Association between inflammatory molecules, nitric oxide metabolites and leg ulcers in individuals with sickle cell anemia

Abstract Introduction Leg ulcers (LUs) are relatively common in patients with sickle cell anemia (SCA). The role of inflammation and nitric oxide (NO) pathways in the pathophysiology of the LU is not understood. Objective The aim of this study was to verify the association between inflammatory molecules and nitric oxide metabolites (NOx) and the occurrence of the LU in patients with SCA. Method It was a cross-sectional study on adult participants with SCA followed at Fundação Hemominas, a public blood center in Brazil. Eligible participants were recruited and included in one of two groups: Group 1, comprised of cases with SCA (Hb SS) and at least one LU at the time of inclusion in the study and Group 2, comprised of controls with SCA without a history of LU, matched by sex and age to cases. Participants were interviewed to obtain sociodemographic data and blood samples were collected. Clinical and laboratory data were abstracted from medical records. Nitric oxide metabolites (NOx) and inflammatory molecules were quantified using an immunoassay and Multiplex xMAP® technology, respectively. Eighty-seven individuals were included, ranging in age from 17 to 61 years (mean 40 ± 10.7 years); 30 had LU and 57 were controls without LU. Results Participants with LU had significantly higher levels of interleukin 8 (IL-8), IL-10, IL-15, NOx and platelet and white blood cell (WBC) counts, when compared to those without LU. Participants with LU had a significantly higher risk of having a history of osteomyelitis and a higher use of antiseptic soap in bathing, when compared to those without LU. Conclusion In conclusion, our results showed that NOx, inflammatory molecules and hematological features were associated with LU in Brazilian adults with SCA.

Association between inflammatory molecules, nitric oxide metabolites and leg ulcers in individuals with sickle cell anemia.

INTRODUCTION: Leg ulcers (LUs) are relatively common in patients with sickle cell anemia (SCA). The role of inflammation and nitric oxide (NO) pathways in the pathophysiology of the LU is not understood. OBJECTIVE: The aim of this study was to verify the association between inflammatory molecules and nitric oxide metabolites (NOx) and the occurrence of the LU in patients with SCA. METHOD: It was a cross-sectional study on adult participants with SCA followed at Fundação Hemominas, a public blood center in Brazil. Eligible participants were recruited and included in one of two groups: Group 1, comprised of cases with SCA (Hb SS) and at least one LU at the time of inclusion in the study and Group 2, comprised of controls with SCA without a history of LU, matched by sex and age to cases. Participants were interviewed to obtain sociodemographic data and blood samples were collected. Clinical and laboratory data were abstracted from medical records. Nitric oxide metabolites (NOx) and inflammatory molecules were quantified using an immunoassay and Multiplex xMAP® technology, respectively. Eighty-seven individuals were included, ranging in age from 17 to 61 years (mean 40 ±â€¯10.7 years); 30 had LU and 57 were controls without LU. RESULTS: Participants with LU had significantly higher levels of interleukin 8 (IL-8), IL-10, IL-15, NOx and platelet and white blood cell (WBC) counts, when compared to those without LU. Participants with LU had a significantly higher risk of having a history of osteomyelitis and a higher use of antiseptic soap in bathing, when compared to those without LU. CONCLUSION: In conclusion, our results showed that NOx, inflammatory molecules and hematological features were associated with LU in Brazilian adults with SCA.

Distinct genetic profiles of Leishmania (Viannia) braziliensis associate with clinical variations in cutaneous-leishmaniasis patients from an endemic area in Brazil.

American tegumentary leishmaniasis (ATL) samples obtained from the lesions of patients with typical (n = 25, 29%), atypical (n = 60, 69%) or both (n = 2%) clinical manifestations were analysed by multilocus enzyme electrophoresis, hsp70 restriction-fragment length polymorphism (PCR-RFLP), hsp70 sequencing and phylogenetics methods. The hsp70 PCR-RFLP analysis revealed two different profiles whose the most samples differed from those expected for Leishmania braziliensis and the other Leishmania species tested: of 39 samples evaluated, two (5%) had a restriction profile corresponding to L. braziliensis, and 37 (95%) had a restriction profile corresponding to a variant pattern. A 1300-bp hsp70 gene fragment was sequenced to aid in parasite identification and a phylogenetic analysis was performed including 26 consensus sequences from the ATL patient's samples and comparing to other Leishmania and trypanosomatids species. The dendrogram allowed to observe a potential population structure of L. braziliensis complex in the studied region, emphasizing that the majority of clinical samples presented a variant genetic profile. Of interest, the L. braziliensis diversity was associated with different clinical manifestations whose parasites with hsp70 variant profile were associated with atypical lesions. The results may be helpful to improve the diagnosis, treatment and control measures of the ATL in endemic areas.

Seroprevalence of Orthopoxvirus in rural Brazil: insights into anti-OPV immunity status and its implications for emergent zoonotic OPV.

BACKGROUND: Bovine vaccinia (BV) is a zoonosis caused by Vaccinia virus, a virus from Orthopoxvirus genus (OPV) that affects mainly cattle herds and humans in rural areas in Brazil. Because most studies have focused on outbreaks situations, data on BV epidemiology is limited. A cross sectional study in Brazilian rural areas during 2012-2013 was conducted to determine the neutralizing antibodies seroprevalence and risk factors for BV. METHODS: A structured questionnaire was applied to elicit demographics data and farming practices considered risk factors for BV exposure. Neutralizing anti-OPV antibodies were investigated using plaque reduction neutralization test. The neutralizing antibodies prevalence rates were calculated and the risk factor analysis was performed using multivariate logistic regression. RESULTS: Two hundred and forty participants were enrolled in this study with a prevalence of neutralizing antibodies of 30.8 % (95 % confidence interval [CI], 25.3-36.9). In multivariate analysis, age > 35 years (Odds Ratio [OR] = 18.2; CI 95 % = 7.7 - 43.2) and previous outbreak in property (OR = 3.9; C I95 % = 1.2 - 12.6) were independently associated with anti-OPV neutralizing antibodies. CONCLUSIONS: In this study, anti-OPV protective immunity (neutralizing antibody titers) was assessed in an endemic BV Brazilian rural area. Our findings indicate that epidemiological surveillance is required and should be applied by public health authorities to create interventions and/or prevention strategies to avoid viral spread causing future outbreaks among individuals who are under risk of infection.

Uso de hemocomponentes em hospital de médio porte em Belo Horizonte, Minas Gerais / Use of blood in mid-sized hospital in Belo Horizonte, Minas Gerais

Introdução: a transfusão de sangue tem como objetivo restaurar o volume de sangue e manter a concentração de oxigênio. Pode ser um procedimento essencial para a melhoria do estado geral e preservação da vida dos pacientes, mas pode ter efeitos indesejáveis e até mesmo letais. Objetivos: avaliar o perfil de transfusão hospitalar e adesão aos protocolospesquisados. Métodos: estudo descritivo de série de casos com amostra coletada de forma sistemática, a partir dos registros de solicitações de hemocomponentes do ano de 2012. A distribuição dos dados coletados foi analisada após soma do total em número absoluto e cálculo da sensibilidade. Resultados: a proporção de solicitações foi maior para pacientes do sexo masculino (54,6%) e a faixa etária com maior prevalência de solicitações de hemotransfusão foi de 60 anos ou mais (40,4% do total). O concentrado de hemácias (CH) foi o componente do sangue com o maior número de pedidos (72,4%), seguido de plasma congelado fresco (PFC) (22,2%) e de concentrados de plaquetas (CP) (6,2%). Entre as indicações de CH, a anemia foi o mais encontrado, seguido por sangramento. O sangramento também foi a principal indicação clínica do PFC. Outras indicações do PFC foram anemia e terapia de coagulopatias/anticoagulação. Pedidos, transfusões e internações não mostraram relação direta temporal durante o ano estudado. Conclusões: o estudo das características da demanda de sangue pode auxiliar na observação e previsão de tendências e desfechos. Considerando a análise temporal da demanda de sangue, por exemplo, destaca-se a importância de manter o banco de sangue com reservas suficientespara atender às exigências. Além disso, foram encontrados registros de uso acima do esperado de PFC, com indicações não preconizadas pelo hospital e literatura nacional.

Introduction: Blood transfusion is aimed to restore blood volume and maintain the oxygen concentration. It can be an essential procedure for improving the general condition and preserve the lives of patients, but it may have unwanted and even lethal effects. Aims: To evaluate the characteristics and adherence to hospital transfusion protocols surveyed. Methods: Descriptive case series study with samples collected systematically from the records of requests for blood products of the year 2012. The distribution of the collected data was analyzed after the totalsum in absolute numbers and calculation of sensitivity. Results: The proportion of requests was higher for male patients (54.3%) and the age group with the highest prevalence of requests for blood transfusion was 60 years or more (40.4% of the total). The packed red blood cells (RBC)was the blood component with the highest number of requests (72.4%), followed by fresh frozen plasma (FFP) (22.2%) and platelet concentrates (PC) (6.2%). Among the indications for RBC, anemia was most frequently found, followed by bleeding. Bleeding was also the main clinical indication FFP. Other indications of the FFP were anemia and coagulopathies / anticoagulation therapy. Orders, transfusions and hospitalizationsdid not show a direct temporal relationship during the year studied. Conclusions: The study of the characteristics of the demand for blood can aid in the detection and prediction of trends and outcomes. Considering the temporal analysis of the demand for blood, for example, emphasize the importance of maintaining the blood bank with sufficient reserves to meet the requirements. Furthermore, usage records higher than expected FFP, with no indications recommended by the hospital and national literature were found.

Neutralizing antibodies associated with exposure factors to Orthopoxvirus in laboratory workers.

Extensive use of Vaccinia virus (VACV) in research has led to associated accidental human exposure in laboratories worldwide. In spite of the social and economic relevance of Bovine Vaccinia outbreaks in Brazil, national data concerning laboratory workers handling these infectious agents are relatively scarce. Therefore, a serological survey was conducted in a Brazilian laboratory to evaluate staff exposure to orthopoxviruses (OPVs). Information concerning direct work with OPVs, vaccination status and laboratory accidents was collected and correlated to serology results. This study presents an opportunity for discussion of routine procedures involving OPVs in laboratories and their intrinsic risks. Aspects of the live attenuated smallpox vaccine are also discussed.

Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis

We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST) patients following in vitro stimulation with staphylococcal enterotoxin A (SEA). We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 secretion in both OST and non-infected individuals (NI). Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months) or "late" osteomyelitis (5-12 months), we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.

Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis.

We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST) patients following in vitro stimulation with staphylococcal enterotoxin A (SEA). We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 secretion in both OST and non-infected individuals (NI). Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months) or "late" osteomyelitis (5-12 months), we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.

Phlebotominae fauna (Diptera: Psychodidae) in an urban district of Belo Horizonte, Brazil, endemic for visceral leishmaniasis: characterization of favored locations as determined by spatial analysis.

Belo Horizonte, the capital of the southeastern state of Minas Gerais, Brazil, and the fourth-largest city in the country, has the highest incidence of human visceral leishmaniasis (VL) together with a high prevalence of canine VL. The Northeast Sanitary District (NSD) of Belo Horizonte has the largest historical average of human VL cases in the metropolitan region, and is classified as a priority area for epidemiological and entomological monitoring of the disease. The objectives of the present study were to determine the seasonal variation in phlebotomine fauna and to describe the environmental situations in the NSD through characterization of peri-domiciles and application of geographical information system analysis. Entomological captures were performed every two weeks during the period July 2006 to June 2007 using HP light traps placed at 16 locations where cases of human VL had been reported in 2005. The environmental characterization of these locations was accomplished using forms and photographic images. Spatial analyses was used to determine the influence of vegetation, hydrography, altitude and pockets of poverty on the occurrence of cases of human and canine VL, and of phlebotomine vectors. A total of 633 phlebotomines belonging to the subtribes Psychodopygina and Lutzomyina were captured and, of these, 75% were identified as Nyssomyia whitmani and 11% as Lutzomyia longipalpis. The majority of the studied peri-domiciles presented inadequate hygienic conditions that would favor the development of phlebotomines. No significant correlations could be established between biogeographical aspects and either the incidence of human and canine VL or the occurrence of phlebotomines. The proximity of areas with vegetation, villages, slums and open watercourses exerted little influence on the incidence of VL. These findings reinforce the urbanization of the VL profile since the disease occurred in locations where conditions that have been classically related to its prevalence were not present. The results reported herein will be important for implementing measures against VL in the study area.

Inaccuracy of enzyme-linked immunosorbent assay using soluble and recombinant antigens to detect asymptomatic infection by Leishmania infantum.

BACKGROUND: One of the most important drawbacks in visceral leishmaniasis (VL) population studies is the difficulty of diagnosing asymptomatic carriers. The aim of this study, conducted in an urban area in the Southeast of Brazil, was to evaluate the performance of serology to identify asymptomatic VL infection in participants selected from a cohort with a two-year follow-up period. METHODOLOGY: Blood samples were collected in 2001 from 136 cohort participants (97 positive and 39 negatives, PCR/hybridization carried out in 1999). They were clinically evaluated and none had progressed to disease from their asymptomatic state. As controls, blood samples from 22 control individuals and 8 patients with kala-azar were collected. Two molecular biology techniques (reference tests) were performed: PCR with Leishmania-generic primer followed by hybridization using L. infantum probe, and PCR with specific primer to L. donovani complex. Plasma samples were tested by ELISA using three different antigens: L. infantum and L. amazonensis crude antigens, and rK39 recombinant protein. Accuracy of the serological tests was evaluated using sensitivity, specificity, likelihood ratio and ROC curve. FINDINGS: The presence of Leishmania was confirmed, by molecular techniques, in all kala-azar patients and in 117 (86%) of the 136 cohort participants. Kala-azar patients showed high reactivity in ELISAs, whereas asymptomatic individuals presented low reactivity against the antigens tested. When compared to molecular techniques, the L. amazonensis and L. infantum antigens showed higher sensitivity (49.6% and 41.0%, respectively) than rK39 (26.5%); however, the specificity of rK39 was higher (73.7%) than L. amazonensis (52.6%) and L. infantum antigens (36.8%). Moreover, there was low agreement among the different antigens used (kappa<0.10). CONCLUSIONS: Serological tests were inaccurate for diagnosing asymptomatic infections compared to molecular methods; this could lead to misclassification bias in population studies. Therefore, studies which have used serological assays to estimate prevalence, to evaluate intervention programs or to identify risk factors for Leishmania infection, may have had their results compromised.

Diagnosing human asymptomatic visceral leishmaniasis in an urban area of the State of Minas Gerais, using serological and molecular biology techniques.

A population-based cross-sectional study was set up in Sabará country, Southeastern Brazil, to identify asymptomatic human visceral leishmaniasis in an urban area of low disease prevalence. Blood was collected on filter paper (n=1,604 inhabitants) and examined by indirect immunofluorescent test, enzyme-linked immunosorbent assay and immunochromatographic strip test. The prevalence rates of infection ranged from 2.4 to 5.6% depending on the test used. One year later, venous blood was collected in a subset of 226 participants (102 seropositive and 124 seronegative). The tests performed were IFAT, ELISA, rk39-ELISA, polymerase chain reaction and hybridization with Leishmania donovani complex probe. No clinical signs or symptoms of leishmaniasis were observed. Using hybridization as a reference test, the sensitivity and specificity of serology were respectively: 24.8 and 71% (ELISA); 26.3 and 76.3% (rk-39); 30.1 and 63.4% (IFAT). Due to disagreements, different criteria were tested to define the infection and hybridization should be considered in epidemiological studies.

Diagnosing human asymptomatic visceral leishmaniasis in an urban area of the State of Minas Gerais, using serological and molecular biology techniques

A population-based cross-sectional study was set up in Sabará country, Southeastern Brazil, to identify asymptomatic human visceral leishmaniasis in an urban area of low disease prevalence. Blood was collected on filter paper (n=1,604 inhabitants) and examined by indirect immunofluorescent test, enzyme-linked immunosorbent assay and immunochromatographic strip test. The prevalence rates of infection ranged from 2.4 to 5.6 percent depending on the test used. One year later, venous blood was collected in a subset of 226 participants (102 seropositive and 124 seronegative). The tests performed were IFAT, ELISA, rk39-ELISA, polymerase chain reaction and hybridization with Leishmania donovani complex probe. No clinical signs or symptoms of leishmaniasis were observed. Using hybridization as a reference test, the sensitivity and specificity of serology were respectively: 24.8 and 71 percent (ELISA); 26.3 and 76.3 percent (rk-39); 30.1 and 63.4 percent (IFAT). Due to disagreements, different criteria were tested to define the infection and hybridization should be considered in epidemiological studies.

Um estudo seccional de base populacional foi desenvolvido no município de Sabará, região sudeste do Brasil, para identificar a leishmaniose visceral humana assintomática em uma área urbana de baixa prevalência da doença. Foi coletado sangue em papel filtro (n=1.604 moradores), sendo examinados pela reação de imunofluoresência indireta, ensaio imunoenzimático e teste imunocromatográfico (strip test). As taxas de prevalência da infecção variaram de 2,4 a 5,6 por cento, dependendo do teste utilizado. Um ano depois foi coletado sangue venoso de um subgrupo de 226 participantes (102 soropositivos e 124 soronegativos). Os testes realizados foram IFAT, ELISA, rk39-ELISA, reação em cadeia da polimerase e hibridização com sonda específica para o complexo Leishmania donovani. Não foi observado nenhum sinal clínico ou sintoma de leishmaniose. Usando a hibridização como teste de referência, a sensibilidade e especificidade dos testes sorológicos foram, respectivamente: 24.8 e 71 por cento (ELISA); 26,3 e 76,3 por cento (rk39-ELISA); 30,1 e 63,4 por cento (IFAT). Devido a discordâncias, diferentes critérios foram testados para definir a presença da infecção e a hibridização deveria ser considerada em estudos epidemiológicos.

Risk factors for Leishmania chagasi infection in an urban area of Minas Gerais State

Com o objetivo de identificar os determinantes da infeccão humana por Leishmania chagasi em uma área urbana, foi realizado um estudo seccional de base populacional utilizando-se métodos moleculares e sorológicos para identificar a infeccão. Os participantes foram entrevistados utilizando-se questionário pré-codificado. Dois critérios foram testados para identificar os fatores de risco: Modelo 1- incluindo todos os participantes positivos na hibridizacão com sonda para o complexo Leishmania donovani; Modelo 2- incluindo todos os participantes positivos na hibridizacão e em pelo menos um teste sorológico. No Modelo 1, as variáveis associadas à infeccão foram: criar pássaros, encontrar-se fora de casa entre 18:00-22:00h e não ter o lixo coletado. No Modelo 2, as variáveis associadas à infeccão foram: família conhecer o vetor, não ter o lixo coletado, lixo não removido ou queimado, criar pássaros, proximidade de áreas erodidas. Os fatores de risco identificados foram associados às condicões das moradias, presenca de animais e probabilidade de contato com flebotomíneos.

Risk factors for Leishmania chagasi infection in an urban area of Minas Gerais State.

In order to understand the determinants of human infection by Leishmania chagasi in an urban area, a cross-sectional population based study was conducted using molecular and serologic methods to identify infection. Participants were interviewed using a pre-coded questionnaire. Two criteria were tested to identify risk factors: Model 1--including all participants positive in hybridization by Leishmania donovani complex probe; Model 2--including all participants positive for hybridization and at least one serologic test. In Model 1, the variables associated with infection were: ownership of birds, time spent outside house between 6:00-10:00 PM and garbage not collected. In Model 2, the variables associated with infection were: family with knowledge of the vector, garbage not collected, garbage not removed or buried, ownership of birds and eroded areas in the neighborhood. The risk factors identified were associated with household conditions, presence of animals and the likelihood of contact with phlebotomine sandflies.

Vigilância epidemiológica no Programa de Controle da Doença de Chagas em Minas Gerais, Brasil (1984-1998) / Epidemiological surveillance in the Chagas disease control program in Minas Gerais State, Brazil (1984-1998)

É apresentada a metodologia utilizada em Minas Gerais para a vigilância no Programa de Controle da Doença de Chagas. O sistema é desenvolvido com a participaçäo das comunidades, desde o planejamento à tomada de decisöes e deve ser gradativamente assumido pelos municípios no processo de municipalizaçäo das açöes e serviços de saúde do Sistema Unico de Saúde (SUS).

Aspectos metodológicos do inquérito sorológico em escolares e estudo caso-controle aninhado para avaliação de eficácia do Programa de Controle da Doença de Chagas / Methodological aspects of sorologic survey in students: evaluation of the Chagas Disease Control Program

Com o objetivo de avaliar a eficácia do Programa de Controle da Doença de Chagas (PCDCh) na redução da infecção humana pelo T. cruzi, a FNS propôs a realização de inquéritos sorológicos periódicos em crianças nascidas após o início das ações de controle nos municípios localizados em áreas de Vigilância Epidemiológica. A Vigilância é fundamentada na participação comunitária, exercida de forma permanente pelos Serviços de Saúde, de Ensino, e comunidades do Município