PCR-based identification of Mycobacterium murphy causing Canine Leproid Granuloma Syndrome in Niterói, southeast Brazil - case report / Identificação baseada em PCR de Mycobacterium murphy causando Síndrome do Granuloma Lepróide Canino em Niterói, sudeste do Brasil ˗ Relato de Caso

Canine Leproid Granuloma Syndrome (CLGS), also known as canine leprosy, is a cutaneous nodular infectious disease caused by Mycobacterium sp.. Despite being reported worldwide, it is still quite unknown and underdiagnosed. Diagnosis may be achieved by cytopathology or histopathology of skin lesions, but identification of the infectious agent is complex, since bacterial in vitro growth is not possible, relying upon molecular techniques such as PCR to confirm Mycobacterium DNA in the sample. We report a CLGS case in Niteroi, Rio de Janeiro state, Brazil, diagnosed by cytopathology and submitted to molecular identification of the agent. PCR amplification of hsp65 gene was performed and revealed 100% genetic homology to M. murphy strain. This is the first CLGS report with molecular identification in Rio de Janeiro state, and this finding should raise awareness about CLGS as a differential diagnosis among granulomatous skin diseases in this region.(AU)

A síndrome de granuloma leproide canino (SGLC), também conhecida como lepra canina, é uma doença infecciosa cutânea nodular causada por Mycobacterium sp. Apesar de ser relatada mundialmente, ainda é bastante desconhecida e subdiagnosticada. O diagnóstico pode ser conseguido por citopatologia ou histopatologia de lesões cutâneas, mas a identificação do agente infeccioso é complexa, uma vez que o crescimento in vitro bacteriano não é possível, dependendo de técnicas moleculares como a PCR para confirmar o DNA de Mycobacterium na amostra. Relatou-se um caso da SGLC em Niterói, estado do Rio de Janeiro, Brasil, diagnosticado por citopatologia e submetido à identificação molecular do agente. Foi realizada amplificação por PCR do gene hsp65, que revelou 100% de homologia genética com a cepa M. murphy. Este é o primeiro relato da SGLC com identificação molecular no estado do Rio de Janeiro, o que mostra a importância de se acrescentar a SGLC ao diagnóstico diferencial das doenças granulomatosas de pele nessa região.(AU)

Leprosy and AIDS: two cases of increasing inflammatory reactions at the start of highly active antiretroviral therapy.

Reported here are the cases of two HIV-positive patients with skin lesions suggestive of leprosy, based on clinical and pathological analysis, which worsened during the few weeks following initiation of highly active antiretroviral therapy. The lesions improved after a few weeks of multidrug therapy for leprosy. Mycobacterium leprae was confirmed by polymerase chain reaction analysis of blood in case 1 and of a biopsy sample in case 2. Neither Mycobacterium avium complex nucleic acid, which is usually associated with immune restoration syndrome, nor mycobacterial cutaneous manifestations were detected in either case.

Molecular epidemiology of methicillin resistant Staphylococcus aureus isolated from newborns in a hospital in Rio de Janeiro, Brazil

Methicillin resistant Staphylococcus aureus (MRSA) is an organism that is frequently transmitted in hospitals and perinatal units. The MRSA is considered a public health problem in neonatology because of its strong potential for dissemination in the wards associated with high rates of morbidity and mortality. In this study we describe the bacteriological, epidemiological and molecular characteristics of MRSA isolated from anterior nares and blood cultures of newborns hospitalized in a public maternity hospital in the city of Rio de Janeiro, Brazil. The frequency of MRSA isolated from nasal swabs of newborns was 47.8 percent (43/90). The genetic analysis of MRSA strains from anterior nares, showed 8 different pulsed field gel electrophoresis patterns (PFGE). Upon analysis of PFGE patterns of the 12 MRSA strains isolated from blood cultures, 8 different patterns were observed, 9 (75 percent) strains were genetic related to nasal secretion isolates patterns. In conclusion, our data demonstrate the importance of screening of newborns for the presence of MRSA in Brazilian hospitals and the usefulness of genetic typing of these pathogen during epidemiologic studies. This should lead to a better knowledge on the significancy and spreading of MRSA in the hospitals

Pathogenesis of nerve damage in leprosy: genetic polymorphism regulates the production of TNF alpha.

Studies carried out over the last decade have strongly suggested that TNF alpha both overtly participates in the cell-mediated immune response against Mycobacterium leprae, and is overproduced during reaction. In addition, reactions are intimately related to the onset of nerve damage. Finally, TNF alpha has been implicated in the pathogenesis of many human and experimental autoimmune peripheral neuropathies that, as in leprosy, result in demyelination and axonal lesions. Because of recent findings associating human TNF alpha mutant alleles at the -308 position with increased production of TNF alpha in many immunological and infectious diseases, an investigation of the role of TNF2 in predisposing leprosy patients to reaction has been undertaken. Analysis of 300 patients with leprosy--210 multibacillary and 90 paucibacillary--has shown that the percentage of reactional patients was similar among both carriers and non-carriers of the TNF2 allele. However, a separate analysis of 57 carriers of TNF2 found that reactions occurred much more frequently among heterozygous than among homozygous patients. Moreover, the frequency of neuritis was somewhat greater among the heterozygous patients than among the non-carriers. Enhanced serum levels of TNF alpha have been noted in both TNF-1 and TNF-2 mutant patients in the course of leprosy reaction. Our observations to date suggest that other factors not related to the presence of the mutant gene may lead to the TNF alpha hyper-responsiveness observed during reaction.

DNA fingerprinting of mycobacterium tuberculis from patients with and without AIDS in Rio de Janeiro

Isolates of Mycobacterium tuberculosis derived from patients with AIDS from a single hospital in Rio de Janeiro were typed using a standardized RFLP technique detecting IS6110 polymorphism. Nineteen isolates were obtained from 15 different patients. Eleven distinct IS6110 patterns were found, with 4 banding patterns shared by 2 patients. The clustering value of 53 percent was much higher in comparison with clustering of M. tuberculosis strains from TB patients without clinical signs for HIV infection from randomly selected health centers. We present these results as preliminary data on M. tuberculosis strain polymorphism in Brazil and on the higher risk for recent transmission amongst patients with AIDS.

Evaluation of chemiluminescence, procoagulant activity and antigen presentation by monocytes from lepromatous leprosy patients with or without reactional episodes.

In this study, we evaluated the activity of peripheral blood mononuclear cells (PBMC), isolated from treated and untreated lepromatous leprosy patients, from lepromatous leprosy patients during and after reactional episodes (erythema nodosum leprosum (ENL) and reversal reaction (RR)), and from normal healthy individuals. We determined reactive oxygen intermediate (ROI) production, procoagulant activity (PCA) and HLA-DR antigen expression of monocytes, besides lymphoproliferation, both in the presence and absence of various stimulatory agents. Phorbol myristate acetate (PMA) stimulated ROI production by monocytes from all the groups studied, with patients during reactional episodes (ENL and RR) showing a significantly higher response (p < 0.009 and p < 0.00001). Irradiated Mycobacterium leprae, although having little effect when added alone, strongly suppressed PMA-stimulated ROI production. Muramyl dipeptide (MDP) had no influence on either basal or on PMA-induced ROI production. Basal monocyte PCA, as well as M. leprae or concanavalin A (ConA)-induced monocyte PCA was comparable in monocytes from all the groups studied. ConA was able to induce mitogenic activity in mononuclear cells isolated from all the groups studied. M. leprae, although stimulatory for normal individuals, did not induce lymphoproliferation in lepromatous leprosy patients, except for cells from patients during RR, which responded equally to M. leprae and to ConA. The absence of M. leprae-induced lymphoproliferation in lepromatous leprosy patients is not caused by the lack of basal HLA-DR expression, as PBMC from all individuals studied showed the same level of this antigen. Our results suggest an increase of spontaneous or PMA-induced monocyte activity, as detected by ROI production, during the reactional episode; addition of M. leprae suppressed this response. The increase in monocyte activity could be correlated with the increase of lymphoproliferation response to M. leprae during RR, but not during ENL. The importance of a possible immune suppressive action of M. leprae is discussed.

Use of PCR-mediated amplification of Mycobacterium leprae DNA in different types of clinical samples for the diagnosis of leprosy.

DNA of Mycobacterium leprae, obtained by a highly efficient nucleic acid extraction procedure, was used for standardisation of the amplification of an M. leprae-specific repetitive sequence by use of the polymerase chain reaction (PCR). With pure DNA, M. leprae-specific amplification was obtained with as low as 100 ag (1 ag = 10(-18) g) of target DNA, a quantity equal to about one-tenth of the bacterial genome. Optimal processing of different types of clinical samples such as biopsy material, blood and lymph fluid, from multibacillary leprosy patients, was studied. Simple freezing-boiling cycles in the presence of Triton X100, with some additional sample-specific modifications such as pre-treatment with NaOH to eliminate PCR inhibitors, was found to be sufficient to yield amplification of bacterial DNA in samples from paucibacillary patients. Clinical samples from 27 untreated leprosy patients, covering the various clinical forms of the disease, and with a bacterial index ranging from 5+ to 0, were collected and processed for PCR analysis. After hybridisation of the amplified material with a specific sequence, 25 of 27 patients analysed gave positive results for M. leprae in at least one of the samples. The potential of PCR for the diagnosis of leprosy is discussed.

In vitro tumor necrosis factor production by mononuclear cells from lepromatous leprosy patients and from patients with erythema nodosum leprosum.

The production of tumor necrosis factor (TNF) by Mycobacterium leprae-stimulated phagocyte cells, isolated from lepromatous leprosy patients (LL) and normal individuals, was evaluated, using the highly TNF-sensitive mouse fibrosarcoma cell line WEHI164cl13. Mononuclear cells, isolated from all individuals studied, showed a low level of spontaneous TNF production, except for patients undergoing erythema nodosum leprosum (ENL), in which we found significantly higher levels of TNF. Addition of M. leprae to the phagocyte cell culture enhanced TNF production in all groups studied, except in the group with untreated leprosy patients. Strongest M. leprae-induced TNF release was found in mononuclear cell cultures derived from ENL patients. Patients in the postreactional state showed significantly higher TNF levels than healthy controls. These findings support the idea that TNF plays a key role in the complex symptomatology of ENL.

Use of PCR for the determination of the frequency of the delta F508 mutation in Brazilian cystic fibrosis patients.

The delta F508 mutation in the cystic fibrosis (CF) gene was studied in a population of 18 Brazilian CF patients and their 17 families by use of PCR and differential hybridization with oligonucleotides. In a total of 34 chromosomes considered, 12 (35%) carried the F508 deletion, a frequency much lower than that reported in most other populations. As a consequence, CF in Brazil would be predominantly caused by mutations different from the F508 deletion.