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AIMS: This study aimed to assess the use of cross-assembled phage (crAssphage) as an endogenous control employing a multivariate normalization analysis and its application as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data normalizer. METHODS AND RESULTS: A total of 188 twelve-hour composite raw sewage samples were obtained from eight wastewater treatment plants (WWTP) during a 1-year monitoring period. Employing the N1 and N2 target regions, SARS-CoV-2 RNA was detected in 94% (177) and 90% (170) of the samples, respectively, with a global median of 5 log10 genomic copies per liter (GC l-1). CrAssphage was detected in 100% of the samples, ranging from 8.29 to 10.43 log10 GC l-1, with a median of 9.46 ± 0.40 log10 GC l-1, presenting both spatial and temporal variabilities. CONCLUSIONS: Although SARS-CoV-2 data normalization employing crAssphage revealed a correlation with clinical cases occurring during the study period, crAssphage normalization by the flow per capita per day of each WWTP increased this correlation, corroborating the importance of normalizing wastewater surveillance data in disease trend monitoring.

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The main objective of this study was to investigate the dynamic of SARS-CoV-2 viral excretion in rectal swab (RS), saliva, and nasopharyngeal swab (NS) samples from symptomatic patients and asymptomatic contacts. In addition, in order to evaluate the replication potential of SARS-CoV-2 in the gastrointestinal (GI) tract and the excretion of infectious SARS-CoV-2 from feces, we investigated the presence of subgenomic nucleoprotein gene (N) mRNA (sgN) in RS samples and cytopathic effects in Vero cell culture. A prospective cohort study was performed to collect samples from symptomatic patients and contacts in Rio de Janeiro, Brazil, from May to October 2020. One hundred and seventy-six patients had samples collected at home visits and/or during the follow up, resulting in a total of 1633 RS, saliva, or NS samples. SARS-CoV-2 RNA was detected in 130 (73.9%) patients who had at least one sample that tested positive for SARS-CoV-2. The presence of replicating SARS-CoV-2 in RS samples, measured by the detection of sgN mRNA, was successfully achieved in 19.4% (6/31) of samples, whilst infectious SARS-CoV-2, measured by the generation of cytopathic effects in cell culture, was identified in only one RS sample. Although rare, our results demonstrated the replication capacity of SARS-CoV-2 in the GI tract, and infectious viruses in one RS sample. There is still a gap in the knowledge regarding SARS-CoV-2 fecal-oral transmission. Additional studies are warranted to investigate fecal or wastewater exposure as a risk factor for transmission in human populations.

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ABSTRACT New viruses of the Picornavirales order have been discovered with the increase in the number of sequences obtained by high-throughput sequencing, as well as human stool-associated RNA virus (husavirus [HuV]), found in human stool samples. However, there is much to be clarified about HuV. Its cellular host, evolutionary history, and other biological characteristics are still unknown. Therefore, samples collected from human beings and environmental samples in a watershed in Southern Brazil were processed for the metagenomic library. Upon metagenomic analysis, we identified a HuV (husavirus LMM_67754 OP019707) genome with 8,846 bp, which was reported for the first time in Southern Brazil. The new genome presents only 37% of nucleotide identity with Brazilian strains and more than 90% with genomes from China, Vietnam, Venezuela, and the Netherlands. The HuV phylogeny presents significant differences among genomes, probably because multiple introductions of the virus may have occurred. Many questions still need to be answered about HuV. Therefore, more sequences and studies on this virus are necessary to improve the comprehension of the unknown origin of Picornavirales.

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Water filtration is a common strategy of water sanitation in resource-poor tropical settings. Here, we assessed the intermediate term effect of this preventive procedure including specific filter-related as well as general hygiene training on the molecular detection of enteric pathogens in stool samples from Colombian Indigenous people. From a total of 89 individuals from an Indigenous tribe called Wiwa, stool samples were assessed by real-time PCR for enteropathogenic microorganisms prior to the implementation of water filtration-based infection prevention. Three years after the onset of the preventive strategy, a follow-up assessment was performed. A significantly beneficial effect of water filtration could be shown for Ascaris spp. only (p = 0.035) and a tendency (p = 0.059) for Hymenolepis nana. No hints for effects on the gastrointestinal shedding of Giardia duodenalis, Entamoeba histolytica, Cryptosporidium spp., Campylobacter spp., Shigella spp./enteroinvasive Escherichia coli, Necator americanus, Strongyloides stercoralis, Trichuris trichiura, and Taenia spp. were seen. In conclusion, the study indicates that water filtration can only be an element of a multi-modal hygiene concept to reduce enteric pathogen carriage in inhabitants of resource-poor tropical settings in spite of tendencies of beneficial effects.

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We described the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in stool samples from patients presenting only acute gastroenteritis (AGE) symptoms. From January to July 2020, 121 AGE stool samples were screened by quantitative reverse-transcription polymerase chain reaction. We detected SARS-CoV-2 in 27.5% of samples received during the epidemic period. No infectious viruses were observed in Vero E6 cells.

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Underdiagnosis of chronic infection with the nematode Strongyloides stercoralis may lead to severe disease in the immunosuppressed. Thus, we have set-up a specific and highly sensitive molecular diagnosis in stool samples. Here, we compared the accuracy of our polymerase chain reaction (PCR)-based method with that of conventional diagnostic methods for chronic infection. We also analyzed clinical and epidemiological predictors of infection to propose an algorithm for the diagnosis of strongyloidiasis useful for the clinician. Molecular and gold standard methods were performed to evaluate a cohort of 237 individuals recruited in Buenos Aires, Argentina. Subjects were assigned according to their immunological status, eosinophilia and/or history of residence in endemic areas. Diagnosis of strongyloidiasis by PCR on the first stool sample was achieved in 71/237 (29.9%) individuals whereas only 35/237(27.4%) were positive by conventional methods, requiring up to four serial stool samples at weekly intervals. Eosinophilia and history of residence in endemic areas have been revealed as independent factors as they increase the likelihood of detecting the parasite according to our study population. Our results underscore the usefulness of robust molecular tools aimed to diagnose chronic S. stercoralis infection. Evidence also highlights the need to survey patients with eosinophilia even when history of an endemic area is absent.

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Approximately 90% of the population in the northwestern Amazonia is composed of indigenous people and their healthcare is still a challenge. The objective of this study was to determine the frequency of parasites in two indigenous ethnic groups (Baré and Baniwa) in northwestern Amazonia. Stool samples from 270 individuals (199 Baniwa and 71 Baré) were analyzed using Richie's method and the spontaneous sedimentation method. Statistical differences among the proportions of infected individuals based on gender, age, and ethnicity were determined. All individuals were infected by protozoans or helminths. The most frequent parasites in the indigenous people were Ascaris lumbricoides (73%), Entamoeba spp. (53%), and Giardia intestinalis (48%). Protozoan parasites were more common among children aged 0-12 years; however, the frequency of helminths, such as hookworms and A. lumbricoides, was higher in adults. There were no significant differences in parasite frequencies between different genders or ethnic groups. Mixed infections by two or more protozoan and/or helminth species were detected in 96% of individuals. One individual was infected by 14 species. A high frequency of intestinal parasites was found in Baré and Baniwa ethnic groups. Improvements to infrastructure and health education programs are required to reduce risk of infection by intestinal parasites.(AU)

Aproximadamente 90% da população no noroeste da Amazônia é composta de grupos indígenas e o acesso deles aos serviços de saúde ainda é um desafio. O objetivo deste estudo foi determinar a frequência de parasitos em dois grupos indígenas (Baré e Baniwa) no noroeste da Amazônia. Amostras de fezes de 270 indivíduos (199 Baniwa e 71 Baré) foram analisadas pelos métodos de Richie e sedimentação espontânea. Foram determinadas diferenças estatísticas entre as proporções de indivíduos infectados com base no sexo, idade e etnia. Todos os indivíduos estavam infectados por protozoários ou helmintos. Os parasitos mais frequentes nos índios foram Ascaris lumbricoides (73%), Entamoeba spp. (53%), e Giardia intestinalis (48%). Protozoários parasitos foram mais comuns entre as crianças com idade entre 0-12 anos; no entanto, a frequência de ancilostomídeos e A. lumbricoides foi maior em adultos. Não houve diferenças significativas nas frequências de parasitos entre os diferentes sexos ou grupos étnicos. Infecções mistas por duas ou mais espécies de protozoários e/ou helmintos foram detectadas em 96% dos indivíduos. Um indivíduo estava infectado por 14 espécies. Uma alta frequência de parasitos intestinais foi encontrada em indígenas dos grupos Baré e Baniwa. Melhorias dos programas de infra-estrutura e educação em saúde são necessárias para reduzir o risco de infecção por parasitos intestinais.(AU)

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Approximately 90% of the population in the northwestern Amazonia is composed of indigenous people and their healthcare is still a challenge. The objective of this study was to determine the frequency of parasites in two indigenous ethnic groups (Baré and Baniwa) in northwestern Amazonia. Stool samples from 270 individuals (199 Baniwa and 71 Baré) were analyzed using Richie's method and the spontaneous sedimentation method. Statistical differences among the proportions of infected individuals based on gender, age, and ethnicity were determined. All individuals were infected by protozoans or helminths. The most frequent parasites in the indigenous people were Ascaris lumbricoides (73%), Entamoeba spp. (53%), and Giardia intestinalis (48%). Protozoan parasites were more common among children aged 0-12 years; however, the frequency of helminths, such as hookworms and A. lumbricoides, was higher in adults. There were no significant differences in parasite frequencies between different genders or ethnic groups. Mixed infections by two or more protozoan and/or helminth species were detected in 96% of individuals. One individual was infected by 14 species. A high frequency of intestinal parasites was found in Baré and Baniwa ethnic groups. Improvements to infrastructure and health education programs are required to reduce risk of infection by intestinal parasites.

Aproximadamente 90% da população no noroeste da Amazônia é composta de grupos indígenas e o acesso deles aos serviços de saúde ainda é um desafio. O objetivo deste estudo foi determinar a frequência de parasitos em dois grupos indígenas (Baré e Baniwa) no noroeste da Amazônia. Amostras de fezes de 270 indivíduos (199 Baniwa e 71 Baré) foram analisadas pelos métodos de Richie e sedimentação espontânea. Foram determinadas diferenças estatísticas entre as proporções de indivíduos infectados com base no sexo, idade e etnia. Todos os indivíduos estavam infectados por protozoários ou helmintos. Os parasitos mais frequentes nos índios foram Ascaris lumbricoides (73%), Entamoeba spp. (53%), e Giardia intestinalis (48%). Protozoários parasitos foram mais comuns entre as crianças com idade entre 0-12 anos; no entanto, a frequência de ancilostomídeos e A. lumbricoides foi maior em adultos. Não houve diferenças significativas nas frequências de parasitos entre os diferentes sexos ou grupos étnicos. Infecções mistas por duas ou mais espécies de protozoários e/ou helmintos foram detectadas em 96% dos indivíduos. Um indivíduo estava infectado por 14 espécies. Uma alta frequência de parasitos intestinais foi encontrada em indígenas dos grupos Baré e Baniwa. Melhorias dos programas de infra-estrutura e educação em saúde são necessárias para reduzir o risco de infecção por parasitos intestinais.

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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.

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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.

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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..(AU)

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Although predominantly associated with health benefits, the gut microbiota has also been shown to harbor genes that promote inflammation. In this work, we report a method for the direct detection and quantification of these pro-inflammatory bacterial genes by PCR and qPCR in DNA extracted from human stool samples. PCR reactions were performed to detect (i) the pks island genes, (ii) tcpC, which is present in some strains of Escherichia coli and (iii) gelE presented in some strains of Enterococcus faecalis. Additionally, we screened for the presence of the following genes encoding cyclomodulins that disrupted mammalian cell division: (iv) cdt (which encodes the cytolethal distending toxin) and (v) cnf-1 (which encodes the cytotoxic necrotizing factor-1). Our results show that 20% of the samples (N = 41) tested positive for detectable amounts of pks island genes, whereas 10% of individuals were positive for tcpC or gelE and only one individual was found to harbor the cnf-1 gene. Of the 13 individuals that were positive for at least one of the pro-inflammatory genes, 5 were found to harbor more than one. A quantitative version of the assay, which used real-time PCR, revealed the pro-inflammatory genes to be in high copy numbers: up to 1.3 million copies per mg of feces for the pks island genes. Direct detection of specific genes in stool could prove useful toward screening for the presence of pro-inflammatory bacterial genes in individuals with inflammatory bowel diseases or colorectal cancer.