Quantification of SARS-CoV-2 in wastewater samples from hospitals treating COVID-19 patients during the first wave of the pandemic in Brazil.

The COVID-19 pandemic has caused a global health crisis, and wastewater-based epidemiology (WBE) has emerged as an important tool to assist public health decision-making. Recent studies have shown that the SARS-CoV-2 RNA concentration in wastewater samples is a reliable indicator of the severity of the pandemic for large populations. However, few studies have established a strong correlation between the number of infected people and the viral concentration in wastewater due to variations in viral shedding over time, viral decay, infiltration, and inflow. Herein we present the relationship between the number of COVID-19-positive patients and the viral concentration in wastewater samples from three different hospitals (A, B, and C) in the city of Belo Horizonte, Minas Gerais, Brazil. A positive and strong correlation between wastewater SARS-CoV-2 concentration and the number of confirmed cases was observed for Hospital B for both regions of the N gene (R = 0.89 and 0.77 for N1 and N2, respectively), while samples from Hospitals A and C showed low and moderate correlations, respectively. Even though the effects of viral decay and infiltration were minimized in our study, the variability of viral shedding throughout the infection period and feces dilution due to water usage for different activities in the hospitals could have affected the viral concentrations. These effects were prominent in Hospital A, which had the smallest sewershed population size, and where no correlation between the number of defecations from COVID-19 patients and viral concentration in wastewater was observed. Although we could not determine trends in the number of infected patients through SARS-CoV-2 concentrations in hospitals' wastewater samples, our results suggest that wastewater monitoring can be efficient for the detection of infected individuals at a local level, complementing clinical data.

Wastewater-based epidemiology for preventing outbreaks and epidemics in Latin America - Lessons from the past and a look to the future.

Wastewater-based epidemiology (WBE) is an approach with the potential to complement clinical surveillance systems. Using WBE, it is possible to carry out an early warning of a possible outbreak, monitor spatial and temporal trends of infectious diseases, produce real-time results and generate representative epidemiological information in a territory, especially in areas of social vulnerability. Despite the historical uses of this approach, particularly in the Global Polio Eradication Initiative, and for other pathogens, it was during the COVID-19 pandemic that occurred an exponential increase in environmental surveillance programs for SARS-CoV-2 in wastewater, with many experiences and developments in the field of public health using data for decision making and prioritizing actions to control the pandemic. In Latin America, WBE was applied in heterogeneous contexts and with emphasis on populations that present many socio-environmental inequalities, a condition shared by all Latin American countries. This manuscript addresses the concepts and applications of WBE in public health actions, as well as different experiences in Latin American countries, and discusses a model to implement this surveillance system at the local or national level. We emphasize the need to implement this sentinel surveillance system in countries that want to detect the early entry and spread of new pathogens and monitor outbreaks or epidemics of infectious agents in their territories as a complement of public health surveillance systems.

Diversity and dynamics of bacterial communities in the drinking water distribution network of a mid-sized city in Brazil.

This study assessed the bacterial community composition of a drinking water system (DWS) serving a mid-sized city (120,000 inhabitants) in Brazil. Water samples, including raw and treated water, were collected at seven points throughout the DWS. DNA was extracted and analysed using high-throughput sequencing (Ion Torrent). Free chlorine and turbidity were measured in situ. Results showed that the highest relative abundance of 16S rRNA genes was from phyla Proteobacteria, followed by Bacteroidetes and Actinobacteria. The next most abundant phylum was Cyanobacteria, represented by Arthronema, Calothrix, and Synechococcus. An interesting observation was that the DNA-based analysis suggested a bacterial community change in the distribution network, with treated reservoir water being very different from the network samples. This suggests active microbiology within the distribution network and a tendency for bacterial diversity to decrease after chlorine disinfection but increase after pipeline distribution. In raw water, a predominance of Proteobacteria was observed with reduced Cyanobacteria, showing a negative correlation. In treated water, Proteobacteria were negatively correlated with Bacteroidetes. Finally, 16S rRNA genes from Firmicutes (especially Staphylococcus) had a high abundance in the chlorinated water, which may indicate the phylum's resistance to chlorine residuals. Opportunistic pathogens, e.g., Mycobacteria, Legionella, and Staphylococcus, were also observed.

Aeration strategies and temperature effects on the partial nitritation/anammox process for nitrogen removal: performance and bacterial community assessment.

The partial nitritation/anammox process (PN/A) could be a promising alternative for nitrogen removal from high-strength wastewater. There is, however, a lack of information about suitable aeration and temperature for PN/A in single-stage reactors for high-strength wastewater, such as food waste (FW) digestate treatment. To this end, a laboratory-scale (10 L) partial nitritation/anammox sequencing batch reactor was operated for more than 230 days under four different intermittent aeration strategies and temperature variations (35°C and ambient temperature - 26-29°C) to investigate the feasibility of nitrogen removal from real FW digestate. High ammonium (NH4+-N) and total nitrogen (TN) removal median efficiencies of 81 and 63%, respectively (corresponding to median NH4+-N and TN loads removed of 76 and 67 g.m-3.d-1), were achieved when the aeration strategy comprised by 7 min/14 min off and an airflow rate of 0.050 L.min-1.Lreactor-1 was applied. Nitrogen removal efficiencies were not affected by temperature variations in southeastern Brazil. COD, chloride and organic nitrogen (520, 239 and 102.8 mg.L-1, respectively) did not prevent PN/A. Changes of the bacterial community in response to aeration strategies were observed. Candidatus Brocadia dominated most of the time being more resistant to aeration and temperature changes than Candidatus Jettenia. This study demonstrated that optimizations of anoxic periods and airflow rate support PN/A with high nitrogen removal from FW digestate.

Puncture points related to the tragal-lateral canthus line in conventional temporomandibular joint arthrocentesis: are they accurate? A cadaver study.

PURPOSE: To evaluate the technical success of conventional TMJ arthrocentesis in cadavers, using the tragal-lateral canthus line as an anatomical reference for the puncture points. METHODS: Ten formalin-treated and 8 fresh cadavers were submitted to the arthrocentesis. A point located 10 mm anteriorly and 2 mm inferiorly to the beginning of the tragal-lateral canthus line (A) and another point 20 mm anteriorly and 10 mm inferiorly (B) were demarked on the cadavers' skin. Following, 1 mL of methylene blue solution was injected through the needle at point A, and then another needle was placed at point B. Saline solution was injected through the first needle, identifying a free flow of bluish solution. RESULTS: Concerning the formalin-treated cadavers, TMJ arthrocentesis was not successfully performed in any case (0%). In the fresh cadavers, the procedure was properly conducted bilaterally in only one case (12.5%) and unilaterally in 3 cases (37.5%), 2 on the left side (25.0%) and one on the right (12.5%). CONCLUSION: The traditional points related to the tragal-lateral canthus line seemed not to be accurate references for the insertion of the needles in conventional TMJ arthrocentesis when 1 mL of solution is used initially and the maximum mouth opening is not achieved.

Nitrogen removal from food waste digestate using partial nitritation-anammox process: Effect of different aeration strategies on performance and microbial community dynamics.

The feasibility of employing anammox and partial nitritation-anammox (PN/A) processes for nitrogen removal from food waste (FW) digestate was investigated in this study. The effects of different aeration strategies on the microbial community were also investigated. To achieve this, after anammox enrichment (Phase 1), the reactor was fed with digestate supplemented with nitrite (Phase 2), and subsequently different aeration strategies were evaluated to establish PN/A. Aeration strategies with high anoxic periods (30 and 45 min) in relation to aerobic periods (15 min) coupled with low air flow rates (0.026 L  min-1. Lreator-1) were found to be better for establishing PN/A, as coefficients of produced nitrate/removed ammonium were closer to those reported previously (0.17 and 0.21). Aeration conditions considerably altered the microbial community. Candidatus Brocadia was replaced by Candidatus Jettenia, after the first aeration strategies. These results support the feasibility of FW digestate treatment using anammox and PN/A processes and provide a better understanding of the effect of aeration on microbial dynamics in PN/A reactors.

Effect of temperature on microbial diversity and nitrogen removal performance of an anammox reactor treating anaerobically pretreated municipal wastewater.

The effects of temperature reduction (from 35 °C to 20 °C) on nitrogen removal performance and microbial diversity of an anammox sequencing batch reactor were evaluated. The reactor was fed for 148 days with anaerobically pretreated municipal wastewater amended with nitrite. On average, removal efficiencies of ammonium and nitrite were high (96%) during the enrichment period and phases 1 (at 35 °C) and 2 (at 25 °C), and slightly decreased (to 90%) when the reactor was operated at 20 °C. Deep sequencing analysis revealed that microbial community structure changed with temperature decrease. Anammox bacteria (Ca. Brocadia and Ca. Anammoximicrobium) and denitrifiers (Burkholderiales, Myxococcales, Rhodocyclales, Xanthomonadales, and Pseudomonadales) were favoured when the temperature was lowered from 35 °C to 25 °C, while Anaerolineales and Clostridiales were negatively affected. The results support the feasibility of using the anammox process for mainstream nitrogen removal from anaerobically pretreated municipal wastewater at typical tropical temperatures.

Identification of Endogenous Reference Genes for RT-qPCR Expression Analysis in Urochloa brizantha Under Abiotic Stresses.

Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4 A, Elongation factor-1 alpha, FTSH protease 4, U2 auxiliary fator, Succinol Co-enzyme A, Tubulin alfa-5, Tubulin beta-6, Ubiquitin conjugating enzyme. Total RNA was extract from leaf tissues of U. brizantha subjected to 6, 12 and 24 h of cold and heat stresses (10 and 45 °C, respectively), and drought, including moderate (-0.5 to -0.7 MPa), severe (-1.1 to -1.8 MPa) and recovery after re-watering. The RefFinder web-based tool was used to rank the most stable reference genes for each stress. Elongation factor-1 alpha, Elongation factor-1 alpha or Ubiquitin conjugating enzyme, and Eukaryotic initiation factor 4 A were the most stable genes for heat, cold and drought stress, respectively. The expression of Rubisco large subunit gene was normalized against the most stable gene selected by ReFfinder for each stress.

Anammox for nitrogen removal from anaerobically pre-treated municipal wastewater: Effect of COD/N ratios on process performance and bacterial community structure.

Long-term effects of COD/N ratios on the nitrogen removal performance and bacterial community of an anammox reactor were evaluated by adding a synthetic medium (with glucose) and real anaerobic effluent to a SBR. At a COD/N ratio of 2.8 (COD, 390mg·L(-1)) ammonium removal efficiency was 66%, while nitrite removal remained high (99%). However, at a COD/N ratio of 5.0 (COD, 300mg·L(-1)), ammonium and nitrite removal efficiencies were high (84% and 99%, respectively). High COD, nitrite, and ammonium removal efficiencies (80%, 90% and 95%, respectively) were obtained on adding anaerobically pre-treated municipal wastewater (with nitrite) to the reactor. DGGE revealed that the addition of anaerobic effluent changed the bacterial community structure and selected for DNA sequences related to Brocadia sinica and Chloroflexi. Adding glucose and anaerobic effluent increased denitrifiers concentration threefold. Thus, the possibility of using the anammox process to remove nitrogen from anaerobically pre-treated municipal wastewater was demonstrated.

Effect of phenol on the nitrogen removal performance and microbial community structure and composition of an anammox reactor.

The effects of phenol on the nitrogen removal performance of a sequencing batch reactor (SBR) with anammox activity and on the microbial community within the reactor were evaluated. A phenol concentration of 300 mg L(-1) reduced the ammonium-nitrogen removal efficiency of the SBR from 96.5% to 47%. The addition of phenol changed the microbial community structure and composition considerably, as shown by denaturing gradient gel electrophoresis and 454 pyrosequencing of 16S rRNA genes. Some phyla, such as Proteobacteria, Verrucomicrobia, and Firmicutes, increased in abundance, whereas others, such as Acidobacteria, Chloroflexi, Planctomycetes, GN04, WS3, and NKB19, decreased. The diversity of the anammox bacteria was also affected by phenol: sequences related to Candidatus Brocadia fulgida were no longer detected, whereas sequences related to Ca. Brocadia sp. 40 and Ca. Jettenia asiatica persisted. These results indicate that phenol adversely affects anammox metabolism and changes the bacterial community within the anammox reactor.

Análise molecular de mycobacterium tuberculosis isolados de pacientes atendidos em Goiânia, Goiás, por meio do RFLP-IS6110 e do 15 loci MIRU-VNTR / Molecular analysis of Mycobacterium tuberculosis isolates from patients in Goiânia, Goiás, by IS6110-RFLP and VNTR-15loci miru

Atualmente, o controle da tuberculose é dependente de vários fatores tais como rápido diagnóstico, terapia adequada e meios de evitar futuras transmissões. Assim, a caracterização de linhagens de Mycobacterium tuberculosis por tipagem molecular por meio da técnica de RFLP-IS6110 representa uma contribuição primordial e tem sido amplamente utilizada nos estudos de genotipagem para que sejam traçadas cadeias de transmissão. No entanto, por causa da complexidade desta técnica e da dificuldade de interpretação dos dados, outras técnicas têm sido propostas. Entre elas, destaca-se o estudo do número variável de unidades repetitivas (MIRU-VNTR) indicado como novo padrão de genotipagem. O presente estudo teve como objetivo analisar as técnicas de RFLP-IS6110 e 15 loci MIRU-VNTR em isolados de pacientes atendidos no município de Goiânia, Goiás. Para isso, foram caracterizados geneticamente os isolados a fim de se estabelecerem possíveis ligações epidemiológicas entre os casos da doença. Também fez-se a comparação entre os resultados encontrados pelas duas técnicas e o cenário do Brasil. Os dados demonstraram que a técnica de 15 loci MIRU-VNTR discriminou mais os isolados que a técnica de RFLP-IS6110. Não foi encontrada associação epidemiológica entre os pacientes estudados. Os resultados validaram o uso da técnica 15 loci MIRU-VNTR para tipagem molecular de M. tuberculosis por apresentar maior poder discriminatório, boa eficiência para caracterizar geneticamente os isolados em Goiânia-GO, podendo, portanto, ser um método usado em estudo epidemiológico isolado ou em conjunto com outras técnicas...

Molecular analysis of Mycobacterium tuberculosis isolated from patients in Goiânia, Brazil, using RFLP-IS6110 and 15 loci MIRU-VNTR techniques.Currently, tuberculosis control is dependent on several factors, such as rapid diagnosis, appropriate therapy and measures to prevent future transmission. Thus, the characterization of strains of Mycobacterium tuberculosis by molecular typing using RFLP-IS6110, provides a major contribution, and has been widely used in genotyping studies in order to trace transmission pathways. However, due to the complexities of the technique and data interpretation, other techniques have been proposed. Among them, the study of the variable number of repeat units (MIRU-VNTR) has been indicated as a new standard method. This study aimed to apply and compare the RFLP-IS6110 and 15 loci MIRU-VNTR techniques for the analysis of isolates from tuberculosis patients treated in Goiânia, Brazil, in order to establish possible molecular epidemiological links between cases of the disease, and also to compare the results found by both techniques against the wider situation in Brazil. The results showed that 15 loci MIRU-VNTR discriminated between the isolates better than the RFLP-IS6110. No epidemiological link was observed among the patients studied. The results validate the use of the 15 loci MIRU-VNTR technique for molecular typing of M. tuberculosis, as it showed greater discriminatory power with good efficiency to genetically characterize the isolates in Goiânia, Goiás. This can be used in epidemiological studies alone or in conjunction with other molecular techniques...

A high prevalence of resistance in new tuberculosis cases of midwestern Brazil.

Tuberculosis (TB) is a serious worldwide public health problem, and drug resistance, particularly multidrug resistance (MDR), is a critical factor involved in TB control. We analyzed Mycobacterium tuberculosis isolates from 132 new TB cases of treatment-naïve patients in Goiás, Brazil by drug susceptibility tests, partial sequencing of the rpoB and katG genes, inhA(C-15T) mutation analysis by PCR, and RFLP-IS6110 genotyping. A high frequency of drug resistance was observed in previously untreated patients (13.6% to at least one antibiotic and 6.1% MDR-TB), and a high rate DNA polymorphism was detected in these strains. These results suggest that the prevalence of resistant TB is underestimated and that resistance in new TB cases was not associated with an outbreak in this region. We recommend routine culture and susceptibility testing for all new TB cases in Goiás for the appropriate treatment and control of this disease.