The rapid spread of Zika virus (ZIKV) represents a global public health problem, especially in areas that harbor several mosquito species responsible for virus transmission, such as Brazil. In these areas, improvement in mosquito control needs to be a top priority, but mosquito viral surveillance occurs inefficiently in ZIKV-endemic countries. Quantitative reverse transcription PCR (qRT-PCR) is the gold standard for molecular diagnostic of ZIKV in both human and mosquito samples. However, the technique presents high cost and limitations for Point-of-care (POC) diagnostics, which hampers its application for a large number of samples in entomological surveillance programs. Here, we developed and validated a one-step reverse transcription LAMP (RT-LAMP) platform for detection of ZIKV in mosquito samples. The RT-LAMP assay was highly specific for ZIKV and up to 10,000 times more sensitive than qRT-PCR. Assay validation was performed using 60 samples from Aedes aegypti and Culex quinquefasciatus mosquitoes collected in Pernambuco State, Brazil, which is at the epicenter of the Zika epidemic. The RT-LAMP had a sensitivity of 100%, specificity of 91.18%, and overall accuracy of 95.24%. Thus, our POC diagnostics is a powerful and inexpensive tool to monitor ZIKV in mosquito populations and will allow developing countries to establish better control strategies for this devastating pathogen.
BackgroundIn Brazil, schistosomiasis is a parasitic disease of public health relevance, mainly in poor areas where Schistosoma mansoni is the only human species encountered and Biomphalaria straminea is one of the intermediate host snails. A nested-PCR based on a specific mitochondrial S. mansoni minisatellite DNA region has been successfully developed and applied as a reference method in Brazil for S. mansoni detection, mainly in host snails for epidemiological studies. The amplification efficiency of LAMP is known to be higher than PCR. The present work aimed to assess the utility of our previously described SmMIT-LAMP assay for S. mansoni detection in human stool and snail samples in a low-transmission area of schistosomiasis in the municipality of Umbuzeiro, Paraíba State, Northeast Region of Brazil.Methodology/Principal findingsA total of 427 human stool samples were collected during June-July 2016 in the municipality of Umbuzeiro and an overall prevalence of 3.04% (13/427) resulted positive by duplicate Kato-Katz thick smear. A total of 1,175 snails identified as Biomphalaria straminea were collected from 14 breeding sites along the Paraíba riverbank and distributed in 46 pools. DNA from human stool samples and pooled snails was extracted using the phenol/chloroform method. When performing the SmMIT-LAMP assay a total of 49/162 (30.24%) stool samples resulted positive, including 12/13 (92.31%) that were Kato-Katz positive and 37/149 (24.83%) previously Kato-Katz negative. By nested-PCR, only 1/46 pooled DNA snail samples was positive. By SmMIT-LAMP assay, the same sample also resulted positive and an additional one was positive from a different breeding site. Data of human and snail surveys were used to build risk maps of schistosomiasis incidence using kernel density analysis.Conclusions/SignificanceThis is the first study in which a LAMP assay was evaluated in both human stool and snail samples from a low-transmission schistosomiasis-endemic area. Our SmMIT-LAMP proved to be much more efficient in detection of S. mansoni in comparison to the 'gold standard' Kato-Katz method in human stool samples and the reference molecular nested-PCR in snails. The SmMIT-LAMP has demonstrated to be a useful molecular tool to identify potential foci of transmission in order to build risk maps of schistosomiasis.
Primers targeting the gene encoding the small subunit rRNA were designed to amplify DNA from Schistosoma mansoni with high specificity. Three PCR systems were developed: conventional PCR, two-step nested PCR (NPCR) and single-tube nested PCR (STNPCR). The limits of detection of parasite DNA for the conventional PCR, NPCR and STNPCR were 10 pg, 0.1 fg and 1 fg, respectively. The assays were highly specific for S. mansoni and did not recognise DNA from closely related non-schistosome trematodes. Using pools of Biomphalaria molluscs, PCR, NPCR and STNPCR were positive in 6/16 (37.5%), 15/16 (93.8%) and 13/16 (81.3%) of the tested samples, respectively, whereas the observation of cercariae shedding after exposure to light was able to detect S. mansoni infection in 6/16 (37.5%) of the pools. Thus, the molecular detection systems had a higher level of sensitivity than standard screening of intermediate hosts by cercarial shedding when DNA was purified from pools of snails collected from endemic areas. These PCR protocols have potential to be used as tools for monitoring of schistosome transmission.
Uncontrolled peripheral urbanisation coupled with environmental degradation has affected the status of schistosomiasis in Pernambuco (PE), Brazil. This endemic disease continues to perpetuate its transmission in rural areas and has also become a cause for concern in coastal towns of the state. The lack of basic infrastructure (sanitation and health programmes) to support the new urban areas leads to faecal contamination of natural aquatic environments, resulting in consequent infection of vector snails and the emergence of new sources of schistosomiasis transmission. In the present paper, we discuss the current epidemiological status of schistosomiasis in PE. We have consolidated and analysed information from parasitological, malacological and morbidity surveys undertaken by the group of researchers at the Laboratory of Schistosomiasis, Centro de Pesquisas Aggeu Magalhães-Fiocruz. The results of our analysis show: (i) the maintenance of the levels of schistosomiasis in the rural Zona da Mata, PE, (ii) the record of the human cases of schistosomiasis and the foci of infected snails detected along the coast of PE through 2007, (iii) the high record of the severe clinical form of schistosomiasis in the metropolitan region of Recife (RMR) and (iv) new breeding sites of schistosomiasis vector snails that were identified in a 2008 survey covering the RMR and the coastal localities of PE.
Staphylococcus aureus is the main human pathogen that colonizes individuals in general population. The objective of the study was evaluate the epidemiological and sensitivity profile of S. aureus lineage, isolated in health care workers (HCW) of a University Hospital in Pernambuco state, Brazil. Biological samples of hands and nasal cavities were sown in agar sheep blood. Colonies under suspicion of being S. aureus were identified using Gram staining, catalase test and coagulase, mannitol-salty agar fermentation and DNAse agar. The resistance to mupirocin was analyzed through the Kirby Bauer technique. In relation to methicillin and vancomycin the determination was by the minimum inhibitory concentration method (E-test). From the 202 HCW evaluated, 52 were colonized by S. aureus (25,7%). The factors associated to the colonization by S. aureus were: age-group, professional category, use of individual protection equipments (frequency and numbers). All S. aureus isolate lineages were sensitive to mupirocin and vancomycin, and three of them were identified as methicillin-resistant. The prevalence of MSSA and MRSA among HCW was considered low and was below the results described in the literature. The isolate S. aureus lineages have shown low resistance profile.
The detection of specific DNA sequences by polymerase chain reaction (PCR) Key words: polymerase chain reaction -molecular diagnosis -schistosomiasis -transmission -snail -Schistosoma Schistosomiasis is a disease transmitted by fresh water snails. It affects more than 200 million people worldwide and is endemic in 74 countries, with more than 80% of infected persons living in Africa. In Brazil, Schistosoma mansoni is the only causative species of schistosomiasis, and there are three species of intermediate hosts: Biomphalaria glabrata, B. straminea, and B. tenagophila. Adult S. mansoni live in the bloodstream. Their eggs pass out of the host with the faeces. In contact with water, free-swimming miracidia emerge from the egg and penetrate the soft tissues of the snail host. Within the snails, miracidia immediately differentiate into sporocysts and, as they migrate through the snail tissues, give rise to cercariae. Cercariae are shed from the snails and are infective to humans by direct penetration of the skin. Thus, molecular approaches can be used to detect DNA of different life cycle stages of S. mansoni by analyzing different biological samples: feces, snail tissues, and infested water bodies, resulting in diagnosis of vertebrate and invertebrate host infections, and identification of transmission sites.The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens (Leal et al.
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