Polymerase chain reaction and nested-PCR approaches for detecting Cryptosporidium in water catchments of water treatment plants in Curitiba, State of Paraná, Brazil

Introduction Cryptosporidium is an important protozoan cause of waterborne disease worldwide of concern to public health authorities. To prevent outbreaks of cryptosporidiosis, the monitoring of this parasite in drinking water is necessary. In the present work, the polymerase chain reaction (PCR) and nested-PCR techniques were used to detect Cryptosporidium in raw water from catchment points of four water treatment plants (WTP) in Curitiba, Paraná, Brazil. Methods First, DNA extraction techniques were tested in samples containing decreasing amount of oocysts in reagent water, and PCR and nested-PCR with specific primers for 18SSU rDNA of Cryptosporidium were conducted to determine their sensitivity. In reagent water, a commercial extraction kit provided the best analytical sensitivity, and PCR and nested-PCR allowed the detection of five and two oocysts, respectively, with the primers XIAOR/XIAOF and XIAO1F/XIAO2R. Results In the spiking experiments, only the PCR with the primers AWA995F/AWA1206R was successful at detecting concentrations of 0.1 oocysts/mL. Two catchments samples of raw water and/or water sludge from four WTPs were contaminated with Cryptosporidium. Conclusions The application of the techniques to monitor Cryptosporidium in water and detect contamination in water catchments of WTPs in Curitiba are discussed in the present work.

Evaluation of parasitological examination, kDNA polymerase chain reaction and rK39-based immunochromatography for the diagnosis of visceral leishmaniasis in seropositive dogs from the screening-culling program in Brazil

Introduction Dogs play a primary role in the zoonotic cycle of visceral leishmaniasis (VL). Therefore, the accurate diagnosis of infected dogs, primarily asymptomatic dogs, is crucial to the efficiency of VL control programs. Methods We investigated the agreement of four diagnostic tests for canine visceral leishmaniasis (CVL): parasite detection, either after myeloculture or by direct microscopic examination of tissue imprints; kinetoplast-deoxyribonucleic acid-polymerase chain reaction (kDNA-PCR); and an immunochromatographic test (ICT). An enzyme-linked immunosorbent assay (ELISA) and an indirect immunofluorescence test (IFAT), both of which were adopted as part of the screening-culling program in Brazil, were used as reference tests. Our sample set consisted of 44 seropositive dogs, 25 of which were clinically asymptomatic and 19 were symptomatic for CVL according to ELISA-IFAT. Results The highest and lowest test co-positivities were observed for ICT (77.3%) and myeloculture (58.1%), respectively. When analyzed together, the overall percentage of co-positive tests was significantly higher for the symptomatic group compared to the asymptomatic group. However, only ICT was significantly different based on the results of a separate analysis per test for each group of dogs. The majority (93.8%) of animals exhibited at least one positive test result, with an average of 2.66 positive tests per dog. Half of the symptomatic dogs tested positive for all four tests administered. Conclusions The variability between test results reinforces the need for more efficient and reliable methods to accurately diagnose canine VL, particularly in asymptomatic animals.

Leishmania, Babesia and Ehrlichia in urban pet dogs: co-infection or cross-reaction in serological methods?

INTRODUCTION: The present study was designed to assess the occurrence of co-infection or cross-reaction in the serological techniques used for detecting the anti-Leishmania spp., -Babesia canis vogeli and -Ehrlichia canis antibodies in urban dogs from an area endemic to these parasites. METHODS: The serum samples from dogs were tested for the Babesia canis vogeli strain Belo Horizonte antigen and Ehrlichia canis strain São Paulo by immunofluorescence antibody test (IFAT) and by anti-Leishmania immunoglobulin G (IgG) antibody detection to assess Leishmania infection. We used the following four commercial kits for canine visceral leishmaniasis: ELISA, IFAT, Dual Path Platform (DPP) (Bio Manguinhos(r)/FIOCRUZ/MS) and a rK39 RDT (Kalazar Detect Canine Rapid Test; Inbios). RESULTS : Of 96 serum samples submitted to serological assays, 4 (4.2%) were positive for Leishmania as determined by ELISA; 12 (12.5%), by IFAT; 14 (14.6%) by rK39 RDT; and 20 (20.8%), by DPP. Antibodies against Ehrlichia and Babesia were detected in 23/96 (23.9%) and 30/96 (31.2%) samples, respectively. No significant association was identified between the results of tests for detecting Babesia or Ehrlichia and those for detecting Leishmania (p-value>0.05). CONCLUSIONS: In the present study, we demonstrated co-infection with Ehrlichia or Babesia and Leishmania in dogs from Minas Gerais (Brazil); we also found that the serological tests that were used did not cross-react.

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.

Validação preliminar de uma versão do Caregiver Reaction Assessment em um contexto de pacientes oncológicos em internação

Objetivo Adaptar para o Brasil uma versão portuguesa do Caregiver Reaction Assessment (CRA) e gerar indicadores preliminares de validade e fidedignidade para sua aplicação em cuidadores de pacientes oncológicos internados. Métodos Participaram voluntariamente 53 cuidadores, que responderam a um questionário sociodemográfico, ao CRA e à Escala de Bem-Estar Psicológico (EBEP). A unidimensionalidade e a homogeneidade dos escores do CRA foram avaliadas por meio de análise de componentes principais e de consistência interna, respectivamente. Correlações de Pearson entre escores do CRA e EBEP foram examinadas e utilizadas como indicadores de validade divergente e de construto. Resultados As cinco escalas que compõem o CRA apresentaram bons níveis de unidimensionalidade e homogeneidade, porém as escalas de impacto nas finanças e impacto na saúde obtiveram alfas insuficientes (< 0,7). O escore total do CRA apresentou alfa elevado (0,886). Correlações entre o CRA e a EBEP produziram coeficientes teoricamente interpretáveis, com magnitudes variando entre nulas e moderadas. Conclusão O CRA apresentou bons indicadores de validade e fidedignidade. Algumas adaptações em relação ao conteúdo de determinados itens se mostram, todavia, necessárias, a fim de serem calibradas ao contexto de pessoas atendidas por serviços subsidiados pelo Sistema Único de Saúde.

Recent brazilian experience on farmer reaction and crop response to fertilizer use

(1) In the period 1965/77 fertilizer consumption in Brazil increased nearly fifteen foild from circa 200,000 tons of N + P2O5 + K2O to 3 million tons. During the fifteen years extending from 1950 to 1964 usage of the primary macronutrients was raised by a factor of 2 only. (2) Several explanations are given for the remarkable increase, namely: an experimental background which supplied data for recommendations of rates, time and type of application; a convenient governmental policy for minimum prices and rural credit; capacity of the industry to meet the demand of the fertilizer market; an adequate mechanism for the diffusion of the practice of fertilizer use to the farmer. (3) The extension work, which has caused a permanent change in the aptitude towards fertilization, was carried out in the traditional way by salesmen supported by a technical staff, as well as by agronomists of the official services. (4) Two new programs were started and conducted in a rather short time, both putting emphasis on the relatively new technology of fertilizer use. (5) The first program, conducted in the Southern part of the country, extended lab and green house work supplemented by a few field trials to small land owners - the so called "operação tatú" (operation armadillo). (6) The seconde program, covering a larger problem area in the Northeast and in Central Brazil, began directly in field as thousands of demonstrations and simple experiments with the participation of local people whose involvement was essential for the success of the initiative; in this case the official extension services, both foreign and national sources of funds, and universities did participate under the leadership of the Brazilian Association for the Diffusion of Fertilizers (ANDA). (7) It is felt that the Brazilian experience gained thereof could be useful to other countries under similar conditions.

Acquired resistance of mice against S. mansoni and lung granulomatous reaction induced by BCG

Studies carried out in Sw outbred mice showed that there is no correlation between the degree of lung granulomatous reaction and the level of acquired resistance against S. mansoni infection induced by BCG.