Influence of canine brain decomposition on laboratory diagnosis of rabies

Canine brains infected with rabies virus were submitted to decomposition by being left at room temperature of 25 to 29oC for up to 168h. At 24h intervals, brain fragments were analyzed by immunofluorescence (IF) and by the mouse intracerebral inoculation (MI) test to confirm the diagnosis of rabies and to measure the putrefaction effect on the accuracy of the diagnosis. Forty eight h after the beginning of the experiment, the MI test showed signs of impairment with four negative results, while after 72h, 100% of the results were negative to the MI test and only one result was negative to the IF test, indicating that the threshold period for accurate diagnosis is 24 to 48h before putrefaction. The authors recommend the shipment of suspected cases of rabies to the laboratory for confirmation, but the use of putrid materials for diagnosis is meaningless because of false-negative results.

Frequency of myocarditis in cases of fatal meningococcal infection in children: observations on 31 cases studied at autopsy

The frequency of myocarditis associated with meningococcal disease in children was reported only in two autopsied series (United States and South Africa). Here we report the frequency of associated myocarditis in 31 children who died of meningoccal infection at Hospital Infantil N.S. da Gl��ria in Vit��ria, Espirito Santo State, Brazil. The diagnosis was confirmed by isolation of Neisseria meningitidis . At least three sections of fragments of both atria and ventricles were studied using the Dallas Criteria for the morphologic diagnosis of myocarditis. The mean age was 47.6 �� 39.8 months and the mean survival time after the onset of symptoms was 46.1 �� 26.5h (12-112h). Myocarditis was present in 13 (41.9%) patients, being of minimal severity in 11 cases and of moderate severity in 2 cases. There were no cases with severe diffuse myocarditis. The frequency of myocarditis was not influenced by sex, presence of meningitis, survival time after the onset of symptoms or use of vasoactive drugs. The frequency of myocarditis reported here was intermediate between the values reported in the only two case series published in the literature (57% in the United States and 27% in South Africa). Although our data confirm the high frequency of myocaditis in meningoccal disease, further investigations are necessary to elucidate the contribution of myocarditis to myocardial dysfunction observed in cases of meningococcal infection in children.

Optimization of randomly amplified polymorphic DNA-polymerase chain reaction for molecular typing of Salmonella enterica serovar Typhi

Optimization of the RAPD reaction for characterizing Salmonella enterica serovar Typhi strains was studied in order to ensure the reproducibility and the discriminatory power of this technique. Eight Salmonella serovar Typhi strains isolated from various regions in Brazil were examined for the fragment patterns produced using different concentrations of DNA template, primer, MgCl2 and Taq DNA polymerase. Using two different low stringency thermal cycle profiles, the RAPD fingerprints obtained were compared. A set of sixteen primers was evaluated for their ability to produce a high number of distinct fragments. We found that variations associated to all of the tested parameters modified the fingerprinting patterns. For the strains of Salmonella enterica serovar Typhi used in this experiment, we have defined a set of conditions for RAPD-PCR reaction, which result in a simple, fast and reproducible typing method.

Lime mortars with heat treated clays and ceramic waste: A review

The formulation and use of lime mortars with ceramic particles has, in the past, been a very common technique. Knowledge of such used techniques and materials is fundamental for the successful rehabilitation and conservation of the built heritage. The durability that these mortars have shown encourages the study of the involved mechanisms, so that they may be adapted to the current reality. The considerable amount of waste from old ceramics factories which is sent for disposal might present an opportunity for the production of reliable improved lime mortars. In this paper a number of studies that characterize old building mortars containing ceramic fragments are reviewed. The most important research undertaken on laboratory prepared mortars with several heat treated clays types is presented, specifically with incorporated ceramic waste. Some studies on the pozzolanicity of heat treated clays are examined and the heating temperatures that seem most likely to achieve pozzolanicity are presented. It was verified that some heating temperatures currently used by ceramic industries might correspond to the temperatures that will achieve pozzolanicity.

Glomerulonephritis in schistosomiasis mansoni: a time to reappraise

INTRODUCTION: The current prevalence of glomerulonephritis in patients with hepatosplenic schistosomiasis mansoni in Brazil was evaluated. METHODS: Sixty three patients (mean age 45.5��11 years) attending the outpatient infectious disease clinic of a University Hospital in Belo Horizonte, Brazil, from 2007 to 2009, were consecutively examined and enrolled in the present investigation. Diagnosis of hepatosplenic schistosomiasis was based on epidemiological, clinical and parasitological data and imaging techniques. Eight patients, who presented >30mg/day albuminuria, were submitted to percutaneous ultrasound guided renal biopsy. Kidney tissue fragments were examined under light, direct immunofluorescence and electron microscopy. RESULTS: All patients showed mesangial enlargement. In five, mesangial hypercellularity was observed and four presented duplication of the glomerular basement membrane. Areas of glomerular sclerosis were diagnosed in four. Deposits of immunoglobulin M and C3 were present in six samples; deposits of IgG in four, IgA in three and C1q in two samples. In all patients, immunoglobulin A was reported in the lumen of renal tubules. Deposits of kappa and lambda were observed in six samples. Electron microscopy revealed dense deposits in the glomerular tissue of three patients. Arterial hypertension, small esophageal varices, slight increases in serum creatinine and decreases in serum albumin were associated with glomerular disease. CONCLUSIONS: Renal disease associated with hepatosplenic schistosomiasis was verified in 12.7% of patients and type I membranoproliferative glomerulonephritis was observed in 50% of them. Schistosomal glomerulopathy still is an important problem in patients with hepatosplenic schistosomiasis in Brazil.

A les��o de isqu��mia/reperfus��o em transplante hep��tico: an��lise gen��tica, morfol��gica e correla����o cl��nica

RESUMO: O transplante hep��tico ortot��pico �� uma terap��utica aceite para casos selecionados de fal��ncia hep��tica terminal. O procedimento tem-�����se aperfei��oado, evidenciado pelo aumento da taxa de sobrevida de 30 para 75% aos 5 anos, mas cerca de 13 a 27% dos enxertos desenvolve fal��ncia prim��ria (PNF) ou disfun����o prim��ria (DF) ap��s o transplante. As consequ��ncias s��o devastadoras para a sobrevida do doente e do enxerto. A sua etiologia �� multifactorial, incluindo factores relacionados com o dador e o receptor, tempos de isqu��mia, agress��es cir��rgicas, bem como caracter��sticas anatomopatol��gicas do enxerto. A les��o de isqu��mia/reperfus��o mantem-�����se como um factor de risco intra operat��rio, com implica����es directas sobre toda a evolu����o do transplante : existe uma rela����o ��ntima entre a PNF e a DF, a preserva����o do enxerto, a les��o de isqu��mia/reperfus��o, e a fal��ncia do transplante. Al��m disso, est�� comprovada evid��ncia que sugere que a les��o de I/R torna um aloenxerto mais vulner��vel por aumento da imunogenicidade, aumentando a probabilidade de epis��dios de rejei����o precoce e tardia. Com base na pr��tica cl��nica quotidiana do CHBPT HCC, estudaram-�����se 54 casos de transplante hep��tico, agrupados segundo grupos por aloca����o do enxerto respectivo: Grupo 1(n=27): dador cad��ver para receptor cirr��tico, Grupo 2 (n=15): dador cad��ver para receptor PAF, Grupo 3 (n=12): dador PAF para receptor cirr��tico. Observaram-�����se as altera����es histol��gicas e moleculares sobre o enxerto at�� ao final da opera����o do receptor, e as suas consequ��ncias cl��nicas,avaliando: -����� As diferentes capacidades de resist��ncia e cada enxerto �� les��o de isqu��mia/reperfus��o. -����� As situa����es em que os factores do receptor se sobrep��em ��s do enxerto na defini����o do progn��stico, e vice versa. -����� A relev��ncia das les��es histol��gicas e moleculares precoces no tecido hep��tico na evolu����o do enxerto e do receptor. Foram colhidas bi��psias por agulha dos 54 enxertos hep��ticos,42 provenientes de cad��ver com cora����o batente(morte cerebral) e 12 provenientes de dador vivo com PAF, em tr��s tempos diferentes do processo de colheita e transplante hep��tico: ����� A primeira(T0)antes da clampagem da aorta do dador -����� A segunda (T1) no final da isqu��mia fria -����� A terceira (T2) ap��s a reperfus��o do enxerto, durante o encerramento da parede abdominal. A estas amostras foi extra��do RNA total, convertido em cDNA por transcri����o reversa e feita a an��lise da express��o dos genes da CTLA4, IL-�����1��, IL-�����4, IL-�����6, IL-�����13, TNF-�������, Perforina, Selectina, (SELE), Fas-�����ligando, Granzima-�����B, Heme-�����Oxigenase 1(HO1)e ��xido N��trico Sintetase(iNOS2A)por PCR quantitativo segundo o m��todo do Ct comparativo, utilizando como refer��ncia a express��o dos genes da amostra n��o-�����isqu��mica ���T0. Os fragmentos de todas as bi��psias foram seccionados, para envio de amostra comparativa para processamento histol��gico habitual, sem qualquer altera����o ao protocolo seguido habitualmente na Unidade de Transplanta����o do Hospital Curry Cabral. A presen��a de alguns par��metros histol��gicos definidos, como esteatose, necrose, vacuoliza����o, congest��o sinusoidal e infiltra����o neutrof��lica, foi registada e contabilizada numa classifica����o num��rica. O seguimento cl��nico e laboratorial, bem como o acompanhamento de eventuais complica����es, foi registado e correlacionado com os dados das colheitas de ��rg��os e com os dados das bi��psias. Foram consideradas as seguintes vari��veis, como as mais relevantes e objectivas para a interpreta����o da evolu����o cl��nica, tendo sido comparadas estatisticamente com os dados recolhidos, laboratoriais e cl��nicos: disfun����o do enxerto, 207 p��s operat��rias, n��mero de internamentos igual ou superior a 2 e rejei����o cr��nica e/ou morte do receptor. Foram identificadas caracter��sticas cl��nicas menos favor��veis, a considerar, nalgumas circunst��ncias: g��nero feminino do receptor (sobretudo associado a enxerto masculino, p=0,077), isqu��mia fria superior a 500 minutos (p=0,074), isqu��mia quente superior a 90 minutos (p=0,099). Na an��lise laboratorial, distinguiram-�����se duas caracter��sticas histol��gicas desfavor��veis e irrevers��veis, como ��ndice de mau progn��stico: a necrose e a baloniza����o (p=0,029); no painel gen��tico escolhido neste estudo,a express��o basal de IL-�����1��(p=0,028), de SELE p=0,013)e de FAS-�����L (p=0,079)relacionaram-�����se com pior progn��stico. Algumas caracter��sticas protectoras intr��nsecas dos enxertos s�� se revelaram indirectamente, como menor infiltra����o neutrof��lica e maior express��o de HO1 e de iNOS nos enxertos PAF, n��o tendo sido poss��vel provar uma interfer��ncia directa nos resultados cl��nicos. N��o se obteve express��o mensur��vel de genes anti-����� inflamat��rios nas biopsias hep��ticas processadas neste estudo, como a IL13 e a I 4: assim, com a metodologia utilizada, n��o foi poss��vel obter um perfil de express��o gen��tica associado a boa evolu����o cl��nica. O perfil inverso foi sugerido apenas pela express��o basal dos 3 genes mencionados (FAS-�����L,IL-�����1�� e SELE)no mesmo painel, com o protocolo seguido neste conjunto de 54 doentes. As caracter��sticas do receptor sobrepuseram-�����se ��s do enxerto no caso de: -����� diagn��stico de PAF no receptor, que determinou uma maior predisposi����o para a disfun����o do enxerto, o que, por sua vez, determina uma menor sobrevida. No entanto, o diagn��stico de PAF no receptor exibe uma curva de sobrevida mais favor��vel. -����� receptores com um baixo balan��o de risco (BAR)definiram caracter��sticas favor��veis para enxertos com n��veis baixos e moderados de esteatose, fazendo que esta caracter��stica, definida como um risco acrescido, n��o s�� n��o se manifestasse clinicamente,como parecesse um factor favor��vel. As caracter��sticas do enxerto sobrepuseram-�����se ��s do receptor no caso de: -����� tempo de isqu��mia fria superior a 500 minutos -����� baloniza����o, necrose, FAS-�����L,IL-�����1�� e SELE em T0 A integra����o dos resultados moleculares e morfol��gicos com a evolu����o cl��nica, real��a o papel da mobiliza����o precoce de neutr��filos nos desempenhos menos favor��veis do enxerto hep��tico. -------------ABSTRACT: Orthotopic liver transplantation is na accepted therapeutic procedure for selected cases of terminal liver failure. The procedure has been improved, evidenced by the rise of survival rates from 30 to 70% at 5 years, but 13 to 27% of the liver grafts develops primary non function (PNF) or primary dysfunction (PDF) after transplantation. The consequences are devastating for the survival of the patient and of the graft. Its etiology is multifactorial, including factos related with the donor and with the recipient, ischemic times, surgical aggressions, as well as the histological characteristics of the graft. The ischemia/reperfusion lesion is still an intraoperative risk factor, with direct implications in the whole transplant outcome: there is a close interrelation between PNF and DF, graft preservation, ischemia / reperfusion lesion and graft failure. Beyond his, there is proved evidence that suggests that I/R lesion turns the allograft more vulnerable by increasing its immunogenity, increasing the probability of precocious and late rejection episodes. Based on the daily clinical practice at CHBPT /HCC, 54 cases of hepatic transplantation have been studied, grouped by allocation of each graft: Group (n=27):deceased do nortocirrhotic recipient, Group 2 (n=15): deceased donor to FAP recipient, Group 3 (n=12): FAP living donor to cirrhotic recipient. The histologic and molecular changes in the liver graft were observed until the end of the recipiente operation,together with its clinical consequences, evaluating:-�����The different capacity of resistance of each graft to the ischemia / reperfusion lesion -����� The situations where the recipiente factos overlap the ones of the graft, in the definition of prognosis, and vice versa.-����� The relevance of the precocious histologic and molecular lesions of the hepatic tissue in the clinical outcome of the graft and the recipient. Needle biopsies were obtained from 54 liver grafts, 42 deceased brain dead donors and 12 from FAP living donors, at three diferente times of the harvesting and the hepatic transplantation: The first one (T0) before clamping the donor aorta -����� The second one (T2) in the end of cold ischemia time -����� The third one (T) after the reperfusion of the graft, during the closure of the abdominal wall. Total RNAwas extracted to these samples, converted to cDNA by reverse transcription and the analysis of gene expression was made for CTLA4,IL-�����1��,IL-�����4,IL-�����6,IL-�����13,TNF-�������,Perforin,E Selectin (SELE),Fas-�����ligand,Granzyme-�����B,Heme-�����oxigenase 1 (HO1) and Nitric Oxide Sintetase (iNOS2A) by quantitative PCR, according with the Ct comparative method, using the expression of the non ischemic sample ��� T0. The fragments of all the biopsies were divided, to send a comparative sample to the usual histologic processement, keeping the same usual protocol at the Transplantation Unit of Curry Cabral Hospital. The presence of some defined histologic parameters, such as steatosis, necrosis, vacuolization, sinusoidal congestion and neutrophilic infiltration, was registered and catalogued in a numeric classification. The clinical and laboratory follow-�����up, as well as the following of eventual complications, was registered and correlated with the data from organ procurement operations and with the data from the biopsies. The following variables were considered as the most relevant and objective ones, to the interpretation of the clinical evolution, being statistically compared with the clinical and laboratorial collected data: graft dysfunction, post-�����operative complications, number of readmissions of 2 or more and chronic rejection and /or recipiente death. There were identified some unfavorable clinical characteristics, to be considered under certain circumstances: recipiente female gender (specially associated with malegraft, p=0,077), cold ischemia time of more than 500 minutes (p=0,074), warm ischemia time of more than 90 minutes (p=0,099). In the laboratory analysis, two histologic characteristics were identified as unfavorable and irreversible, associated with bad prognosis: necrosis and balonization (p=0,029); in the gene panel selected in this study, the basal expression of IL-�����1�� (p=0,028), SELE (p=0,013) and FAS-�����L (p=0,079)were related with worse prognosis.Some intrinsic protective characteristics of the grafts were only indirectly revealed, such as less neutrophilic infiltration and bigger expression of HO1 and iNOS in FAP grafts, being impossible to prove any direct inte ference in the clinical results. A relevant and measurable expression of the anti inflammatory genes IL13 and IL4 was not obtained: with the used methodology, it was impossible to obtain a gene expression profile associated with a favorable clinical outcome.The inverse profile was suggested only by the basal expression of the three mentioned genes (FAS-�����L, IL-����� 1�� e SELE) in the same gene panel, according with the followed protocol in this group of 54 patients. The characteristics of the recipient overlapped those from the graft, in the case of :-����� FAP diagnosis in the recipient, which determined a bigger predisposition to graft dysfunction, which by itself determines a shorter survival. However, FAP diagnosis in the recipiente depicts a more favorable survival curve. -����� Recipients with a low balance risk ��ndex (BAR) defined favorable characteristics to grafts with low and moderate grades of steatosis, making that this characteristic, associated with bad prognosis, looked like a favorable factor, and with no clinical interference. The graft characteristics overlapped those from the receptor in the case of: -����� Cold ischemic time more than 500 minutes -����� Balonization, necrosis, FAS-�����L, IL-�����1�� and SELE at T0. The integration of molecular and morphologic results with the clinical evolution, stresses the role of a precocious neutrophils mobilization in the worse outcomes of liver grafts.

Differentiation between Candida albicans and Candida dubliniensis using hypertonic Sabouraud broth and tobacco agar

INTRODUCTION: Opportunistic fungal infections in immunocompromised hosts are caused by Candida species, and the majority of such infections are due to Candida albicans. However, the emerging pathogen Candida dubliniensis demonstrates several phenotypic characteristics in common with C. albicans, such as production of germ tubes and chlamydospores, calling attention to the development of stable resistance to fluconazole in vitro. The aim of this study was to evaluate the performance of biochemistry identification in the differentiating between C. albicans and C. dubliniensis, by phenotyping of yeast identified as C. albicans. METHODS: Seventy-nine isolates identified as C. albicans by the API system ID 32C were grown on Sabouraud dextrose agar at 30��C for 24-48h and then inoculated on hypertonic Sabouraud broth and tobacco agar. RESULTS: Our results showed that 17 (21.5%) isolates were growth-inhibited on hypertonic Sabouraud broth, a phenotypic trait inconsistent with C. albicans in this medium. However, the results observed on tobacco agar showed that only 9 (11.4%) of the growth-inhibited isolates produced characteristic colonies of C. dubliniensis (rough colonies, yellowish-brown with abundant fragments of hyphae and chlamydospores). CONCLUSIONS: The results suggest that this method is a simple tool for screening C. albicans and non-albicans yeast and for verification of automated identification.

Notification of the first isolation of Cacipacore virus in a human in the State of Rond��nia, Brazil

Flavivirus is a genus of arthropod-transmitted viruses of the family Flaviviridae, and in Brazil, up to eleven different Flavivirus have been isolated. We collected blood from farmers in the municipality of Theobroma, which is located 320km from the City of Porto Velho, the former capital of the Brazilian State of Rond��nia. For viral isolation, we used newborn mouse brain, followed by RT-PCR with specific universal Flavivirus primers. We obtained fragments 958bp and 800bp in length. Based on BLAST, these sequences were 91% similar to a sequence of Cacipacore virus.

Drug resistance of Mycobacterium tuberculosis strains in southern Brazil

INTRODUCTION: The aim of this work was to evaluate the prevalence of Mycobacterium tuberculosis (MT) strains with mutations that could result in resistance to the main drugs used in treatment in a region with one of the highest numbers of tuberculosis (TB) cases in southern Brazil. METHODS: Deoxyribonucleic acid (DNA) from 120 sputum samples from different patients suspicious of pulmonary tuberculosis who attended the Municipal Public Laboratory for Mycobacterium sp. diagnosis was directly amplified and analyzed by PCR-SSCP. The DNA was amplified in known hotspot mutation regions of the genes rpoB, ahpC, embB, katG, inhA, and pncA. RESULTS: The percentage of samples positive by culture was 9.2% (11/120); 5% (6/120) were positive by bacilloscopy and MT-PCR, and DNA fragments of the aforementioned resistance genes could be amplified from seven (7) of the eleven (11) samples with positive results, either by culture or PCR/bacilloscopy. All presented a SSCP pattern similar to a native, nonresistant genotype, with the ATCC strain 25177 as control, except for one sample (0.01%), which presented a SSCP profile demonstrating mutation at the embB gene. CONCLUSIONS: These results are consistent with the empirical observations by physicians treating TB patients in our region of a low occurrence of cases that are refractory to conventional treatment schemes, in contrast to other parts of the country. Continued surveillance, especially molecular, is essential to detect and monitor the outbreak of MT-resistant strains.

Prevalence of enterotoxin-encoding genes and antimicrobial resistance in coagulase-negative and coagulase-positive Staphylococcus isolates from black pudding

INTRODUCTION: Staphylococcal species are pathogens that are responsible for outbreaks of foodborne diseases. The aim of this study was to investigate the prevalence of enterotoxin-genes and the antimicrobial resistance profile in staphylococcus coagulase-negative (CoNS) and coagulasepositive (CoPS) isolates from black pudding in southern Brazil. METHODS: Two hundred typical and atypical colonies from Baird-Parker agar were inoculated on mannitol salt agar. Eighty-two mannitol-positive staphylococci were submitted to conventional biochemical tests and antimicrobial susceptibility profiling. The presence of coagulase (coa) and enterotoxin (se) genes was investigated by polymerase chain reaction. RESULTS: The isolates were divided into 2 groups: 75.6% (62/82) were CoNS and 24.4% (20/82) were CoPS. The biochemical tests identified 9 species, of which Staphylococcus saprophyticus (37.8%) and Staphylococcus carnosus (15.9%) were the most prevalent. Antimicrobial susceptibility tests showed resistance phenotypes to antibiotics widely administered in humans, such as gentamicin, tetracycline, chloramphenicol, and erythromycin. The coa gene was detected in 19.5% (16/82) of the strains and 4 polymorphic DNA fragments were observed. Five CoNS isolates carrying the coa gene were submitted for 16S rRNA sequencing and 3 showed similarity with CoNS. Forty strains were positive for at least 1 enterotoxin-encoding gene, the genes most frequently detected were sea (28.6%) and seb (27.5%). CONCLUSIONS: The presence of antimicrobial resistant and enterotoxin-encoding genes in staphylococci isolates from black pudding indicated that this fermented food may represent a potential health risk, since staphylococci present in food could cause foodborne diseases or be a possible route for the transfer of antimicrobial resistance to humans.

Schistosoma mansoni granuloma in late evolutive phase, in a case of tumoral form in man

INTRODUCTION: Authors describe human schistosomal granuloma in late chronic phase, from the morphological and evolutionary viewpoints. METHODS: The study was based on a histological analysis of two fragments obtained from a surgical biopsy of peritoneum and large intestine of a 42-year-old patient, with a pseudotumoral form mimicking a peritoneal carcinomatosis associated to the schistosomiasis hepatointestinal form. RESULTS: Two hundred and three granulomas were identified in the pseudotumor and 27 in the intestinal biopsy, with similar morphological features, most in the late chronic phase, in fibrotic healing. A new structural classification was suggested for granulomas: zone 1 (internal), 2 (intermediate) and 3 (external). CONCLUSIONS: Regarding granuloma as a whole, we may conclude that fibrosis is likely to be controlled by different and independent mechanisms in the three zones of the granuloma. Lamellar fibrosis in zone 3 seems to be controlled by matrix mesenchymal cells (fibroblasts and myoepithelial cells) and by inflammatory exudate cells (lymphocytes, plasmocytes, neutrophils, eosinophils). Annular fibrosis in zone 2, comprising a dense fibrous connective tissue, with few cells in the advanced phase, would be controlled by epithelioid cells involving zone 1 in recent granulomas. In zone 1, replacing periovular necrosis, an initialy loose and tracery connective neoformation, housing stellate cells or with fusiform nuclei, a dense paucicellular nodular connctive tissue emerges, probably induced by fibroblasts. In several granulomas, one of the zones is missing and granuloma is represented by two of them: Z3 and Z2, Z3 and Z1 or Z2 and Z1 and, ultimately, by a scar.

CD8+ T cells in situ in different clinical forms of human cutaneous leishmaniasis

Introduction Leishmania braziliensis infection induces a large spectrum of lesions that clinically manifest as nodules or papules that progress to ulcers. Although it is already known that T helper cells predominate in the lesions, cytotoxic T cells have also been reported to be present, and their role in leishmaniasis immunopathogenesis is not well known. This study investigated the amounts of CD8+ and granzyme B+ cells in different clinical forms of human cutaneous leishmaniasis (CL). Methods Forty tissue fragments from early (E-CL) and late CL (L-CL) lesions and from disseminated leishmaniasis (DL) - papules and ulcers - were characterized. The inflamed area per fragment was calculated, and the CD8 and granzyme B expression levels in the infiltrates were quantified by counting positive cells in 15 fields. The localization of CD8 and granzyme B was graded subjectively. Results Inflammation was higher in L-CL and DL ulcers. CD8 expression was increased in late ulcerated lesions compared to recent lesions. The increase in CD8+ cells also correlated with the duration of the lesion. Papules had a higher frequency of granzyme B+ cells than E-CL lesions, although the frequency was similar to those for late and DL ulcers. CD8+ cells were mostly found in the papillary dermis. Conclusions CD8+ T and granzyme B+ cells are present in the inflammatory infiltrates of CL and DL and may participate in the immunopathogenesis of Leishmania infection.

Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays

Introduction During a diagnostic evaluation of canine visceral leishmaniasis (VL), two of seventeen dogs were found to be co-infected by Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi. Methods Specific polymerase chain reaction (PCR) and restriction fragment length polymorphism-PCR (RFLP-PCR) assays were performed. Results PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia) braziliensis and Leishmania (Leishmania) chagasi in those fragments. Conclusions This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

IntroductionKala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar.MethodsTo determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification.ResultsThe nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival.ConclusionsNAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

Polymerase chain reaction-based method for the identification of Leishmania (Viannia) braziliensis and Leishmania (Viannia) guyanensis in mucosal tissues conserved in paraffin

ABSTRACTINTRODUCTION: In the Americas, mucosal leishmaniasis is primarily associated with infection by Leishmania (Viannia) braziliensis. However, Leishmania (Viannia) guyanensis is another important cause of this disease in the Brazilian Amazon. In this study, we aimed at detecting Leishmaniadeoxyribonucleic acid (DNA) within paraffin-embedded fragments of mucosal tissues, and characterizing the infecting parasite species.METHODS: We evaluated samples collected from 114 patients treated at a reference center in the Brazilian Amazon by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analyses.RESULTS: Direct examination of biopsy imprints detected parasites in 10 of the 114 samples, while evaluation of hematoxylin and eosin-stained slides detected amastigotes in an additional 17 samples. Meanwhile, 31/114 samples (27.2%) were positive for Leishmania spp. kinetoplast deoxyribonucleic acid (kDNA) by PCR analysis. Of these, 17 (54.8%) yielded amplification of the mini-exon PCR target, thereby allowing for PCR-RFLP-based identification. Six of the samples were identified as L. (V.) braziliensis, while the remaining 11 were identified as L. (V.) guyanensis.CONCLUSIONS: The results of this study demonstrate the feasibility of applying molecular techniques for the diagnosis of human parasites within paraffin-embedded tissues. Moreover, our findings confirm that L. (V.) guyanensisis a relevant causative agent of mucosal leishmaniasis in the Brazilian Amazon.