Molecular detection of vector borne pathogens in anemic and thrombocytopenic dogs in southern Brazil / Detecção molecular de patógenos transmitidos por vetores em cães anemicos e trombocitopenicos no Sul do Brasil

Abstract Arthropod-borne pathogens are medically important because of their ability to cause diseases in their hosts. The purpose of this study was to detect the occurrence of Ehrlichia spp., piroplasmids and Hepatozoon spp. in dogs with anemia and thrombocytopenia in southern Brazil. EDTA-whole blood was collected from 75 domestic dogs presenting anemia or/and thrombocytopenia from Guarapuava, state of Paraná, Brazil. DNA samples were subjected to conventional PCR assays for Ehrlichia spp. (dsb), piroplasmids (18S rRNA) and Hepatozoon spp. (18S rRNA), followed by sequencing and phylogenetic analyses. Among the 75 dogs, one (1.33%) was positive for Hepatozoon sp. and six (8%) were positive for piroplasmids in 18S rRNA cPCR assays. None of the dogs showed positive results in Ehrlichia spp.-cPCR targeting dsb gene. The phylogenetic analyses revealed that three piroplasm sequences were clustered with Rangellia vitalii, while one sequence was grouped with B. vogeli. The only sequence obtained from Hepatozoon spp.-PCR protocol was pooled with H. canis. Therefore, there is urgent need for differential molecular diagnosis of the two piroplasm species cited as etiological agents in clinical cases of canine hemoparasitic diseases, given the higher pathogenic potential of R. vitalii than of B. vogeli.

Resumo Agentes transmitidos por artrópodes têm grande importância na medicina veterinária devido à sua capacidade de causar doenças graves em seus hospedeiros. O presente estudo objetivou investigar a ocorrência de três patógenos transmitidos por vetores, Ehrlichia canis, Rangelia vitalii e Hepatozoon canis, em cães na região sul do Brasil. Foram coletadas amostras de sangue total de 75 cães domésticos que apresentavam anemia e/ou trombocitopenia, em Guarapuava, Paraná, Brasil. As amostras de DNA foram submetidas à técnica de PCR convencional para E. canis (dsb), piroplasmídeos (18S rRNA) e Hepatozoon spp. (18S rRNA), seguida de sequenciamento e análises filogenéticas. Das 75 amostras, uma (1,33%) foi positiva para Hepatozoon spp. e seis (8%) foram positivas para Babesia spp. Nenhuma amostra mostrou resultados positivos para Ehrlichia spp. utilizando a detecção pelo gene dsb. As análises filogenéticas revelaram que três sequências obtidas foram agrupadas no mesmo clado que R. vitalii , enquanto uma foi agrupada juntamente com B. vogeli. A única sequência obtida pelo protocolo de PCR para Hepatozoon spp. foi agrupada juntamente com H. canis. Assim, é justificada necessidade de diferenciação das espécies de piroplasmas, através do diagnóstico molecular, como agentes etiológicos nos casos clínicos de hemoparasitose canina, considerando o potencial patogênico de R. vitalii quando comparado à B. vogeli.

Fecal microbiota analysis of children with small intestinal bacterial overgrowth among residents of an urban slum in Brazil / Análise da microbiota fecal de crianças com sobrecrescimento bacteriano no intestino delgado de moradoras de uma favela urbana no Brasil

Abstract Objective: To analyze the fecal microbiota composition of children living in an urban slum in Brazil, with or without small intestinal bacterial overgrowth, and to investigate the occurrence of stunting and anemia. Methods: A total of 100 children were studied, aged 5-11 years, from the municipality of Osasco, São Paulo. Small intestinal bacterial overgrowth was screened through hydrogen and methane breath test with lactulose. Weight and height were measured, and the height-for-age and body mass-for-age anthropometric indexes were calculated. The occurrence of anemia was investigated by capillary hemoglobin. Analysis of bacterial phylum, genus, and species was performed by real-time polymerase chain reaction in fecal samples. Results: Small intestinal bacterial overgrowth was identified in 61.0% of the children. A lower mean of height-for-age Z-score ([−0.48 ± 0.90] vs. [−0.11 ± 0.97]; p = 0.027), as well as capillary hemoglobin ([12.61 ± 1.03 g/dL] vs. [13.44 ± 1.19 g/dL]; p < 0.001) was demonstrated in children with SIBO when compared with children without small intestinal bacterial overgrowth. Children with small intestinal bacterial overgrowth presented a higher frequency of Salmonella spp., when compared to those without small intestinal bacterial overgrowth (37.7% vs. 10.3%; p = 0.002). Higher counts of total Eubacteria (p = 0.014) and Firmicutes (p = 0.038) were observed in children without small intestinal bacterial overgrowth; however, a higher count of Salmonella (p = 0.002) was found in children with small intestinal bacterial overgrowth. Conclusion: Children who lived in a slum and were diagnosed with small intestinal bacterial overgrowth showed lower H/A Z-scores and hemoglobin levels. Furthermore, differences were observed in the fecal microbiota of children with small intestinal bacterial overgrowth, when compared to those without it; specifically, a higher frequency and count of Salmonella, and lower counts of Firmicutes and total Eubacteria.

Resumo Objetivo: Analisar a composição da microbiota fecal de crianças moradoras de uma favela urbana no Brasil, com e sem sobrecrescimento bacteriano no intestino delgado, e investigar a ocorrência de déficit de crescimento e anemia. Métodos: Foram estudadas 100 crianças, com idade entre 5 e 11 anos, na cidade de Osasco, São Paulo. Sobrecrescimento bacteriano no intestino delgado foi pesquisado por teste respiratório do hidrogênio e metano no ar expirado com lactulose. Foram mensurados peso, estatura e calculados os índices antropométricos estatura para idade e índice de massa corporal para idade. Foi investigada a ocorrência de anemia, pela avaliação da hemoglobina capilar. A análise dos filos, gêneros e espécies bacterianas em amostras de fezes foi realizada por polymerase chain reaction em tempo real. Resultados: Sobrecrescimento bacteriano no intestino delgado foi diagnosticado em 61,0% das crianças avaliadas. Foi verificada menor média do escore Z do índice estatura para idade (-0,48±0,90 vs.-0,11±0,97 DP) e de hemoglobina capilar (12,61±1,03 vs. 13,44±1,19 g/dL) no grupo de crianças com sobrecrescimento bacteriano no intestino delgado, quando comparadas àquelas sem sobrecrescimento bacteriano no intestino delgado (p < 0,05). Nas crianças com sobrecrescimento bacteriano no intestino delgado foi observada maior frequência de Salmonella spp., quando comparadas àquelas sem sobrecrescimento bacteriano no intestino delgado (37,7% vs. 10,3%; p = 0,002). Maior contagem de Eubactérias totais (p = 0,014) e Firmicutes (p = 0,038) foi observada nas crianças sem sobrecrescimento bacteriano no intestino delgado, enquanto que as crianças com sobrecrescimento bacteriano no intestino delgado apresentaram maior contagem de Salmonella (p = 0,002). Conclusão: Nas crianças com diagnóstico de sobrecrescimento bacteriano no intestino delgado verificaram-se menores valores de estatura para idade e de hemoglobina. Foram constatadas diferenças na microbiota fecal das crianças com sobrecrescimento bacteriano no intestino delgado, especificamente, maior frequência e contagem de Salmonella spp. e menores contagens de Firmicutes e Eubactérias totais.

Disseminated histoplasmosis: A comparative study of the clinical features and outcome among immunocompromised and immunocompetent patients.

Background. Disseminated histoplasmosis is a chronic granulomatous disease caused by the dimorphic fungus, Histoplasma capsulatum. Clinical presentation can vary from the acute pulmonary to the chronic disseminated form. In India, disseminated histoplasmosis often presents with pyrexia of unknown origin with a presentation similar to ‘disseminated tuberculosis’ involving the adrenal glands and bone marrow. Due to rarity of the disease, data are lacking regarding its clinical presentation and outcome among immunocompromised and immunocompetent patients. Methods. During January 2000 to December 2010, we identified 37 patients of disseminated histoplasmosis and attempted to characterize the differences between immunocompromised and immunocompetent patients. Demographic characteristics, clinical presentation, risk factors, laboratory findings, diagnostic yield, treatment received and prognosis were noted and compared between the two groups. Results. Eleven of 37 patients with disseminated histoplasmosis were immunocompromised and 26 were immunocompetent. Comparison of their clinical features showed a higher frequency of skin lesions in the immunocompromised compared to the immunocompetent group (54.5% v. 11.5%). Pancytopenia and anaemia were more common among the immunocompromised (81.8%) compared to the immunocompetent (46.2%) group. In the immunocompromised patients, the diagnosis was made most often by bone marrow aspirate and culture (72.7%) compared to the immunocompromised group where the diagnosis was most often obtained by adrenal gland biopsy and fungal cultures (57.7%). The cure rate was significantly higher in the immunocompetent group (73% v. 45%). Conclusion. The clinical presentation and outcome of patients with disseminated histoplasmosis differs among immunocompromised and immunocompetent patients.

Increased hepcidin expression in multibacillary leprosy

Iron is essential for all organisms and its availability can control the growth of microorganisms; therefore, we examined the role of iron metabolism in multibacillary (MB) leprosy, focusing on the involvement of hepcidin. Erythrograms, iron metabolism parameters, pro-inflammatory cytokines and urinary hepcidin levels were evaluated in patients with MB and matched control subjects. Hepcidin expression in MB lesions was evaluated by quantitative polymerase chain reaction. The expression of ferroportin and hepcidin was evaluated by immunofluorescence in paucibacillary and MB lesions. Analysis of hepcidin protein levels in urine and of hepcidin mRNA and protein levels in leprosy lesions and skin biopsies from healthy control subjects showed elevated hepcidin levels in MB patients. Decreases in haematologic parameters and total iron binding capacity were observed in patients with MB leprosy. Moreover, interleukin-1 beta, ferritin, soluble transferrin receptor and soluble transferrin receptor/log ferritin index values were increased in leprosy patients. Hepcidin was elevated in lepromatous lesions, whereas ferroportin was more abundant in tuberculoid lesions. In addition, hepcidin and ferroportin were not colocalised in the biopsies from leprosy lesions. Anaemia was not commonly observed in patients with MB; however, the observed changes in haematologic parameters indicating altered iron metabolism appeared to result from a mixture of anaemia of inflammation and iron deficiency. Thus, iron sequestration inside host cells might play a role in leprosy by providing an optimal environment for the bacillus.

Anaemia & expression levels of CD35, CD55 & CD59 on red blood cells in Plasmodium falciparum malaria patients from India.

Background & objectives: Severe anaemia in Plasmodium falciparum (Pf) associated malaria is a leading cause of death despite low levels of parasitaemia. In an effort to understand the pathogenesis of anaemia we studied expression level of RBC complement regulatory proteins, CR1 (CD35), CD55 and CD59 with haemoglobin status in a group of malaria cases from Assam, Goa and Chennai, and in healthy controls. Methods: Flowcytometry was used to study expression of CR1, CD55 and CD59 in 50 Pf cases and 30 normal healthy volunteers. Giemsa stained thick and thin blood films were used for microscopic detection and identification of malarial parasites and parasite count. Results: No correlation was found between degree of expression of RBC surface receptors CR1, CD55 and CD59 with haemoglobin level. However, expression of CD55 was less in malaria cases than in healthy controls. Interpretation & conclusions: The present findings indicate that malaria infection changes the expression profile of complement regulatory protein CD55 irrespective of severity status of anaemia. Further studies are needed to explore the pathophysiology of anaemia in malaria cases in Assam where expression of RBC complement receptors appears to be low even in normal healthy population.

Antibody response in typhoid fever in endemic Indonesia and the relevance of serology and culture to diagnosis.

Culture and serology were performed on blood and serum samples collected at or shortly after admission from 473 patients presented with suspected clinical typhoid. Clinical symptoms at first presentation including confusion, hepatomegaly, splenomegaly, abdominal pain, anemia, and gastrointestinal bleeding were non-specific as they were observed even more often in non-typhoid patients. Culture confirmed the diagnosis in 65.3% of the patients with typhoid fever as the final diagnosis. The sensitivity (58%) and specificity (98.1%) of a rapid dipstick assay for the detection of S. typhi-specific immunoglobulin M were somewhat lower than those of culture but higher than those of the Widal test. The dipstick assay thus may well be used in the serodiagnosis of typhoid in situation where culture facilities are not available. Combination of test results of dipstick and culture improved sensitivity to 82.5%. In laboratories that perform blood culture the dipstick assay may be used as a rapid screening tests to facilitate a rapid diagnosis. Sensitivity of the dipstick assay strongly increased with duration of illness and was higher for culture positive than for culture negative patients. Duration of illness, and different pathogen and host factors including dose of infection, pathogenicity and antigenicity, and prior antibiotic use are likely to influence the immune response, therefore the result of the dipstick assay. Duration of illness and presence of S. typhi in the blood are major factors that determine severity of disease.

Brucellosis in children in south Jordan

Retrospectively we evaluated the records of 68 children with brucellosis. We found 58.2% had consumed unpasteurized milk and dairy products. Nonspecific manifestations included: arthralgia [78%], fever [75%] and sweating [60%]. Localized manifestations included limping [75%] and arthritis [54%]. Leukopenia was found in 51% of children and anaemia in 24%. Brucella species was cultured for blood of 16 [23.5%] patients. Combination therapy containing streptomycin was more effective than gentamicin combinations

Prevalence of tuberculosis in Kishtwar Tehsil of Jammu region in Jammu and Kashmir State.

A study of prevalence of tuberculosis in Kishtwar tehsil of Jammu region was conducted from June 1991 to May 1992. A total of one thousand two hundred ninety-eight rural as well as urban population suffering from various types of lung diseases was studied for prevalence of tuberculosis and a sizeable number (98) of patients were found to be positive of various types of tuberculosis, viz, pulmonary tuberculosis, tuberculous pleural effusion and miliary tuberculosis. Of the population studied 7.55% were found to be positive for tuberculosis and among them 88.76% patients were anaemic and the commonest type of anaemia was normocytic normochromic and normocytic hypochromic. The ESR was raised in almost of all the patients. Of the population, 80.61% were seen to be positive on skiagram chest and 58.16% were found to be positive on other diagnostic tests like Mantoux's tests, sputum for acid-fast bacilli, etc. Mantoux's test was positive in 27.55% cases and sputum was positive in 30.61% cases. All the cases studied were subjected to special investigations. The changes in parameters are consistent with the diagnosis of tuberculosis. Large family size, poverty, excessive smoking, illiteracy, etc, are the major contributing factors.

Diagnóstico diferencial de la microcitemia en pacientes hospitalarios / Differential, diagnosis of the microcytosis in hospitalized patients

En el presente estudio, se pudo comprobar que los índices eritrocitarios VCM (Volumen Corpuscular Medio) y RDW (Amplitud Distribución Eritrocitaria), en unión del computo absoluto de eritrocitos, son indicadores de buena sensibilidad para la clásica B-talasemia (9,4 por ciento) y la anemia por defiencia de hierro (56 por ciento), condiciones que constituyeron más del 65 por ciento de microcitemia encontrada. El nivel de la PP-Zn evidenció en forma rápida la deficiencia de hierro y la correspondiente micrositosis. En los casos considerados como propios de una enfermedad crónica, se demostró una anormalidad de la PP-Zn en aproximadamente la mitad de los casos. Fue evidente que en comparación con anemia ferropriva las B-tal menores resultan ser más microcíticas que hipocrómicas, y que el diagnóstico de la alfa talasemia menor es asunto difícil si se exige para ello la presencia de cuerpos de inclusión de Hb H. Finalmente, se considera necesario ampliar la experiencia que se ha acumulado en torno al uso de los autoanalizadores, en especial para conocer mejor la utilidad del índice RDW en la discriminación de la microcitemia