Parasitoses de interesse clínico em sedimento de rio: uma abordagem na Saúde Pública / Parasitoses of clinical interest in sediment of rio: an approach to Public Health

Falta de saneamento básico facilita a disseminação de doenças parasitárias que impactam à saúde humana, sendo o sedimento capaz de albergar esses microrganismos. Objetivo do presente estudo foi avaliar a presença de parasitas patogênicos ao ser humano em sedimentos de borda de rio, abordando os riscos de infecções parasitárias na questão de saúde pública. Avaliaram-se pela técnica de HPJ 80 amostras de sedimentos dos rios Paranhana e Caí. Obtiveram-se 53 amostras positivas (66,2%) com diferentes parasitas, Ancylostoma sp., Strongyloides sp., Endolimax nana, Ascaris lumbricoides, Toxocara canis, Giardia lamblia, Entamoeba coli, Entamoeba histolytica/dispar, Trichiuris trichiura e Taenia sp. Locais com maior urbanização apresentaram 60% de amostras positivas e maior número de espécies. O sedimento de borda de rio indicou ser um meio apropriado para a manutenção das formas infectantes de parasitas. Faz-se necessário um saneamento adequado, a fim de minimizar a contaminação ambiental bem como o risco à saúde da população.

Lack of basic sanitation facilitates the spread of parasitic diseases that impact human health, with the sediment being able to house these microorganisms. The present study aims to evaluate the presence of pathogenic parasites to humans in riverbank sediments, addressing the risks of parasitic infections in public health. Eighty samples of sediments from the Paranhana and Caí rivers were evaluated using the HPJ technique. Fifty-three positive samples (66.2%) were obtained with different parasites, Ancylostoma sp., Strongyloides sp., Endolimax nana, Ascaris lumbricoides, Toxocara canis, Giardia lamblia, Entamoeba coli, Entamoeba histolytica/dispar, Trichiuris trichiura and Taenia sp. Places with greater urbanization presented 60% of positive samples and a greater number of species. Riverbank sediments indicated to be an appropriate means for the maintenance of infectious forms of parasites. Adequate sanitation is necessary in order to minimize environmental contamination as well as the population's health risk.

Low occurrence of Hepatitis A virus in water samples from an urban area of Southern Brazil.

Hepatitis A virus (HAV), a member of Picornaviridae family, is the main causative agent of acute viral hepatitis in the world, mainly in developing countries. HAV may be present in contaminated water and food and its presence is often associated to a lesser extent with socioeconomic factors and environmental quality. The main goals in the present study were to standardize a cell culture combined to a polymerase chain reaction protocol for the detection and quantification of viral viability and analyze whether the virus could be found in water samples collected in four urban streams of Sinos River watershed. Virus recovery was assayed from known virus concentrations measured in experimentally contaminated raw and ultrapure water (MilliQ®). Recovery rates ranged from 270% in raw water to 15,000% in ultrapure water. In a second step, a qPCR coupled to a previous passage in cells, demonstrated more analytical sensitivity when compared to samples assayed without a previous passage in cell cultures. HAV genome was detected in only 1 of 84 samples analyzed, pointing to a very low occurrence of HAV in water samples in the studied region. These findings are remarkable, since no more than 5% of the domestic sewage in this area is treated pointing to a low occurrence of HAV in the population living nearby during the study period.

Evaluation of virus recovery methods and efficiency of tannin-derived coagulants in removing total coliforms, E. coli and enteric viruses in effluents of a domestic sewage treatment plant.

In the present study, nine coagulants having potential to be used for sewage treatment were compared to assess their efficiency in removing total coliform bacteria, Escherichia coli and adenovirus. The coagulants tested were metallic and organic and their efficiency was compared when treating samples of raw and treated sewage (activated sludge). Before the efficiency tests of the coagulants, viral concentration methods were compared. Coagulation tests were carried out by using the jar-test system and the doses used ranged from 100 ppm to 1,000 ppm. Viral DNA was extracted and subjected to real-time polymerase chain reaction (qPCR) using primers for the gene of AdV hexon. Aluminum sulfate (1,000 ppm) presented the best results for raw sewage among metal coagulants whereas Acquapol® C118 and WW (1,000 ppm) had the most satisfactory results among organic coagulants, both reducing up to 7 logs for coliforms and 4 logs for virus. For the treated effluent, FeCl2 (1,000 ppm) presented best results for metal coagulants, whereas, from organic coagulants, the best removal rates were for Acquapol® 893/11 (1,000 ppm), both reducing up to 3 logs for coliforms and 4 logs for virus.

Enteric viruses and adenovirus diversity in waters from 2016 Olympic venues.

Rio de Janeiro's inner and coastal waters are heavily impacted by human sewage pollution for decades. Enteric viruses, including human adenoviruses (HAdV), human enterovirus (EV), group A rotavirus (RV) and hepatitis A virus (HAV) are more likely to be found in contaminated surface waters. The present work aimed to assess the frequency and loads of EV, HAdV-C and -F species, RV and HAV in sand and water samples from venues used during the 2016 Summer Olympics and by tourists attending the event. Sixteen monthly collections were carried out from March 2015 to July 2016 in 12 different sites from Rio de Janeiro, Brazil. Total and thermotolerant coliform counting was performed along molecular detection of virus was performed using quantitative polymerase chain reaction (qPCR). Analyses of all samples were further investigated by integrated cell culture PCR to check about the presence of HAdV infectious virus particles. The results show that 95.9% of water samples showed contamination with at least one type of virus. Regarding the viruses individually (% for water and sand respectively): HAdV-C (93.1%-57.8%), HAdV-F (25.3%-0%), RV (12.3%-4.4%), EV (26.7%-8.8%) and HAV (0%). The viral loads ranged from 103gc/L up to 109gc/L (water), and 103gc/g to 106gc/g (sand). In the phylogenetic tree, were classified into four main clusters, referring to species C, D, F and BAdV. And up to 90% of sites studied presented at least once presence of infectious HAdV-C. The most contaminated points were the Rodrigo de Freitas Lagoon, where Olympic rowing took place, and the Marina da Glória, the starting point for the sailing races, demonstrating serious problem of fecal contamination of water resources and threatens the health of Olympic athletes, tourists and residents.

Endogenous plasma and salivary uracil to dihydrouracil ratios and DPYD genotyping as predictors of severe fluoropyrimidine toxicity in patients with gastrointestinal malignancies.

OBJECTIVE: The aim of this study was to evaluate the use of plasma and saliva uracil (U) to dihydrouracil (UH2) metabolic ratio and DPYD genotyping, as a means to identify patients with dihydropyrimidine dehydrogenase (DPD) deficiency and fluoropyrimidine toxicity. METHODS: Paired plasma and saliva samples were obtained from 60 patients with gastrointestinal cancer, before fluoropyrimidine treatment. U and UH2 concentrations were measured by LC-MS/MS. DPYD was genotyped for alleles *7, *2A, *13 and Y186C. Data on toxicity included grade 1 to 4 neutropenia, mucositis, diarrhea, nausea/vomiting and cutaneous rash. RESULTS: 35% of the patients had severe toxicity. There was no variant allele carrier for DPYD. The [UH2]/[U] metabolic ratios were 0.09-26.73 in plasma and 0.08-24.0 in saliva, with higher correlation with toxicity grade in saliva compared to plasma (rs=-0.515 vs rs=-0.282). Median metabolic ratios were lower in patients with severe toxicity as compared to those with absence of toxicity (0.59 vs 2.83 saliva; 1.62 vs 6.75 plasma, P<0.01). A cut-off of 1.16 for salivary ratio was set (AUC 0.842), with 86% sensitivity and 77% specificity for the identification of patients with severe toxicity. Similarly, a plasma cut-off of 4.0 (AUC 0.746), revealed a 71% sensitivity and 76% specificity. CONCLUSIONS: DPYD genotyping for alleles 7, *2A, *13 and Y186C was not helpful in the identification of patients with severe DPD deficiency in this series of patients. The [UH2]/[U] metabolic ratios, however, proved to be a promising functional test to identify the majority of cases of severe DPD activity, with saliva performing better than plasma.

Detection and quantification of human adenovirus genomes in Acanthamoeba isolated from swimming pools.

Acanthamoeba is the most common free-living environmental amoeba, it may serve as an important vehicle for various microorganisms living in the same environment, such as viruses, being pathogenic to humans. This study aimed to detect and quantify human adenoviruses (HAdV) in Acanthamoebas isolated from water samples collected from swimming pools in the city of Porto Alegre, Southern Brazil. Free-living amoebae of the genus Acanthamoeba were isolated from water samples, and isolates (n=16) were used to investigate the occurrence of HAdVs. HAdV detection was performed by quantitative real-time polymerase chain reaction (qPCR). HAdVs were detected in 62.5% (10/16) of Acanthamoeba isolates, ranging from 3.24x103 to 5.14x105 DNA copies per milliliter of isolate. HAdV viral loads found in this study are not negligible, especially because HAdV infections are associated with several human diseases, including gastroenteritis, respiratory distress, and ocular diseases. These findings reinforce the concept that Acanthamoeba may act as a reservoir and promote HAdV transmission through water.

Human adenovirus spread, rainfalls, and the occurrence of gastroenteritis cases in a Brazilian basin.

Climate variables may interfere with the environmental persistence and spread of pathogenic microorganisms. This study aimed to investigate the occurrence of human adenovirus (HAdV) and total and thermotolerant coliforms in treated and untreated water and report gastroenteritis cases in seven cities located in the hydrographic basin of the Sinos River (HBSR), Southern Brazil. The data on water quality from samples collected at catchment areas of HBSR from March to December 2011 were compared with precipitation records, virus detection rates and viral loads, and information on enteric diseases among residents of the region. There was a marked increase in precipitation intensity in April, July, and August and a decrease in May and November. The number of HAdV genome copies (gc) in untreated water ranged from 2.1×10(8) gc/L in June to 7.8×10(1) gc/L in December, and in treated water, from 6.3×10(4) gc/L in September to 4.1×10(1) gc/L in November. The most probable number (MPN) of total coliforms ranged from 5×10(1) MPN/100 mL in December to 2.4×10(5) MPN/100 mL in July, and thermotolerant coliforms ranged from 1×10(1) MPN/100 mL in August to 6.9×10(4) MPN/100 mL in July. A total of 79 hospital admissions due to gastroenteritis were registered in the cities studied. The results for coliforms in untreated water demonstrate deficits in sanitation and wastewater treatment. These findings also indicate a possible relationship between the occurrence of rainfalls after dry periods and an increase in the number of gastroenteritis cases and in HAdV load quantified in surface water collected for conventional potabilization.

QUANTITATIVE VS. CONVENTIONAL PCR FOR DETECTION OF HUMAN ADENOVIRUSES IN WATER AND SEDIMENT SAMPLES.

Human Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

Bioaccumulation of animal adenoviruses in the pink shrimp.

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

Seasonal variation on the presence of adenoviruses in stools from non-diarrheic patients.

Human adenoviruses (HAdV), members of the Adenoviridae family, are excreted through the fecal route and may be present in the feces of humans consuming contaminated food or water. The presence of HAdV from different serotypes in the feces of healthy individuals was already reported using conventional polymerase chain reaction; however, real-time PCR (qPCR) may reveal not only the rates of detection as well as demonstrate the viral loads excreted by healthy persons. Aiming to identify and characterize the presence of adenoviruses in stool samples, 147 fecal samples from patients with no records of diarrhea were analyzed (74 from winter season and 73 from summer) by Real-Time PCR (qPCR) assay and conventional PCR. HAdV genome was present in 43.8% (32/73) of stools samples collected during summer season and 21.6% (16/74) during winter. The rate of detection of genomic copies (gc) ranged from 4.04×10(2) to 6.72×10(5)gc/g of feces among the 147 samples analyzed, of which the ranged of genomic copies of DNA HAdV was major in summer. All samples were negative when tested for rotaviruses (RV) and noroviruses (NoV) by PCR conventional and qPCR respectively. HAdV is excreted constantly by infected individuals in the absence of clinical signs and the occurrence may vary seasonally.

Bioaccumulation of animal adenoviruses in the pink shrimp

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems..

Bioaccumulation of animal adenoviruses in the pink shrimp

Adenoviruses are among the most promising viral markers of fecal contamination. They are frequently found in the water, sediment and soil of regions impacted by human activity. Studies of the bioaccumulation of enteric viruses in shrimp are scarce. The cities located in the northern coast of the lake systems in Southern Brazil have high urbanization and intensive farming rates, and poor sewage collection and treatment. One hundred (n = 100) Farfantepenaeus paulensis pink-shrimp specimens and 48 water samples were collected from coastal lagoons between June 2012 and May 2013. Water samples were concentrated and the shrimp, mashed. After DNA extraction, samples were analyzed by real time polymerase chain reaction (qPCR) in order to detect and quantify viral genomes. Thirty-five percent of shrimp samples were positive for contamination, predominantly by avian adenoviruses. A total of 91.7% of water samples contained adenoviruses DNA, with the human form being the most frequent. Our results provided evidence of significant bioaccumulation of adenoviruses in shrimp, showing the extent of the impact of fecal pollution on aquatic ecosystems.

Seasonal variation on the presence of adenoviruses in stools from non-diarrheic patients

Human adenoviruses (HAdV), members of the Adenoviridae family, are excreted through the fecal route and may be present in the feces of humans consuming contaminated food or water. The presence of HAdV from different serotypes in the feces of healthy individuals was already reported using conventional polymerase chain reaction; however, real-time PCR (qPCR) may reveal not only the rates of detection as well as demonstrate the viral loads excreted by healthy persons. Aiming to identify and characterize the presence of adenoviruses in stool samples, 147 fecal samples from patients with no records of diarrhea were analyzed (74 from winter season and 73 from summer) by Real-Time PCR (qPCR) assay and conventional PCR. HAdV genome was present in 43.8% (32/73) of stools samples collected during summer season and 21.6% (16/74) during winter. The rate of detection of genomic copies (gc) ranged from 4.04×10² to 6.72×10⁵gc/g of feces among the 147 samples analyzed, of which the ranged of genomic copies of DNA HAdV was major in summer. All samples were negative when tested for rotaviruses (RV) and noroviruses (NoV) by PCR conventional and qPCR respectively. HAdV is excreted constantly by infected individuals in the absence of clinical signs and the occurrence may vary seasonally.

Quantitative vs conventional PCR for detectionof human adenoviruses in water and sediment samples / PCR quantitativa versus convencional para a detecção de adenovírus humano em amostras de água e sedimento

SUMMARYHuman Adenoviruses (HAdV) are notably resistant in the environment. These agents may serve as effective indicators of fecal contamination, and may act as causative agents of a number of different diseases in human beings. Conventional polymerase chain reaction (PCR) and, more recently, quantitative PCR (qPCR) are widely used for detection of viral agents in environmental matrices. In the present study PCR and SYBR(r)Green qPCR assays were compared for detection of HAdV in water (55) and sediments (20) samples of spring and artesian wells, ponds and streams, collected from dairy farms. By the quantitative methodology HAdV were detected in 87.3% of the water samples and 80% of the sediments, while by the conventional PCR 47.3% and 35% were detected in water samples and sediments, respectively.

RESUMOOs adenovírus humanos (HAdV) são notavelmente resistentes ao ambiente. Estes agentes podem servir como indicadores efetivos de contaminação fecal, tanto quanto podem atuar como agentes causadores de diferentes doenças em seres humanos. A reação em cadeia da polimerase (PCR) e mais recentemente a PCR quantitativa (qPCR) são amplamente usadas para detecção de agentes virais em matrizes ambientais. No presente estudo, PCR e SYBR(r)Green qPCR foram comparadas para a detecção de HAdV em amostras de água (55) e sedimento (20) provenientes de nascentes, poços, açudes e arroios coletadas em propriedades leiteiras. A metodologia quantitativa detectou HAdV em 87,3% das amostras de água e 80% dos sedimentos, enquanto por PCR convencional a detecção foi de 47,3% e 35%, respectivamente.

Surface water quality in the Sinos River basin, in Southern Brazil: tracking microbiological contamination and correlation with physicochemical parameters.

Around the world, enteric viruses are often found in surface waters. This study set out to evaluate the occurrence of adenoviruses (AdVs) in water samples, and its relation to different physical, chemical, and bacteriological parameters [total coliform (TC) and fecal coliform (FC), represented by Escherichia coli]. Monthly samples of 500 ml of raw water were collected from May 2011 to June 2013 in eight abstraction points water treatment stations along three stretches of the Sinos River Basin (SRB), in Southern Brazil and, subsequently, were analyzed using real-time polymerase chain reaction (qPCR). AdVs from different species, from human (HAdV), and from other animals (CAV1-2, BAdV, PAdV, and AvAdV) were detected along the three stretches of the basin, indicating fecal contamination from different sources and proving the inefficiency of the wastewater treatment in the waters of the SRB and intensifying the strong influence of human activities that can contribute to the presence of inhibitory substances such as organic acids in surface of these waters. Statistical analyses revealed no significant correlations between the concentrations of TC and FC and the concentrations of AdVs. We observed a small, nonconstant, and unstable correlation between viruses and physicochemical parameters. These correlations were not sufficiently consistent to establish a reliable association; therefore, this study corroborates that only the viral assay itself is reliable for the diagnosis of fecal contamination by viruses in environmental samples.

Molecular detection of human adenovirus in sediment using a direct detection method compared to the classical polyethylene glycol precipitation.

Various effective methods have been developed to measure the concentration of viruses in sediment samples. However, there is need to standardize less laborious and simpler techniques. The objective of the present study was to compare two different methods to measure the concentration of viruses in soil samples. The use of polyethylene glycol (PEG) was compared with a direct extraction of viral nucleic acids from the samples diluted in modified Eagle's minimal essential medium (E-MEM). The presence of adenovirus in the samples was detected by real-time quantitative polymerase chain reaction (qPCR). Only six samples (30%) were positive for adenovirus when PEG technique was used. The direct method showed 16 (80%) samples positive for adenovirus. Therefore, direct detection (i.e. without previous concentration) demonstrated a higher rate of detection, better effectiveness, and shorter execution time. Furthermore, direct detection uses reagents that are often readily available in virology laboratories. Thus, it is an attractive alternative to other methods of detection of virus particles in sediments.

MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR) ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS / PCR em Tempo Real (qPCR) multiplex utilizando SYBR® Green para a detecção e diferenciação de Bartonella henselae e Bartonella clarridgeiae em gatos

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

Um novo teste baseado na reação em cadeia da polimerase em tempo real (qPCR) com SYBR ® Green foi desenvolvido para detectar duas espécies de Bartonella, B. henselae e B. clarridgeiae, diretamente em amostras de sangue. Este teste foi utilizado em amostras de sangue obtidas de gatos que vivem em abrigos de animais do sul do Brasil. Os resultados foram comparados aos obtidos pelo PCR convencional utilizado para a detecção de Bartonella spp. Das 47 amostras analisadas, oito foram positivas no PCR convencional e 12 foram positivas para qPCR. A reação de qPCR, permitiu a detecção da presença simultânea de B. henselae e B. clarridgeiae em duas destas amostras. Os resultados mostram que a qPCR aqui descrita pode ser uma ferramenta confiável para a detecção e diferenciação de duas espécies importantes de Bartonella spp.

Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats.

A novel SYBR® green-real time polymerase chain reaction (qPCR) was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

Prevalence of Bartonella henselae and Bartonella clarridgeiae in cats in the south of Brazil: a molecular study.

Bartonella spp are the causative agent of cat scratch disease in humans. Cats are the natural reservoir of these bacteria and may infect humans through scratches, bites or fleas. Blood samples from 47 cats aged up to 12 months were collected for this study. All animals were lodged in municipal animal shelters in the Vale do Sinos region, Rio Grande do Sul, Brazil. Bartonella spp were detected by genus-specific polymerase chain reaction (PCR) and when the PCR was positive, the species were determined by DNA sequencing. A Giemsa-stained blood smear was also examined for the presence of intraerythrocytic elements suggestive of Bartonella spp infection. Phylogenetic analysis was also performed for all positive samples. Using molecular detection methods, Bartonella spp were detected in 17.02% (8/47) of the samples. In seven out of eight samples confirmed to be positive for Bartonella spp, blood smear examination revealed the presence of intraerythrocytic elements suggestive of Bartonella spp. Phylogenetic analysis characterized positive samples as Bartonella henselae (5) or Bartonella clarridgeiae (3). To the best of our knowledge, this is the first molecular study demonstrating the presence of Bartonella spp in cats from the Southern Region of Brazil.