswaldo Cruz. The A.A. isolated Pneumococcus from 10 out the 20 cases, Gonococcus from 2, Staphylococcus from 2, Streptococcus from 5 and Friedlaender bacil¬lus from 1 . The A.A. mainly lay stress on the necessity of the bacteriological exa¬minations being made by a specialist, owing to the difficulties sometimes met with and to the consequences of a detailed examination of the isolated germ.

Tentativas de transmissão da lepra ao homem, por meio de Triatomídeos infectados em doentes lepromatosos: (nota prévia)

The A.A. started a new series of experiments upon the transmission of Leprosy to man by means of one of the more widespread hematophagi of the hinterland of Brazil, the Triatomidae. Two species of these insects were found naturally infected with Hansen's bacillus in huts of lepers in the interior of the State of Minas Gerais and one of the writers (S.A.) upon feeding the same insects on lepromatous cases could obtain two strains of acid-fast bacilli cultures smearing Lowenstein medium with the intestinal contains of the same. The first phase of the experiments lasted five months and the results, partially positive, are here describe. More than one hundred Triatomidae (Triatoma infestans and Panstrongylus megistus) bred in the Institute Oswaldo Cruz and fed in normal pigeons until convenient growth were put on lepromatous lesions, which they sucked many times, and them after one or more days they were put to be fed on selected regions of the skin of four negativated cases of leprosy. The arguments in favour and against the possibility of obtaining new lesions of leprosy in such burnet out patients were discussed. The A.A. are not authorized to draw any definite conclusions, but the few facts registered are worth of divulgation, in orther that other workers send their suggestions. Three out of the four volunteers showed moderate local reactions between 1 to 4 days after being sucked by the infected insectes. After five months experiments subcutanous lymph were obtained from the points where the insects have bitten. A very few acid-fast bacilli were found in such material. The patients, being kept in separation from infectious cases, will be followed up during months or a year in order to be detected any suspicious experimental lesions of leprosy.

A Leprolina "Souza-Araujo" no tratamento da lepra

The A. summarizes clinical records of four lepers treated with Leprolin “Souza-Araujo”, as follow: ist – Male, 28 years old, lepromatous case, treated during about 3 years, who received 80 injections of the antigen, being 52 by vene, 18 by muscle and 10 intradermally. Total about 100 cm³. Results: Absence of clinical signs, routine examinations negative for acidfast bacilli. Histopathology: from lepromatous became incaracteristic, with a few bacilli. Mitsuda test from negative became positive. 2nd – Male, 33 ys. old, lepromatous case. From September 1944 to February 1948 received 110 intravenous injections (57 cm³) and 10 cm³ perineurally to control neurites. His Mitsuda test was negative in 1944 and in 1945 became positive and remained so until 1948 (4 times controlled). Since Sept. 1946 became bacillus negative in his mucous, ear lobe and lymph nodes. Out of his lepromatous symptoms remained only mild atrophy of his hands. 3rd- Male, 30 ys. old, lepromatous case, treated since 1944. From 1946 to Feb. 1948 received 80 intravenous injections (high dosage: from 2 to 5 cm³), total 310 cm³ and 140 cm³ intramuscularly. His Mitsuda test was negative in 1943; became positive (from 1945 till 1948) after Leprolin treatment. Bacilloscopy became negative even in sections. Histopathology: the lesions from lepromatous changed to tuberculoid. 4th – Male, 17 years old, lepromatous case, received, from Dec. 1944 to Feb. 1948 only 88 intravenous injections of small doses of Leprolin (0,1 to 1,0 cm³). His Mitsuda remained negative from 1944 to 1946 and in 1947 became positive. Routine examinations negative for bacilli. Histopathology: the lepromataus lesions regressed to incaracteristic, without bacillus.

Estudo bacteriológico de escarros de leprosos-tuberculosos em tratamento com a Estreptomicina

Fourteen samples of sputum from fourteen lepers with pulmonary tuberculosis, were treated by PETROFF method and its sediments were smeared on LOEWENSTEIN medium and incubated at 37°C. These fourteen patients are under treatment by Streptomycin. They are advanced cases of active leprosy associated with pulmonary tuberculosis, according to X ray diagnosis. Between 15 to 45 days thirteen out of fourteen (92,85%) sputa gave cultures of acid-fast bacilli with all characteristics of KOCH'S bacillus, eugonic type. Nine out of thirteen positive cases produced eugonic colonies in all ten tubes smeared with each sample. These facts proved that Streptomycin did not affect the pulmonary flora. Three out of fourteen patients died within two months after positive cultures of KOCH'S bacillus. New fact - Three out of those thirteen positive patients gave non-chromogenic cultures, eugonic type, associated with chromogenic ones, quite similar to cultures of acid-fast bacilli isolated previously by the author from leprous material. One of the three patients who died showed in smear of fresh sputum only characteristics globies (globies of MARCHOUX not globi of NEISSER) of HANSEN'S Bacillus. Probably he died from leprous-pneumonia. The eugonic type cultures are being inoculated in guinea-pigs and the choromogenic ones, similar to leprosy-culture, will be inoculated in white rats and mice.

Isolamento e estudo experimental de duas novas culturas de bacilos ácido-alcool resistentes de muco nasal de leprosos

The A. described on December 14, 1951, his first culture of acid-fast bacillus isolated from nasal mucus of a leprous girl. in this paper the A. describes two new strains of acid-fast bacilli gotten also from nasal mucus of other two leprous girls, L3 cases. The first patient (layse S) had her mucus treated by petroff's method on December 16, 1952 and sown onto three tubes of loewenstein medium and in glycerin broth. After two weeks incubation at 37°C all three tubes of Loewenstein showed many punctiforme and pin-head yellowish colonies, whose microscopic examination proved to be of a pure acid-fast bacillus culture. This sample inoculated in rats and mice produced, after 55 days incubation, small tumors from which the culture was easily recovered. On April, 9, 1953 a new sample of nasal mucus of the same patient was sown in three tubes of Loewenstein. After two weeks incubation at 37°C all 3 tubes showed germination of small yellowish colonies of acid-fast bacilli. Within four months being gotten two samples of identical cultures in all smeared tubes of Loewenstein medium sown, proved that such cultures were not an ordinary ambient contamination. Second patient: - Maria N. After various sowing of different kinds of material from her, february 26, 1953 her nasal mucus treated by soda and sown onto Loewenstein medium, after 25 days incubation showed in only one tube, one small round colony, at first white, becoming creamy after three months. Transplants in various media grew at first slowly and after 2 or 3 generations grew faster. The "Layse" strain produced pellicle in glycerin broth and Dubos medium; the "Maria N." strain did not produce as yet. Both strains (Layse I and II, and Maria N.) gave weak positive Dubos test in half-an-hour and negative after 24 hours reading. Both were strongly positive when stained by Gram, Ziehl-Neelsen and Fontes methods. Both strains gave also positive fluoroscopy. These cultures are being studied. The A. concludes that, according to his experience, the slower growing cultures of acid-fast bacilli isolated from leprosy material, are the more suitable for experimental work. Aknowledgement. The A. thanks to Miss MARIA DE LOURDES SANTANA for her valuable collaboration in the studies of the described cultures.

Morfologia do Mycobacterium leprae hominis e do M. leprae muris: estudo baseado na microscopia electrônica e de contraste de fases

Hansen's Bacillus: By electron microscopy this bacillus shows membrane and halo, this being more visible when sorrounding the globi or bundles of bacilli; shows, also, free granules of various sizes which were before considered as dust of the dyes; shows external granules bound with the membrane and some times branching. By phases contrast microscopy examining leproma suspensions and subcataneous lymph at 400 x we saw many free granules with intense rotatory movement; granulated bacilli with screw, skip or stroke motion, producing slow progressive motion. All such elementes are surrounded by a halo, corresponding to the classical gloea. By a patient and delayed examination we were able to see that the internal granules are motile and help the progression of the bacilli, giving the impression that the cytoplasm is liquid. By a lasting observation we could see the larger granules form prolapse, like a pseudopode and abandon the bacilli and going in very rapid rotatory movement. There are branched bacilli; there are pedunculated fred granules like comets. The addition of a drop of formol at the preparation stops all movements. Stefansky's Bacillus: Repeated examination by RCA electron microscope, type EMU-25 of fresh suspensions of rat lepromas, led us to confirm the close relationship between human and murine leprosy agents. We examined also material from carabo (Lepra bubalorum) from Java, but due to fixation, the material was unsuitable for comparative studies. The Stefansky's bacilli showed also emmbranes and halos, internal or external granules (smaller than those of Hansen's bacillus). The bacilli shaded by chromium look thicker and shorter than those of Hansen. Due to electron bombardment both, Hansen's and Stefansky's baccilli suffer considerable alterations in their structure, showing black barrs of chromatin condensation at their extremities as also in their centers. By phase microscopy the Stefansky's bacilli showed elements with 1, 2 (bipolar), 3 or more internal small granules, developing identical movements as those of Hansen. The globi seem to be non-motile but the free bacilli appearing around the globi show intense movement. At 1000 x the examination is less satisfactory than at 400 x. The addition of formol solution in the preparation suppresses all movements, even the brownian, but the material becomes more suitable for the study of static morphology of the bacilli. CONCLUSION - The electron and phases contrast microscopy of leprous material from different types and phases of the disease may explain some of the unknown aspects of the biology and morphology of the bacilli.

Integrated control measures against Culex quinquefasciatus, the vector of filariasis in Recife

Integrated control measures against Culex quinquefasciastus have been implemented in a pilot urban area in Recife, Brazil. About 3,000 breeding sites found within the operational area were responsible for very high mosquito densities recorded during the pretrial period. Physical control measures have been applied to cess pits before starting a series of 37 treatments of the other sites with Bacillus sphaericus strain 2362, over 27 months. In spite of the difficulties due to environmental conditions, very significant reductions in preimaginal population of C. quinquefasciatus were achieved and, as a consequence, low adult mosquito densities were maintained for a relatively long period of time. Entomological and environmental data gathered in this pilot project can contribute to design an integrated mosquito control program in Recife city.

The associated microflora to the larvae of human bot fly Dermatobia hominis L. Jr. (Diptera: Cuterebridae) and its furuncular lesions in cattle

The microflora associated to furuncular lesions, larvae and pupae of Dermatobia hominis, as well as the relationships between parasite, host and microflora associated, as a comprehensive microsystem, has been studied. One hundred and two furuncular myiasis due to D. hominis larvae in several breeds of cattle were studied and the following bacterial species were significant: Staphylococcus aureus, S. epidermidis, S. warneri, Bacillus subtilis and Escherichia coli. Closely related, the microflora associated to 141 samples from first, second, third instar larva and both external surface and larval cavities has been studied. The representative associated microflora to the larvae were: S. aureus, B. subtilis, S. hycus and Moraxella phenylpiruvica, Moerella wisconsiensis, Proteus mirabilis and P. vulgaris, M. phenylpiruvica, M. wisconsiensis, P. mirabilis and P. rettgeri were the representative microflora associated to 64 pupae of D. hominis.

Evaluation of Three Mycobacterium leprae Monoclonal Antibodies in Mucus and Lymph Samples from Ziehl-Neelsen Stain Negative Leprosy Patients and their Household Contacts in an Indian Community

Mucus and lymph smears collected from leprosy patients (9) and their household contacts (44) in the Caño Mochuelo Indian Reservation, Casanare, Colombia, were examined with monoclonal antibodies (MoAb) against Mycobacterium leprae. The individuals studied were: 5 borderline leprosy (BB) patients, 4 with a lepromatous leprosy (LL), all of whom were undergoing epidemiological surveillance after treatment and 44 household contacts: 21 of the LL and 23 contacts of the BB patients. The MoAb were reactive with the following M. leprae antigens: 65 kd heat shock protein, A6; soluble antigen G7 and complete antigen, E11. All the samples were tested with each of the MoAb using the avidin-biotin-peroxidase technique and 3,3 diaminobenzidine as chromogen. The patients and household contacts studied were all recorded as Ziehl-Neelsen stain negative. The MoAb which showed optimal reaction was G7, this MoAb permited good visualization of the bacilli. Five patients with BB diagnosis and one with LL were positive for G7; of the BB patients' household contacts, 9 were positive for G7; 7 of the LL patients' household contacts were positive for the same MoAb. MoAb G7 allowed the detection of bacillar Mycobacterium spp. compatible structures in both patients and household contacts. G7 permited the visualization of the complete bacillus and could be used for early diagnosis and follow-up of the disease in patients.

Biological screening of Brazilian medicinal plants

In this study, we screened sixty medicinal plant species from the Brazilian savanna ("cerrado") that could contain useful compounds for the control of tropical diseases. The plant selection was based on existing ethnobotanic information and interviews with local healers. Plant extracts were screened for: (a) molluscicidal activity against Biomphalaria glabrata, (b) toxicity to brine shrimp (Artemia salina L.), (c) antifungal activity in the bioautographic assay with Cladosporium sphaerospermum and (d) antibacterial activity in the agar diffusion assay against Staphylococcus aureus, Escherichia coli, Bacillus cereus and Pseudomonas aeruginosa. Forty-two species afforded extracts that showed some degree of activity in one or more of these bioassays.

Screening of some plants used in the Brazilian folk medicine for the treatment of infectious diseases

Extracts of 13 Brazilian medicinal plants were screened for their antimicrobial activity against bacteria and yeasts. Of these, 10 plant extracts showed varied levels of antibacterial activity. Piper regnellii presented a good activity against Staphylococus aureus and Bacillus subtilis, a moderate activity on Pseudomonas aeruginosa, and a weak activity against Escherichia coli. Punica granatum showed good activity on S. aureus and was inactive against the other standard strains. Eugenia uniflora presented moderate activity on both S. aureus and E. coli. Psidium guajava,Tanacetum vulgare, Arctium lappa, Mikania glomerata, Sambucus canadensis, Plantago major and Erythrina speciosa presented some degree of antibacterial activity. Spilanthes acmella, Lippia alba, and Achillea millefolium were considered inactive. Five of the plant extracts presented compounds with Rf values similar to the antibacterial compounds visible on bioautogram. Of these, three plants belong to the Asteraceae family. This may mean that the same compounds are responsible for the antibacterial activity in these plants. Anticandidal activity was detected in nine plant extracts (P. guajava, E. uniflora, P. granatum, A. lappa, T. vulgare, M. glomerata, L. alba, P. regnellii, and P. major). The results might explain the ethnobotanical use of the studied species for the treatment of various infectious diseases.

Study of the safety, immunogenicity and efficacy of attenuated and killed Leishmania (Leishmania) major vaccines in a rhesus monkey (Macaca mulatta) model of the human disease

We have compared the efficacy of two Leishmania (Leishmania) major vaccines, one genetically attenuated (DHFR-TS deficient organisms), the other inactivated [autoclaved promastigotes (ALM) with bacillus Calmete-Guérin (BCG)], in protecting rhesus macaques (Macaca mulatta) against infection with virulent L. (L.) major. Positive antigen-specific recall proliferative response was observed in vaccinees (79% in attenuated parasite-vaccinated monkeys, versus 75% in ALM-plus-BCG-vaccinated animals), although none of these animals exhibited either augmented in vitro gamma interferon (IFN-g) production or positive delayed-type hypersensitivity (DTH) response to the leishmanin skin test prior to the challenge. Following challenge, there were significant differences in blastogenic responses (p < 0.05) between attenuated-vaccinated monkeys and naïve controls. In both vaccinated groups very low levels of antibody were found before challenge, which increased after infective challenge. Protective immunity did not follow vaccination, in that monkeys exhibited skin lesion at the site of challenge in all the groups. The most striking result was the lack of pathogenicity of the attenuated parasite, which persisted in infected animals for up to three months, but were incapable of causing disease under the conditions employed. We concluded that both vaccine protocols used in this study are safe in primates, but require further improvement for vaccine application.

In vitro antibacterial activity of a 7-O-beta-D-glucopyranosyl-nutanocoumarin from Chaptalia nutans (Asteraceae)

Ethanolic crude extracts from the roots of Chaptalia nutans, traditionally used in Brazilian folk medicine, were screened against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa by using the disk diffusion test technique. S. aureus with 14 mm inhibition zone was considered susceptible. E. coli and P. aeruginosa without such a zone were considered resistant. As a result of this finding, the ethanolic crude extract was fractionated on silica gel column chromatography into five fractions. The ethyl acetate fraction was active against S. aureus and Bacillus subtilis. Further column chromatography separation of the ethyl acetate fraction afforded 30 fractions, which were assayed against S. aureus. Fractions 16 and 17 showed inhibition zones with S. aureus, indicating the presence of active compounds, and were subjected to purification by repeated preparative thin layer chromatography. The pure compound 7-O-beta-D-glucopyranosyl-nutanocoumarin inhibited B. subtilis and S. aureus at concentrations of 62.5 µg/ml and 125 µg/ml, respectively. The antibacterial property of C. nutans appears to have justified its use for the treatment of wounds, which are contaminated through bacterial infections.

Antibacterial xanthones from Kielmeyera variabilis mart. (Clusiaceae)

The bioassay-guided fractionation of stems from Kielmeyera variabilis, traditionally used in Brazilian folk medicine, yielded assiguxanthone-B (1), kielcorin (4), 2,5-dihydroxybenzoic acid (3), and a mixture of xanthones containing assiguxanthone-B (1) and 1,3,5,6-tetrahydroxy-2-prenylxanthone (2) (1:1 w/w). The xanthone mixture inhibited Staphylococcus aureus and Bacillus subtilis at a concentration of 6.25 µg/ml. When tested alone, the minimal inhibitory concentration of assiguxanthone-B was 25 µg/ml against B. subtilis. Kielcorin and 2,5-dihydroxybenzoic acid were inactive against both strains. None of the fractions was active against Escherichia coli or Pseudomonas aeruginosa. Viable cells of S. aureus were reduced by a 1-3 log CFU/ml within 12 h after exposure of one to eight times the MIC of the xanthone mixture. It is not known whether the tetrahydroxy-2-prenylxanthone or other components of the xanthone mixture are responsible for the main antibacterial activity or whether additive or synergistic action is involved