Molecular model for studying the uncultivated fungal pathogen Lacazia loboi.

Lacazia loboi is an uncultivated fungal pathogen of humans and dolphins that causes cutaneous and subcutaneous infections only in the tropical areas of the Americas. It was recently found by phylogenetic analysis that this unusual pathogen is closely related to Paracoccidioides brasiliensis and to the other fungal dimorphic members of the order Onygenales. That original phylogenetic study used universal primers to amplify well-known genes. However, this approach cannot be applied to the study of other proteins. We have developed a strategy for studying the gene encoding the gp43 homologous protein of P. brasiliensis in L. loboi. The gp43 protein was selected because it has been found that this P. brasiliensis antigen strongly reacts when it is used to test sera from patients with lacaziosis. The principle behind this idea was to obtain the gp43 amino acid sequence of P. brasiliensis and other homologous fungal sequences from GenBank and design primers from their aligned conserved regions. These sets of primers were used to amplify the selected regions with genomic DNA extracted from the yeast-like cells of L. loboi from experimentally infected mice. Using this approach, we amplified 483 bp of the L. loboi gp43-like gene. These sequences had 85% identity at the nucleotide level and 75% identity with the deduced amino acid sequences of the P. brasiliensis gp43 protein. The identity of the 483-bp DNA fragment was confirmed by phylogenetic analysis. This analysis revealed that the L. loboi gp43-like deduced amino acid sequence formed a strongly supported (100%) sister group with several P. brasiliensis gp43 sequences and that this taxon in turn was linked to the other fungal sequences used in this analysis. This study shows that the use of a molecular model for investigation of the genes encoding important proteins in L. loboi is feasible.

Isolamento de bactérias produtoras de betalactamase de espectro estendido (ESBL) em amostras clínicas de origem ambulatorial e enfermaria / Isolation of bacteria producing extended-spectrum beta-lactamase (ESBL) in clinical samples of ambulatory and nursing home

A resistencia a antibioticos beta-lactamicos na familia Enterobacteriaceae, tem aumentado tanto no ambiente hospitalar quanto na comunidade. A inativacao desses antimicrobianos por enzimas como betalactamases, é o principal mecanismo que leva a resistaencia em bacilos Gram negativos. As bacterias produtoras de beta-lactamase de espectro estendido (ESBL), representam um dos mais importantes problemas de resistencia bacteriana nos hospitais brasileiros. A necessidade da implantacao de teste que apresentam alta acuracia e baixa custo é de suma importancia devido a dificuldade na deteccao de ESBL. As ESBL produzidas, principalmente por Klebsiella sp e E. coli, constituem um problema cada vez mais comum e com serias consequencias terapeuticas.

Persistencia de bacilos viaveis em pacientes de hanseniase multibacilar altamente baciliferos apos doze doses do esquema poliquimioterapico (PQT/OMS) / Persistence of viable bacilli in highly bacilliferous multibacillary leprosy patients after the twelve doses of multidrug therapy WHO/MDT

Trabalho de investigacao, com o obejtivo de avaliar a eficacia do tratamento de doze doses com o esquema poliquimioterapico proposto pela Organizacao Mundial da Saude em 10 pacientes de hanseniase virchoviana em indice baciloscopico igual ou maior que 4. Os pacientes foram submetidos ao tratamento por 12 meses, regularmente. Apos o final do tratamento, foi feita retirada de material e inoculacao em coxim plantar de camundongos, conforme a tecnica de Shepard. Os resultados mostraram persistencia de bacilos viaveis em tres pacientes. Os autores, apesar da amostra pequena, observam que os mesmos resultados tem sido encontrados por outros autores surgerindo que talves seja necessario tratar os pacientes com altos indices baciloscopico no inicio do tratamento, por mais tempo, na tentativa de evitar possiveis recidivas.

Persistence of viable bacilli in highly bacilliferous multibacillary leprosy patients after the twelve doses of multidrug therapy WHO/MDT

The present study evaluate efficacy of twelve doses of multidrug therapy, as suggested by World Health Organization, in ten lepromatous leprosy patients with bacilloscopic index equal or higher than 4. The patients have taken the doses in twelve month-doses regularly. At the end od treatment a specimen was collected by punch biopsy and inoculated into mouse hind footpad acoording to Shepard's technique. Persistence of viable bacilli was demonstrated in three patients. In spite of the small number of patients, same results have been found by other authors, showing that in highly bacilliferous leprosy patients, at the beginning of treatment more doses may be required to prevent relapses.

Comparative experimental infection of Lacazia loboi in BALB/c and B10.A mice.

Both hind foot pads of BALB/c and B10.A mice strains, were inoculated with a fungal suspension of Lacazia loboi obtained from a Jorge Lobo's disease patient. The suspension had 9 x 105 cells/ml and its viability index was 45%. The animals were sacrificed at different time periods varying from 24 h to 18 months after inoculation. The BALB/c mice developed an extensive granulomatous infiltrate, similar to the disease in humans, that progressively evolved. The number of fungal elements also increased as the disease progressed, and after the seventh month of inoculation, macroscopic changes of the foot pads were evident. Although the B10.A mice developed an exuberant granulomatous infiltrate, macroscopic changes were not detected. The number of fungal cells in the infected tissues increased in number, but they were lower then the numbers found in the BALB/c strain. The viability indexes were also lower for the B10.A strain. Considering the histopathological findings, the presence of macroscopic changes and the great amount of fungal cells in the infected tissues, the authors concluded that the BALB/c mice strain was more susceptible to L. loboi infection than the B10.A strain.

Evaluation of the vital staining method for Lacazia loboi through the experimental inoculation of BALB/c mice

The viability of the currently unculturable fungal pathogen Lacazia loboi can be determined by means of fluorescein diacetate-ethidium bromide (FD-EB) staining. This technique can be used in experimental study of the mycosis, in attempts to cultivate the fungus and in attempts to gauge the success of therapies. In the present study, the potential applications of FD-EB vital staining were studied using a proposed murine experimental model of lobomycosis. BALB/c mice were inoculated in the footpads with an L. loboi suspension that appeared in FD-EB staining to have lost viability after being held for 15 days at room temperature, whereas a control group of mice was inoculated with apparently viable fungi. The animals were killed after 1, 2, 4, 6, 8, 11 and 13 months. Both inoculated footpads were excised, one for determination of viability and the other for histological examination. In the group injected with nonviable material, no active infection was noted; inoculation sites showed small quantities of macrophage-laden infiltrate and no viable fungal cells. In the control group, the infection progressed with exuberant infiltrates surrounding copious fungal growth, most of which consisted of cells staining as viable in FD-EB. These results suggest that the FD-EB vital staining is a sensitive and specific method that can reliably be used for viability determination in L. loboi

Estudo comparativo de inoculação do Lacazia loboi em camundongos BALB/c e B10. A, a partir da analise histopatológica, número de fungos e índice de viabilidade / Comparative study of inoculation Lacazia loboi in BALB / c mice and B10. A from the histopathological analysis of number and viability of fungi indices

O presente estudo teve por objetivos avaliar a susceptibilidade de camundongos isogenicos das linhagens BALB/c e B10.A a infeccao pelo fungo Lacazia loboi (L. loboi), mediante estudo do aspecto histopatologico das lesoes produzidas, numero de fungos e indice de viabilidade obtidos nos diferentes periodos de sacrificio e tambem verificar se o padrao de resposta aos parametros avaliados, nos animais isogenicos, e homogeneo, de modo a permitir correlacao dos resultados entre si. Ambos coxins plantares traseiros de quarenta camundongos BALB/c e quarenta e quatro B10.A foram intradermicamente inoculados com 0,03ml de uma suspencao fungica. A suspensao foi obtida a partir de um unico fragmento de lesao, coletado de um paciente portador da doenca de Jorge Lobo.

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 percent 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.

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

Prevalência de sulfono resistência em pacientes hansenianos do município de Bauro, estado de Säo Paulo / Prevalence of resistence sulphone in leprosy patients of the municipally of Bauro, state of Säo Paulo

O presente teve como objetivo determinar a prevalência de sulfono-resistência na populaçäo de pacientes hansenianos virchovianos do município de Bauru, Estado de Säo Paulo. Em uma populaçäo total de 349 pacientes virchovianos, em registro ativo, dos quais 205 já estavam com mais de 5 anos de tratamento (pacientes com risco de resistência), 30 (14,63 por cento) apresentaram suspeita clínica de resistência (Indice Baciloscópico igual ou maior que 3+). 10 pacientes mostraram seus bacilos resistentes a diamino difenil sulfona (DDS), evidenciando uma proporçäo de 2,86 por cento. Em funçäo dos dados os autores concluem e recomendam a urgência no processo de implantaçäo da poliquimioterapia no país

Curva de crescimento padräo do Mycobacterium leprae em coxim plantar de camundongos Mus musculus, estirpe BALB/c, isogênicos, do biotério do Instituto Lauro de Souza Lima, Bauru, Estado de Säo Paulo / The standard growth curve of the Mycobacterium leprae in plantar cushion of Mus musculus mice, BALB/c strain, isogenic, at the vivarium of Instituto Lauro de Souza Lima, Bauru, Säo Paulo state

Os autores exibem a curva de crescimento do Mycobacterium leprae em coxim plantar de camundongos Mus musculus, estirpe BALB/c, isogênicos, nas condiçöes ambientais do biotério do Instituto Lauro de Souza Lima, em Bauru, estado de Säo Paulo. Os resultados evidenciaram um retardo no início do crescimento, o que pode ter ocorrido em funçäo da ausência de uma temperatura ótima de crescimento para o M. leprae