Whole Genome Sequencing of Mycobacterium tuberculosis under routine conditions in a high-burden area of multidrug-resistant tuberculosis in Peru.

Whole Genome Sequencing (WGS) is a promising tool in the global fight against tuberculosis (TB). The aim of this study was to evaluate the use of WGS in routine conditions for detection of drug resistance markers and transmission clusters in a multidrug-resistant TB hot-spot area in Peru. For this, 140 drug-resistant Mycobacterium tuberculosis strains from Lima and Callao were prospectively selected and processed through routine (GenoType MTBDRsl and BACTEC MGIT) and WGS workflows, simultaneously. Resistance was determined in accordance with the World Health Organization mutation catalogue. Agreements between WGS and BACTEC results were calculated for rifampicin, isoniazid, pyrazinamide, moxifloxacin, levofloxacin, amikacin and capreomycin. Transmission clusters were determined using different cut-off values of Single Nucleotide Polymorphism differences. 100% (140/140) of strains had valid WGS results for 13 anti-TB drugs. However, the availability of final, definitive phenotypic BACTEC MGIT results varied by drug with 10-17% of invalid results for the seven compared drugs. The median time to obtain results of WGS for the complete set of drugs was 11.5 days, compared to 28.6-52.6 days for the routine workflow. Overall categorical agreement by WGS and BACTEC MGIT for the compared drugs was 96.5%. Kappa index was good (0.65≤k≤1.00), except for moxifloxacin, but the sensitivity and specificity values were high for all cases. 97.9% (137/140) of strains were characterized with only one sublineage (134 belonging to "lineage 4" and 3 to "lineage 2"), and 2.1% (3/140) were mixed strains presenting two different sublineages. Clustering rates of 3.6% (5/140), 17.9% (25/140) and 22.1% (31/140) were obtained for 5, 10 and 12 SNP cut-off values, respectively. In conclusion, routine WGS has a high diagnostic accuracy to detect resistance against key current anti-TB drugs, allowing results to be obtained through a single analysis and helping to cut quickly the chain of transmission of drug-resistant TB in Peru.

Bacteriophages as Biotechnological Tools.

Bacteriophages are ubiquitous organisms that can be specific to one or multiple strains of hosts, in addition to being the most abundant entities on the planet. It is estimated that they exceed ten times the total number of bacteria. They are classified as temperate, which means that phages can integrate their genome into the host genome, originating a prophage that replicates with the host cell and may confer immunity against infection by the same type of phage; and lytics, those with greater biotechnological interest and are viruses that lyse the host cell at the end of its reproductive cycle. When lysogenic, they are capable of disseminating bacterial antibiotic resistance genes through horizontal gene transfer. When professionally lytic-that is, obligately lytic and not recently descended from a temperate ancestor-they become allies in bacterial control in ecological imbalance scenarios; these viruses have a biofilm-reducing capacity. Phage therapy has also been advocated by the scientific community, given the uniqueness of issues related to the control of microorganisms and biofilm production when compared to other commonly used techniques. The advantages of using bacteriophages appear as a viable and promising alternative. This review will provide updates on the landscape of phage applications for the biocontrol of pathogens in industrial settings and healthcare.

Characterization of Planktochlorella nurekis Extracts and Virucidal Activity against a Coronavirus Model, the Murine Coronavirus 3.

Certain members of the Coronaviridae family have emerged as zoonotic agents and have recently caused severe respiratory diseases in humans and animals, such as SARS, MERS, and, more recently, COVID-19. Antivirals (drugs and antiseptics) capable of controlling viruses at the site of infection are scarce. Microalgae from the Chlorellaceae family are sources of bioactive compounds with antioxidant, antiviral, and antitumor activity. In the present study, we aimed to evaluate various extracts from Planktochlorella nurekis in vitro against murine coronavirus-3 (MHV-3), which is an essential human coronavirus surrogate for laboratory assays. Methanol, hexane, and dichloromethane extracts of P. nurekis were tested in cells infected with MHV-3, and characterized by UV-vis spectrophotometry, nuclear magnetic resonance (NMR) spectroscopy, ultraperformance liquid chromatography-mass spectrometry (UPLC-MS), and the application of chemometrics through principal component analysis (PCA). All the extracts were highly efficient against MHV-3 (more than a 6 Log unit reduction), regardless of the solvent used or the concentration of the extract, but the dichloromethane extract was the most effective. Chemical characterization by spectrophotometry and NMR, with the aid of statistical analysis, showed that polyphenols, carbohydrates, and isoprene derivatives, such as terpenes and carotenoids have a more significant impact on the virucidal potential. Compounds identified by UPLC-MS were mainly lipids and only found in the dichloromethane extract. These results open new biotechnological possibilities to explore the biomass of P. nurekis; it is a natural extract and shows low cytotoxicity and an excellent antiviral effect, with low production costs, highlighting a promising potential for development and implementation of therapies against coronaviruses, such as SARS-CoV-2.

Evaluation of the broth microdilution plate methodology for susceptibility testing of Mycobacterium tuberculosis in Peru.

BACKGROUND: Tuberculosis (TB) is a communicable, preventable and curable disease caused by the bacterium Mycobacterium tuberculosis (MTB). Peru is amongst the 30 countries with the highest burden of multidrug-resistant tuberculosis (MDR-TB) worldwide. In the fight against drug-resistant tuberculosis, the UKMYC6 microdilution plate was developed and validated by the CRyPTIC project. The objective of the study was to evaluate the use of the broth microdilution (BMD) plate methodology for susceptibility testing of drug-resistant MTB strains in Peru. METHODS: MTB strains isolated between 2015 and 2018 in Peru were used. 496 nationally-representative strains determined as drug-resistant by the routine 7H10 Agar Proportion Method (APM) were included in the present study. The Minimum Inhibitory Concentration (MIC) of 13 antituberculosis drugs were determined for each strain using the UKMYC6 microdilution plates. Diagnostic agreement between APM and BMD plate methodology was determined for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. Phenotypes were set using binary (or ternary) classification based on Epidemiological cut-off values (ECOFF/ECV) proposed by the CRyPTIC project. Whole Genome Sequencing (WGS) was performed on strains with discrepant results between both methods. RESULTS: MIC distributions were determined for 13 first- and second-line anti-TB drugs, including new (bedaquiline, delamanid) and repurposed (clofazimine, linezolid) agents. MIC results were available for 80% (397/496) of the strains at 14 days and the remainder at 21 days. The comparative analysis determined a good agreement (0.64 ≤ k ≤ 0.79) for the drugs rifampicin, ethambutol, ethionamide and kanamycin, and the best agreement (k > 0.8) for isoniazid and levofloxacin. Overall, 12% of MIC values were above the UKMYC6 plate dilution ranges, most notably for the drugs rifampicin and rifabutin. No strain presented MICs higher than the ECOFF/ECV values for the new or repurposed drugs. Discrepant analysis using genotypic susceptibility testing by WGS supported half of the results obtained by APM (52%, 93/179) and half of those obtained by BMD plate methodology (48%, 86/179). CONCLUSIONS: The BMD methodology using the UKMYC6 plate allows the complete susceptibility characterization, through the determination of MICs, of drug-resistant MTB strains in Peru. This methodology shows good diagnostic performances for rifampicin, isoniazid, ethambutol, ethionamide, kanamycin and levofloxacin. It also allows for the characterization of MICs for other drugs used in previous years against tuberculosis, as well as for new and repurposed drugs recently introduced worldwide.

Classical and Next-Generation Vaccine Platforms to SARS-CoV-2: Biotechnological Strategies and Genomic Variants.

Several coronaviruses (CoVs) have been identified as human pathogens, including the α-CoVs strains HCoV-229E and HCoV-NL63 and the ß-CoVs strains HCoV-HKU1 and HCoV-OC43. SARS-CoV, MERS-CoV, and SARS-CoV-2 are also classified as ß-coronavirus. New SARS-CoV-2 spike genomic variants are responsible for human-to-human and interspecies transmissibility, consequences of adaptations of strains from animals to humans. The receptor-binding domain (RBD) of SARS-CoV-2 binds to receptor ACE2 in humans and animal species with high affinity, suggesting there have been adaptive genomic variants. New genomic variants including the incorporation, replacement, or deletion of the amino acids at a variety of positions in the S protein have been documented and are associated with the emergence of new strains adapted to different hosts. Interactions between mutated residues and RBD have been demonstrated by structural modelling of variants including D614G, B.1.1.7, B1.351, P.1, P2; other genomic variants allow escape from antibodies generated by vaccines. Epidemiological and molecular tools are being used for real-time tracking of pathogen evolution and particularly new SARS-CoV-2 variants. COVID-19 vaccines obtained from classical and next-generation vaccine production platforms have entered clinicals trials. Biotechnology strategies of the first generation (attenuated and inactivated virus-CoronaVac, CoVaxin; BBIBP-CorV), second generation (replicating-incompetent vector vaccines-ChAdOx-1; Ad5-nCoV; Sputnik V; JNJ-78436735 vaccine-replicating-competent vector, protein subunits, virus-like particles-NVX-CoV2373 vaccine), and third generation (nucleic-acid vaccines-INO-4800 (DNA); mRNA-1273 and BNT 162b (RNA vaccines) have been used. Additionally, dendritic cells (LV-SMENP-DC) and artificial antigen-presenting (aAPC) cells modified with lentiviral vector have also been developed to inhibit viral activity. Recombinant vaccines against COVID-19 are continuously being applied, and new clinical trials have been tested by interchangeability studies of viral vaccines developed by classical and next-generation platforms.

SARS-CoV-2 in Human Sewage and River Water from a Remote and Vulnerable Area as a Surveillance Tool in Brazil.

In the present study, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was monitored in environmental samples from rural and vulnerable areas (a presidio, worker accommodation units, and river waters upstream and downstream of a rural community) from Minas Gerais State region, Southern Brazil, in August 2020. The sampling was performed prior to official declaration of the coronavirus disease (COVID-19) cases in those sites. SARS-CoV-2 RNA was detected in the presidio and workers accommodation units (3.0 × 104 virus genome copies (GC)/mL and 4.3 × 104 GC/mL of sewage, respectively). While SARS-CoV-2 was not detected in the river water upstream of the rural community, SARS-CoV-2 RNA was detected in downstream river waters (1.1 × 102 SARS-CoV-2 GC/mL). The results obtained in this study highlight the utility of SARS-CoV-2 monitoring in wastewater and human sewage as a non-invasive early warning tool to support health surveillance in vulnerable and remote areas, particularly in development countries.

Uses of Bacteriophages as Bacterial Control Tools and Environmental Safety Indicators.

Bacteriophages are bacterial-specific viruses and the most abundant biological form on Earth. Each bacterial species possesses one or multiple bacteriophages and the specificity of infection makes them a promising alternative for bacterial control and environmental safety, as a biotechnological tool against pathogenic bacteria, including those resistant to antibiotics. This application can be either directly into foods and food-related environments as biocontrol agents of biofilm formation. In addition, bacteriophages are used for microbial source-tracking and as fecal indicators. The present review will focus on the uses of bacteriophages like bacterial control tools, environmental safety indicators as well as on their contribution to bacterial control in human, animal, and environmental health.

Detection of Enteric Viruses and Core Microbiome Analysis in Artisanal Colonial Salami-Type Dry-Fermented Sausages from Santa Catarina, Brazil.

Microbial fermentation plays an important role in the manufacturing of artisanal sausages and can have major effects on product quality and safety. We used metagenomics and culture-dependent methods to study the presence of Hepatitis E virus (HEV) and Rotavirus-A (RV-A), and fungal and bacterial communities, in artisanal Colonial salami-type dry-fermented sausages in Santa Catarina state, Brazil. Lactic acid bacteria (LAB) and yeast dominated the microbiome. Latilactobacillus sakei and Debaryomyces hansenii were ubiquitous and the most abundant species. The DNA of some foodborne pathogens was found in very low concentrations although viable cells of most of these species were undetectable by cultivation methods. The characteristics of the raw material and hygiene of the artisanal sausage manufacturing process resulted in high loads of beneficial microorganisms and the absence of HEV and RV-A viruses as determined by RT-qPCR assays. In conclusion, high LAB load in sausages was more relevant to preventing pathogen growth than the ripening time and/or physicochemical characteristics. However, the presence of Clostridium spp. and other pathogens in some samples must be taken into account for the development of future preservation methods; appropriate LAB starter cultures and health surveillance are required in the production process to prevent foodborne outbreaks.

Salmonella enterica Serovar Enteritidis Control in Poultry Litter Mediated by Lytic Bacteriophage Isolated from Swine Manure.

We report the use of bacteriophages for control of Salmonella Enteritidis in poultry production. Phage was isolated by the double-agar plate assay from agricultural waste samples, and one isolate, named SM1, was selected and propagated for application in poultry litter. Two experimental protocols were tested: single treatment and repeated treatment (re-application of phage SM1 after 6 h and 12 h). Each treatment cycle involved 25 g of poultry litter placed in plastic boxes and contaminated with 105 Colony Forming Units mL-1 (CFU mL-1) of S. Enteritidis, in independent duplicates. The contaminated litter was treated with 106 Plaque Forming Units mL-1 (PFU mL-1) of SM1 phage by dripping. Repeated application of phage SM1 reduced Salmonella counts by over 99.9%; the phage persisted in poultry litter for over 35 days. This study illustrates the application of SM1 treatment as a promising technology for bacterial control in production matrices that could allow safe and sustainable use of agricultural waste products as biofertilizers.

Bioactive Compounds from Mangrove Endophytic Fungus and Their Uses for Microorganism Control.

Mangroves are ecosystems with unique characteristics due to the high salinity and amount of organic matter that house a rich biodiversity. Fungi have aroused much interest as they are an important natural source for the discovery of new bioactive compounds, with potential biotechnological and pharmacological interest. This review aims to highlight endophytic fungi isolated from mangrove plant species and the isolated bioactive compounds and their bioactivity against protozoa, bacteria and pathogenic viruses. Knowledge about this type of ecosystem is of great relevance for its preservation and as a source of new molecules for the control of pathogens that may be of importance for human, animal and environmental health.

Whole genome analysis of extensively drug resistant Mycobacterium tuberculosis strains in Peru.

Peru has the highest burden of multidrug-resistant tuberculosis in the Americas region. Since 1999, the annual number of extensively drug-resistant tuberculosis (XDR-TB) Peruvian cases has been increasing, becoming a public health challenge. The objective of this study was to perform genomic characterization of Mycobacterium tuberculosis strains obtained from Peruvian patients with XDR-TB diagnosed from 2011 to 2015 in Peru. Whole genome sequencing (WGS) was performed on 68 XDR-TB strains from different regions of Peru. 58 (85.3%) strains came from the most populated districts of Lima and Callao. Concerning the lineages, 62 (91.2%) strains belonged to the Euro-American Lineage, while the remaining 6 (8.8%) strains belonged to the East-Asian Lineage. Most strains (90%) had high-confidence resistance mutations according to pre-established WHO-confident grading system. Discordant results between microbiological and molecular methodologies were caused by mutations outside the hotspot regions analysed by commercial molecular assays (rpoB I491F and inhA S94A). Cluster analysis using a cut-off ≤ 10 SNPs revealed that only 23 (34%) strains evidenced recent transmission links. This study highlights the relevance and utility of WGS as a high-resolution approach to predict drug resistance, analyse transmission of strains between groups, and determine evolutionary patterns of circulating XDR-TB strains in the country.

The presence of SARS-CoV-2 RNA in human sewage in Santa Catarina, Brazil, November 2019.

Human sewage from Florianopolis (Santa Catarina, Brazil) was analyzed for severe acute respiratory syndrome coronavirus-2 (SARS-CoV2) from October 2019 until March 2020. Twenty five ml of sewage samples were clarified and viruses concentrated using a glycine buffer method coupled with polyethylene glycol precipitation, and viral RNA extracted using a commercial kit. SARS-CoV-2 RNA was detected by RT-qPCR using oligonucleotides targeting N1, S and two RdRp regions. The results of all positive samples were further confirmed by a different RT-qPCR system in an independent laboratory. S and RdRp amplicons were sequenced to confirm identity with SARS-CoV-2. Genome sequencing was performed using two strategies; a sequence-independent single-primer amplification (SISPA) approach, and by direct metagenomics using Illumina's NGS. SARS-CoV-2 RNA was detected on 27th November 2019 (5.49 ± 0.02 log10 SARS-CoV-2 genome copies (GC) L-1), detection being confirmed by an independent laboratory and genome sequencing analysis. The samples in the subsequent three events were positive by all RT-qPCR assays; these positive results were also confirmed by an independent laboratory. The average load was 5.83 ± 0.12 log10 SARS-CoV-2 GC L-1, ranging from 5.49 ± 0.02 log10 GC L-1 (27th November 2019) to 6.68 ± 0.02 log10 GC L-1 (4th March 2020). Our findings demonstrate that SARS-CoV-2 was likely circulating undetected in the community in Brazil since November 2019, earlier than the first reported case in the Americas (21st January 2020).

[Genetic structure of drug-resistant Mycobacterium tuberculosis strains in Peru based on haplotypes obtained from a line probe assay]. / Estructura genética de cepas drogorresistentes de Mycobacterium tuberculosis en Perú basada en haplotipos obtenidos de un ensayo con sondas en línea.

OBJECTIVE.: To determine the genetic structure of drug-resistant strains of Mycobacterium tuberculosis that circulated throughout Peru during the years 2011-2015, by using haplotypes obtained from a line probe assay. MATERIALS AND METHODS.: A total of 6589 samples that were admitted to the Instituto Nacional de Salud for routine diagnosis using the GenoType® MTBDRplus v2 assay were analyzed during the study period. Resistant haplotypes were created by concatenating 21 polymorphic sites of the evaluated genes using the line probe assay; and the association analysis was carried out with phenotypes obtained by the 7H10 agar ratio method. RESULTS.: The most frequent mutations were: rpoB S531L (55.4%) and rpoB D516V (18.5%) for rifampicin resistance, and katG S315T (59.5%) and inhA c-15t (25.7%) for isoniazid resistance. We obtained 13 representative haplotypes (87.8% of analyzed samples), 6 corresponded to the multidrug-resistant genotype, 4 to the isoniazid mono-resistant genotype and 3 to the rifampicin mono-resistant genotype. Eighteen regions and the province of Callao showed high haplotype diversity; four showed moderate diversity and two showed low diversity. CONCLUSIONS.: Most regions showed high haplotype diversity; in addition, most drug-resistant strains of Mycobacterium tuberculosis were concentrated in the cities of Lima and Callao. Likewise, drug-resistant Mycobacterium tuberculosis strains circulating in Peru mainly contain the genetic markers with the highest prevalence worldwide, which are associated with resistance to rifampicin and isoniazid.

OBJETIVO.: Determinar la estructura genética de las cepas drogorresistentes de Mycobacterium tuberculosis que circularon en todo el Perú durante los años 2011-2015 a través de haplotipos obtenidos de un ensayo con sondas en línea. MATERIALES Y MÉTODOS.: Se analizaron 6589 muestras que ingresaron al Instituto Nacional de Salud para el diagnóstico rutinario mediante el ensayo GenoType® MTBDRplus v2, durante el periodo de estudio. Se crearon haplotipos resistentes mediante la concatenación de 21 sitios polimórficos de los genes evaluados por el ensayo con sondas en línea, y se realizó el análisis de asociación con fenotipos obtenidos por el método de proporciones agar 7H10. RESULTADOS.: Las mutaciones de mayores frecuencias fueron: rpoB S531L (55,4%) y rpoB D516V (18,5%) para la resistencia a rifampicina, y katG S315T (59,5%) e inhA c-15t (25,7%) para la resistencia a isoniacida. Se obtuvieron 13 haplotipos representativos (87,8% de muestras analizadas) de los cuales seis correspondieron al genotipo multidrogorresistente, cuatro al genotipo monorresistente a isoniacida y tres al genotipo monorresistente a rifampicina. Dieciocho departamentos, y la provincia del Callao, presentaron una alta diversidad haplotípica; cuatro presentaron moderada diversidad y dos presentaron baja diversidad. CONCLUSIONES.: Existe una alta diversidad haplotípica en la mayoría de los departamentos, además de una concentración de las cepas de Mycobacterium tuberculosis drogorresistentes en las ciudades de Lima y Callao. Asimismo, las cepas de Mycobacterium tuberculosis con perfil drogorresistente que circulan en el Perú contienen principalmente los marcadores genéticos de mayor prevalencia a nivel mundial asociados con la resistencia frente a rifampicina e isoniacida.

Broad Spectrum Algae Compounds Against Viruses.

The pharmaceutical industry is currently trying to develop new bioactive compounds to inactivate both enveloped and non-enveloped viruses for therapeutic purposes. Consequently, microalgal and macroalgal bioactive compounds are being explored by pharmaceutical, as well as biotechnology and food industries. In this review, we show how compounds produced by algae include important candidates for viral control applications. We discuss their mechanisms of action and activity against enveloped and non-enveloped viruses, including those causing infections by enteric, parenteral, and respiratory routes. Indeed, algal products have potential in human and animal medicine.

Hepatitis E Virus in Manure and Its Removal by Psychrophilic anaerobic Biodigestion in Intensive Production Farms, Santa Catarina, Brazil, 2018-2019.

Hepatitis E virus (HEV) is an important enteric agent that can circulate in swine; it is excreted in manure, and of zoonotic interest. The present study investigated, by RT-qPCR, the circulation of HEV in swine manure from different types of pig farms (maternity, nursery, and grow-finish farms) in Santa Catarina State, the major pig production area of Brazil, and also evaluated the HEV removal efficiency of psychrophilic anaerobic biodigesters (PABs). While HEV was consistently detected in manure from grow-finish pig farms (>4 log HEV genome copies (GC) L-1), the virus was not detected in manure from maternity and nursery farms. These findings suggest a potential high biosafety status during primary-swine production, with a subsequent contamination in grow-finish production. The anaerobic biodigestion process reduced more than 2 log10 HEV GC in the processed swine manure. However, the virus concentration in final effluent remained high, with an average value of 3.85 log10 HEV GC L-1. Consequently, our results demonstrate that PABs can be a robust tool for effective inactivation of HEV, while reinforcing the need for sanitary surveillance and legislation of swine manure-derived biofertilizers, to avoid the spread of zoonotic enteric pathogens such as HEV.

Aplicación de la secuenciación masiva y la bioinformática al diagnóstico microbiológico clínico / Bioinformatics of next generation sequencing in clinical microbiology diagnosis

Resumen La aparición de secuenciadores masivos que permiten leer en paralelo de millones a miles de millones de secuencias o fragmentos del ADN (reads) ha revolucionado la microbiología, la cual ha pasado de un ámbito exclusivamente laboratorial a uno computacional, con la aplicación ineludible de la bioinformática. La posibilidad de efectuar estudios de la microbiota, el microbioma y el metagenoma de una muestra clínica de manera rápida y a un coste reducido permite avanzar más rápidamente en el diagnóstico de enfermedades, en el conocimiento de la taxonomía y la epidemiología de los agentes involucrados, así como de su virulencia. También posibilita la realización de estudios de genómica comparada y el descubrimiento de genes o variantes de interés, lo que puede llevar a que enfermedades tradicionalmente consideradas como de carácter no microbiano sean asociadas a la presencia de microrganismos. En esta revisión se aclara la terminología usada en este campo, y se describen las principales tecnologías de secuenciación y su utilidad en el análisis microbiano. Asimismo, se señalan diversos programas de código libre, pipelines de análisis, bases de datos y plataformas web que permiten que la bioinformática se integre exitosamente al ámbito de la microbiología clínica y al estudio de las enfermedades infecciosas.

Abstract Massive parallel sequencing (High-Throughput Sequencing [HTS]) allows to read millions or billions of DNA sequences or fragments (reads) in parallel and is revolutionizing microbiology research, moving from laboratory methods to computed-assisted analyses, with the compelling use of Bioinformatics. The time and cost reduction in studies on the microbiota, microbiome and metagenome, allows to rapidly progress in diagnosis, taxonomy, epidemiology, comparative genomics, virulence, discovery of genes or variants of interest and the association of microorganisms with traditionally considered non-microbial diseases. In this review, the terminology, the sequencing technologies and their applications are described for microbial analysis using open-source bioinformatics software, analysis pipelines, databases and web platforms that allow a user-friendly bioinformatics approach affordable by the clinical microbiologist and infectious disease practitioners.

Nutritional, Energy and Sanitary Aspects of Swine Manure and Carcass Co-digestion.

Renewable energy can assist the management of the effects of population growth and rapid economic development on the sustainability of animal husbandry. The primary aim of renewable energy is to minimize the use of fossil fuels via the creation of environmentally friendly energy products from depleted fossil fuels. Digesters that treat swine manure are extensively used in treatment systems; and inclusion of swine carcasses can increase the organic loading rate (OLR) thereby improving biogas yield and productivity on farms. However, the characteristics of the components including animal residues, proteins, lipids, remains of undigested feed items, antimicrobial drug residues, pathogenic microorganisms and nutrient contents, are complex and diverse. It is therefore necessary to manage the anaerobic process stability and digestate purification for subsequent use as fertilizer. Efficient methane recovery from residues rich in lipids is difficult because such residues are only slowly biodegradable. Pretreatment can promote solubilization of lipids and accelerate anaerobic digestion, and pretreatments can process the swine carcass before its introduction onto biodigesters. This review presents an overview of the anaerobic digestion of swine manure and carcasses. We analyze the characteristics of these residues, and we identify strategies to enhance biogas yield and process stability. We consider energy potential, co-digestion of swine manure and carcasses, physical, chemical, and biological pretreatment of biomass, sanitary aspects of swine manure and co-digestates and their recycling as fertilizers.

[Bioinformatics of next generation sequencing in clinical microbiology diagnosis]. / Aplicación de la secuenciación masiva y la bioinformática al diagnóstico microbiológico clínico.

Massive parallel sequencing (High-Throughput Sequencing [HTS]) allows to read millions or billions of DNA sequences or fragments (reads) in parallel and is revolutionizing microbiology research, moving from laboratory methods to computed-assisted analyses, with the compelling use of Bioinformatics. The time and cost reduction in studies on the microbiota, microbiome and metagenome, allows to rapidly progress in diagnosis, taxonomy, epidemiology, comparative genomics, virulence, discovery of genes or variants of interest and the association of microorganisms with traditionally considered non-microbial diseases. In this review, the terminology, the sequencing technologies and their applications are described for microbial analysis using open-source bioinformatics software, analysis pipelines, databases and web platforms that allow a user-friendly bioinformatics approach affordable by the clinical microbiologist and infectious disease practitioners.

Household-based biodigesters promote reduction of enteric virus and bacteria in vulnerable and poverty rural area.

The present study evaluated the river water quality improvement by implementation of household-based biodigesters in vulnerability and poverty rural area, in Minas Gerais State-Brazil. For that, 78 household-based biodigesters were installed for domestic wastewater treatment. Wastewater was collected before and after treatment and the physicochemical parameters and pathogens removal (human adenovirus (HAdV), hepatitis A (HAV) virus, Salmonella sp. and Escherichia coli) were evaluated; Additionally, river water was sampled before and after the household-based biodigesters implementation, to verify the contamination reduction and the positive impact of domestic wastewater treatment on waterborne pathogen reduction, considering HAdV, HAV, Salmonella sp. and E. coli quantification. The applicability in real-scale of decentralized treatment systems using household-based biodigesters promoted reduction of 90, 99, 99.99 and 99.999% from HAV, Salmonella sp., E. coli and HAdV from domestic wastewater, respectively; The river water quality improvement before the wastewater treatment application was highlight in the present study, considering that the reduction of waterborne pathogens in this water in 90, 99.99 and 99.999% of E. coli, HAV and HAdV, respectively (Salmonella sp. was not detected in river water). In general, this is an important study for encouraging the decentralized sanitation in vulnerable and poverty area, as well in rural sites, considering the positive impact of this implementation on public health.

Mineral Waste Containing High Levels of Iron from an Environmental Disaster (Bento Rodrigues, Mariana, Brazil) is Associated with Higher Titers of Enteric Viruses.

Although the effects of heavy metals on the behavior, including infectivity, of bacteria have been studied, little information is available about their effects on enteric viruses. We report an investigation of effects on the biosynthesis of human adenoviruses (HAdV) and hepatitis A (HAV) of waters contaminated with mineral waste following an environmental disaster in Mariana City, Minas Gerais State, Brazil. The study area was affected on November 5, 2015, by 60 million m3 of mud (containing very high concentrations of iron salts) from a mining reservoir (Fundão), reaching the Gualaxo do Norte River (sites evaluated in this study), the "Rio Doce" River and finally the Atlantic Ocean. We found substantial counts of infectious HAdV and HAV (by qPCR) in all sampled sites from Gualaxo do Norte River, indicating poor basic sanitation in this area. The effects of iron on viral infection processes were evaluated using HAdV-2 and HAV-175, as DNA and RNA enteric virus models, respectively, propagated in the laboratory and exposed to this contaminated water. Experiments in field and laboratory scales found that the numbers of plaque forming units (PFU) of HAdV and HAV were significantly higher in contaminated water with high iron concentrations than in waters with low iron concentration (< 20 µg/L of iron). These findings indicate that iron can potentiate enteric virus infectivity, posing a potential risk to human and animal health, particularly during pollution disasters such as that described here in Mariana, Brazil.