Molecular detection of human astrovirus in an urban sewage treatment plant in Rio de Janeiro, Brazil

The objective of this study was to evaluate the prevalence and dissemination of human astroviruses (HAstV) in the environment by analyzing urban sewage samples from a wastewater treatment plant in the city of Rio de Janeiro, Brazil. A one-year study was performed with a total of 48 raw and treated sewage composite samples, which were collected biweekly from an activated sludge plant. Virus particles were concentrated by the adsorption-elution method using negatively charged membranes associated to a Centriprep Concentrator® 50 (Nihon Millipore). HAstV were detected in 16.7% of the samples in raw and treated sewage by using both qualitative and quantitative reverse transcriptase-polymerase chain reactions (RT-PCR and qPCR, respectively). Positive untreated sewage sample exhibited mean values of 1.1 x 10(4) gEq/mL. The qPCR sensitivity was 18 gEq/reaction. Through utilization of qPCR, a HAstV recovery efficiency of 4.2% and 4.3% was demonstrated for raw and treated sewage samples, respectively. The presence of HAstV in both the raw and treated sewage samples demonstrated the dissemination of these viruses in the environment as well as viral permanence after sewage treatment. There was a reduction in the total and faecal coliform levels, indicating efficiency of the wastewater treatment plant.

The molecular detection of different Leishmania species within sand flies from a cutaneous and visceral leishmaniasis sympatric area in Southeastern Brazil

Over the last 20 years, there has been an increase in the number of leishmaniasis cases in Brazil. Belo Horizonte (BH) is one of the most highly populated Brazilian cities that is affected by visceral leishmaniasis (VL). The health services in BH are coordinated by a central nucleus that is subdivided into nine sanitary districts. Historically, the highest level of human VL cases was found in the northeast sanitary district (NSD). The objective of our study was to detect Leishmania infection in the phlebotomine sand flies collected in the NSD by dissection and molecular approaches. Following the occurrence of human VL cases in 2005, entomological captures were performed from July 2006-June 2007. Out of the 245 sand flies dissected, only three Lutzomyia longipalpis spp contained flagellates. The female sand flies were grouped into 120 pools according to date, collection site and species, with approximately 10 individual sand flies in each pool. Subsquently, the DNA was extracted and Leishmania spp and other parasites were detected and identified by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorfism. Leishmania infantum was present in at least 19% of the Lu. longipalpis collected, in 3.8% of the Nyssomiya whitmani collected, in 33.3% of the Evandromiya termitophila collected and in 14.3% of the Nyssomiya intermedia collected. When the females of the cortelezzii complex were compared with each other, 3.2% of the females were infected with Leishmania braziliensis, whereas 3.2% of the females were infected with trypanosomatids.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.

Molecular detection of Mayaro virus during a dengue outbreak in the state of Mato Grosso, Central-West Brazil

Mayaro virus (MAYV) is frequently reported in Pan-Amazonia. The aim of this study was to investigate the circulation of alphaviruses during a dengue outbreak in the state of Mato Grosso, Brazil. Serum samples from dengue-suspected patients were subjected to multiplex semi-nested reverse transcriptase polymerase chain reaction for 11 flaviviruses and five alphaviruses, to nucleotide sequencing and to viral isolation. MAYV was detected in 15 (2.5%) of 604 patients. Twelve were co-infected with dengue virus 4, which was isolated from 10 patients. The molecular detection of MAYV in dengue-suspected patients suggests that other arboviruses may be silently circulating during dengue outbreaks in Brazil.

Evaluation of the speed-oligo direct Mycobacterium tuberculosis assay for molecular detection of mycobacteria in clinical respiratory specimens.

We present the first evaluation of a novel molecular assay, the Speed-oligo Direct Mycobacterium tuberculosis (SO-DMT) assay, which is based on PCR combined with a dipstick for the detection of mycobacteria and the specific identification of M. tuberculosis complex (MTC) in respiratory specimens. A blind evaluation was carried out in two stages: first, under experimental conditions on convenience samples comprising 20 negative specimens, 44 smear- and culture-positive respiratory specimens, and 11 sputa inoculated with various mycobacterium-related organisms; and second, in the routine workflow of 566 fresh respiratory specimens (4.9% acid-fast bacillus [AFB] smear positives, 7.6% MTC positives, and 1.8% nontuberculous mycobacteria [NTM] culture positives) from two Mycobacterium laboratories. SO-DMT assay showed no reactivity in any of the mycobacterium-free specimens or in those with mycobacterium-related organisms. Compared to culture, the sensitivity in the selected smear-positive specimens was 0.91 (0.92 for MTC and 0.90 for NTM), and there was no molecular detection of NTM in a tuberculosis case or vice versa. With respect to culture and clinical data, the sensitivity, specificity, and positive and negative predictive values for the SO-DMT system in routine specimens were 0.76 (0.93 in smear positives [1.0 for MTC and 0.5 for NTM] and 0.56 in smear negatives [0.68 for MTC and 0.16 for NTM]), 0.99, 0.85 (1.00 in smear positives and 0.68 in smear negatives), and 0.97, respectively. Molecular misidentification of NTM cases occurred when testing 2 gastric aspirates from two children with clinically but not microbiologically confirmed lung tuberculosis. The SO-DMT assay appears to be a fast and easy alternative for detecting mycobacteria and differentiating MTC from NTM in smear-positive respiratory specimens.

Molecular detection of enteropathogenic Escherichia coli in asymptomatic captive psittacines

Psittaciformes are one of the most endangered groups of birds, and several Brazilian species are classified between vulnerable and critically endangered. It is thus necessary to identify agents that cause infections in captive wild animals and to assess the risks posed thereof and to design interventions to minimize the possibility of disease outbreaks, leading to the conservation of endangered species. The purpose of this study was to identify enteropathogenic Escherichia coli (EPEC) cloacal isolates from asymptomatic psittacines in captivity and evaluate the distribution of the EPEC pathotype. Cloacal swabs were obtained from 46 asymptomatic birds, and resulting isolates were tested by polymerase chain reaction (PCR) for the presence of the attaching and effacing gene (eae) and bundle-forming pilus structural gene (bfpA) of EPEC. Samples from several species were tested, and three samples were found to be positive for the eae and bfpA genes and characterized as typical EPEC. This is the first report of this pathotype in asymptomatic psittacines. Although certain E. coli strains are more pathogenic than others, various factors should be considered when determining the potential of E. coli isolates to cause disease in captive psittacines. Birds that are positive for the EPEC (typical) strain could be zoonotic sources of infection, and may have acquired these strains through contact with humans or domestic animals. These findings may also be valuable for the long-term management of endangered species ex situ as one EPEC sample was isolated from a Red-tailed Amazon (Amazona brasiliensis).

Molecular detection of Neorickettsia risticii in Brazilian free-tailed bats (Tadarida brasiliensis) from Buenos Aires , Argentina

Neorickettsia risticii is the causative agent of Potomac Horse Fever, a severe febrile disease affecting horses, transmitted by trematodes species with a complex life cycle. A total of 30 insectivorous bats (Brazilian free-tailed bat Tadarida brasiliensis) were analyzed by PCR for presence of genus Anaplasma, Ehrlichia, Neorickettsia and Rickettsia. Three samples showed positive reactions for genus Anaplasma, Ehrlichia and Neorickettsia, and the sequences were 99.67% identical to Neorickettsia risticii. The role of bats in the life cycle of N. risticii has yet to be elucidated; however bats may be reservoirs for this bacterium. To our knowledge, this is the first evidence of N. risticii in Argentina.

Analysis of hematologic and serum chemistry values of Spheniscus magellanicus with molecular detection of avian malarial parasites (Plasmodium spp.)

Magellanic penguins (Spheniscus magellanicus) routinely migrate from their breeding colonies to Southern Brazil often contracting diseases during this migration, notably avian malaria, which has been already reported in Brazil and throughout the world. Detection of Plasmodium spp. in blood smears is the routine diagnostic method of avian malaria, however it has a low sensitivity rate when compared to molecular methods. Considering the negative impact of avian malaria on penguins, the aim of this study was to detect the presence of Plasmodium spp. in Magellanic penguins using Polymerase Chain Reaction (PCR) and by verifying clinical, hematological, and biochemical alterations in blood samples as well as to verify the likely prognosis in response to infection. Blood samples were obtained from 75 penguins to determine packed cell volume (PCV), red blood cell (RBC) and white blood cell (WBC) counts, mean corpuscular volume (MCV), uric acid, total protein, albumin, globulin and aspartate aminotransferase (AST) activity levels. Whole blood samples were used for PCR assays. Plasmodium spp. was detected in 32.0% of the specimens using PCR and in 29.3% using microscopic analyses. Anorexia, diarrhea and neurological disorders were more frequent in penguins with malaria and a significant weight difference between infected and non-infected penguins was detected. PCV and MCV rates showed no significant difference. RBC and WBC counts were lower in animals with avian malaria and leukopenia was present in some penguins. Basophil and lymphocyte counts were lower in infected penguins along with high monocyte counts. There was no significant difference in AST activities between infected and non-infected animals. There was a significant increase in uric acid values, however a decrease in albumin values was observed in infected penguins. Based on this study, we concluded that Plasmodium spp. occurs in Magellanic penguins of rehabilitation centers in Southeastern Brazil, compromising the weight of infected animals with clinical alterations appearing in severe cases of this disease. It was also noted that, although the hematological abnormalities presented by these animals may not have been conclusive, leukopenia, monocytosis and the decrease of basophils and lymphocytes revealed an unfavorable prognosis, and Plasmodium spp. infections may progress with elevated uric acid concentration and low albumin levels.

Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice

AbstractMycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Molecular detection of enterotoxins in environmental strains of Aeromonas hydrophila and Aeromonas jandaei

Aeromonas species are widely distributed in aquatic environments and recent studies include the genus in the emergent pathogens group because of its frequent association with local and systemic infections in immunocompetent humans. Aiming to search for virulence genes in environmental strains of Aeromonas hydrophila and Aeromonas jandaei, we designed specific primers to detect act/hly A/aer complex and alt genes. Primers described elsewhere were used to detect ast. Eighty-seven strains previously identified using phenotypic and genotypic tests as A. hydrophila (41) and A. jandaei (46) were analysed for the presence of the virulence genes using PCR. DNA fragments of expected size were purified and directly sequenced. Among the 41 strains of A. hydrophila 70.7% (29), 97.6% (40) and 26.8% (11) possessed act/hly A/aer complex, ast and alt genes, respectively. Among the 46 strains of A. jandaei, 4.4% (2), 0% (0) and 32.6% (15) were positive for act/hly A/aer complex, ast and alt genes, respectively. Sequencing allowed for the confirmation of amplified products using BLAST. The present work proposes a specific and rapid diagnostic method to detect the main virulence determinants of Aeromonas, a genus potentially pathogenic to humans.

Molecular detection of hemoplasma infection among cats from São Luís island, Maranhão, Brazil

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Molecular detection of tick-borne bacterial agents in Brazilian and exotic captive carnivores

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Molecular detection of Streptococcus agalactiae in bovine raw milk samples obtained directly from bulk tanks

Mastitis is the most common infectious disease affecting dairy cattle; in addition, it remains the most economically important disease of dairy industries around the world. Streptococcus agalactiae, a contagious pathogen associated with subclinical mastitis, is highly infectious. This bacterium can cause an increase in bulk tank bacterial counts (BTBC) and bulk tank somatic cell counts (BTSCC). The microbiological identification of S. agalactiae in samples from bulk tanks is an auxiliary method to control contagious mastitis. Thus, there are some limitations for time-consuming cultures or identification methods and additional concerns about the conservation and transport of samples. Bulk tank samples from 247 dairy farms were cultured and compared through polymerase chain reaction (PCR), directed to 16S rRNA genes of S. agalactiae, followed by BTBC and S. agalactiae isolation. The mean value of BTBC was 1.08 x 10(6) CFU mL(-1) and the bacterium was identified through the microbiological method in 98 (39.7%; CI95% = 33.8-45.9%) and through PCR in 110 (44.5%; CI95% = 38.5-50.8%) samples. Results indicated sensitivity of 0.8571 +/- 0.0353 (CI95% = 0.7719-0.9196) and specificity of 0.8255 +/- 0.0311 (CI95% = 0.7549-0.8827). The lack of significant difference between microbiological and molecular results (kappa = 0.6686 +/- 0.0477 and CI95% = 0.5752-0.7620) indicated substantial agreement between the methods. This suggests that PCR can be used for bulk tank samples to detect contagious mastitis caused by S. agalactiae. (C) 2011 Elsevier Ltd. All rights reserved.

Molecular detection of Leishmania sp. in cats (Felis catus) from Andradina Municipality, São Paulo State, Brazil

The aim of this work was to molecularly detect Leishmania species in 52 cats from Andradina Municipality, São Paulo State, Brazil. The direct parasitological test was performed by using imprints of poplited lymph node, bone marrow and spleen to verify amastigote forms of Leishmania spp. The samples that were positive parasitological tests were subjected to molecular analysis (PCR) and sequencing. Infection was detected for 5.76% (3/52) of the examined cats and two had presence of amastigote forms of Leishmania spp. in lymph nodes. Polymerase chain reaction (PCR) of kinetoplast minicircle DNA, indicated positive amplification for samples of spleen and lymph nodes and the sequencing resulted in 97% similarity with Leishmania (L) chagasi. This study proved the occurrence of infection with Leishmania (L) chagasi in felines from Andradina municipality, São Paulo State. (C) 2010 Elsevier B.V. All rights reserved.