Risk factors for moderate acute malnutrition among children with acute diarrhoea in India and Tanzania: a secondary analysis of data from a randomized trial.

BACKGROUND: Moderate acute malnutrition (MAM) affects over 30 million children aged < 5 years worldwide. MAM may confer a greater risk of developing severe malnutrition and even mortality in children. Assessing risk factors for MAM may allow for earlier recognition of children at risk of deleterious health outcomes. OBJECTIVE: To determine risk factors associated with the prevalence and development of MAM among children aged 6 to 59 months with acute diarrhoea who received treatment with oral rehydration solution and zinc supplementation. METHODS: We conducted a secondary analysis of data from a randomized, dose-finding trial of zinc among children with acute diarrhoea in India and Tanzania. We used regression models to assess risk factors for prevalent MAM at the start of diarrhoea treatment and to identify risk factors associated with the development of MAM at 60 days. MAM was defined as weight for length (or height) Z score ≤-2 and > -3 or mid-upper arm circumference < 12.5 and ≥ 11.5 cm. RESULTS: A total of 4,500 children were enrolled; 593 (13.2%) had MAM at the baseline. MAM at baseline was significantly less common among children in Tanzania than in India (adjusted risk ratio [aRR] 0.37, 95% confidence interval [CI]: 0.30, 0.44, P < 0.001), in children aged 24- < 60 months versus 6- < 12 months (aRR 0.46, 95% CI: 0.38, 0.56, P < 0.001), and in families with household wealth index higher than the median (aRR 0.79, 95% CI: 0.68, 0.92, P = 0.002). Sixty days after outpatient treatment and follow-up, 87 (2.5%) children developed MAM. When compared to children aged 6- < 12 months, children aged 24- < 60 months had a 52% lower risk of developing MAM. Every one unit increase in weight for length (or height) Z score at enrolment was associated with a 93% lower risk of developing MAM during follow-up. CONCLUSIONS: Among children with diarrhoea, younger children and those from households with lower wealth were at greater risk of MAM. These children may benefit from targeted interventions focusing on feeding (targeted nutrition support for at-risk households) and follow up in order to reduce the occurrence of MAM and its consequences.

Proteogenomic Refinement of the Neomegalonema perideroedesT Genome Annotation.

Neomegalonema perideroedes (formerly Meganema perideroedes) str. G1 is the type strain and only described isolate of the genus Neomegalonema (formerly Meganema) which belongs to the Alphaproteobacteria. N. perideroedes is distinguished by the ability to accumulate high amounts of polyhydroxyalkanoates and has been associated with bulking problems in wastewater treatment plants due to its filamentous morphology. In 2013, its genome was sequenced as part of the Genomic Encyclopedia of Bacteria and Archaea (GEBA), which aims to improve the sequencing coverage of the poorly represented regions of the bacterial and archaeal branches of the tree of life. As N. perideroedes str. G1 is relatively distantly related to well described species-being the only sequenced member of its proposed family-the in silico prediction of genes by nucleotide homology to reference genes might be less reliable. Here, a proteomic dataset for the refinement of the N. perideroedes genome annotations is generated which clearly indicates the shortcomings of high-throughput in silico genome annotation.

Identification of active denitrifiers in full-scale nutrient removal wastewater treatment systems.

Denitrification is essential to the removal of nitrogen from wastewater during treatment, yet an understanding of the diversity of the active denitrifying bacteria responsible in full-scale wastewater treatment plants (WWTPs) is lacking. In this study, stable-isotope probing (SIP) was applied in combination with microautoradiography (MAR)-fluorescence in situ hybridization (FISH) to identify previously unrecognized active denitrifying phylotypes in a full-scale WWTP with biological N and P removal. Acknowledging that different denitrifiers will have specific carbon source preferences, a fully (13)C-labelled complex substrate was used for SIP incubations, under nitrite-reducing conditions, in order to maximize the capture of the potentially metabolically diverse denitrifiers likely present. Members of the Rhodoferax, Dechloromonas, Sulfuritalea, Haliangium and Thermomonas were represented in the 16S rRNA gene clone libraries from DNA enriched in (13)C, with FISH probes optimized here for their in situ characterization. FISH and MAR confirmed that they were all active denitrifiers in the community. The combined approach of SIP and MAR-FISH represents an excellent approach for identifying and characterizing an un-described diversity of active denitrifiers in full-scale systems.

Antibiotic resistance trends for common bacterial aetiologies of childhood diarrhoea in low- and middle-income countries: A systematic review.

Background: Diarrhoea is the second most common cause of death among children under the age of five worldwide. The World Health Organization (WHO) recommends treating diarrhoea with oral rehydration therapy, intravenous fluids for severe dehydration, and zinc supplements. Antibiotics are only recommended to treat acute, invasive diarrhoea. Rising antibiotic resistance has led to a decrease in the effectiveness of treatments for diarrhoea. Methods: A systematic literature review in PubMed, Web of Science, and EMBASE was conducted to identify articles relevant to antibiotic-resistant childhood diarrhoea. Articles in English published between 1990 to 2020 that described antibiotic resistance patterns of common pathogens causing childhood diarrhoea in low- and middle-income countries were included. The studies were limited to papers that categorized children as 0-5 years or 0-10 years old. The proportion of isolates with resistance to major classes of antibiotics stratified by major WHO global regions and time was determined. Results: Quantitative data were extracted from 44 articles that met screening criteria; most focused on children under five years. Escherichia coli isolates had relatively high resistance rates to ampicillin and tetracycline in the African (AFR), American (AMR), and Eastern Mediterranean Regions (EMR). There was moderate to high resistance to ampicillin and third generation cephalosporins among Salmonella spp in the AFR, EMR, and the Western Pacific Region (WPR). Resistance rates for ampicillin, co-trimoxazole, and chloramphenicol for Shigella in the AFR started at an alarmingly high rate ( ~ 90%) in 2006 and fluctuated over time. There were limited antibiotic resistance data for Aeromonas, Yersinia, and V. cholerae. The 161 isolates of Campylobacter analysed showed initially low rates of fluoroquinolone resistance with high rates of resistance in recent years, especially in the Southeast Asian Region. Conclusions: Resistance to inexpensive antibiotics for treatment of invasive diarrhoea in children under ten years is widespread (although data on 6- to 10-year-old children are limited), and resistance rates to fluoroquinolones and later-generation cephalosporins are increasing. A strong regional surveillance system is needed to carefully monitor trends in antibiotic resistance, future studies should include school-aged children, and interventions are needed to reduce inappropriate use of antibiotics for the treatment of community-acquired, non-invasive diarrhoea. Registration: This systematic review was registered in Prospero (registration number CRD42020204004) in August 2020.

Radiolabeled Risperidone microSPECT/CT Imaging for Intranasal Implant Studies Development.

The use of intranasal implantable drug delivery systems has many potential advantages for the treatment of different diseases, as they can provide sustained drug delivery, improving patient compliance. We describe a novel proof-of-concept methodological study using intranasal implants with radiolabeled risperidone (RISP) as a model molecule. This novel approach could provide very valuable data for the design and optimization of intranasal implants for sustained drug delivery. RISP was radiolabeled with 125I by solid supported direct halogen electrophilic substitution and added to a poly(lactide-co-glycolide) (PLGA; 75/25 D,L-Lactide/glycolide ratio) solution that was casted on top of 3D-printed silicone molds adapted for intranasal administration to laboratory animals. Implants were intranasally administered to rats, and radiolabeled RISP release followed for 4 weeks by in vivo non-invasive quantitative microSPECT/CT imaging. Percentage release data were compared with in vitro ones using radiolabeled implants containing either 125I-RISP or [125I]INa and also by HPLC measurement of drug release. Implants remained in the nasal cavity for up to a month and were slowly and steadily dissolved. All methods showed a fast release of the lipophilic drug in the first days with a steadier increase to reach a plateau after approximately 5 days. The release of [125I]I- took place at a much slower rate. We herein demonstrate the feasibility of this experimental approach to obtain high-resolution, non-invasive quantitative images of the release of the radiolabeled drug, providing valuable information for improved pharmaceutical development of intranasal implants.

Infection-specific PET imaging with 18F-fluorodeoxysorbitol and 2-[18F]F-ρ-aminobenzoic acid: An extended diagnostic tool for bacterial and fungal diseases.

Introduction: Suspected infectious diseases located in difficult-to-access sites can be challenging due to the need for invasive procedures to isolate the etiological agent. Positron emission tomography (PET) is a non-invasive imaging technology that can help locate the infection site. The most widely used radiotracer for PET imaging (2-deoxy-2[18F] fluoro-D-glucose: [18F]FDG) shows uptake in both infected and sterile inflammation. Therefore, there is a need to develop new radiotracers able to specifically detect microorganisms. Methods: We tested two specific radiotracers: 2-deoxy-2-[18F]-fluoro-D-sorbitol ([18F]FDS) and 2-[18F]F-ρ-aminobenzoic acid ([18F]FPABA), and also developed a simplified alternative of the latter for automated synthesis. Clinical and reference isolates of bacterial and yeast species (19 different strains in all) were tested in vitro and in an experimental mouse model of myositis infection. Results and discussion: Non-lactose fermenters (Pseudomonas aeruginosa and Stenotrophomonas maltophilia) were unable to take up [18F]FDG in vitro. [18F]FDS PET was able to visualize Enterobacterales myositis infection (i.e., Escherichia coli) and to differentiate between yeasts with differential assimilation of sorbitol (i.e., Candida albicans vs. Candida glabrata). All bacteria and yeasts tested were detected in vitro by [18F]FPABA. Furthermore, [18F]FPABA was able to distinguish between inflammation and infection in the myositis mouse model (E. coli and Staphylococcus aureus) and could be used as a probe for a wide variety of bacterial and fungal species.

A novel [89Zr]-anti-PD-1-PET-CT to assess response to PD-1/PD-L1 blockade in lung cancer.

Background: Harnessing the anti-tumor immune system response by targeting the program cell death protein (PD-1) and program cell death ligand protein (PD-L1) axis has been a major breakthrough in non-small cell lung cancer (NSCLC) therapy. Nonetheless, conventional imaging tools cannot accurately assess response in immunotherapy-treated patients. Using a lung cancer syngeneic mouse model responder to immunotherapy, we aimed to demonstrate that [89Zr]-anti-PD-1 immuno-PET is a safe and feasible imaging modality to assess the response to PD-1/PD-L1 blockade in NSCLC. Materials and methods: A syngeneic mouse model responder to anti-PD-1 therapy was used. Tumor growth and response to PD-1 blockade were monitored by conventional 2-deoxy-2-[18F]fluoro-D-glucose ([18F]-FDG) PET scans. Additionally, tumor lymphocyte infiltration was analyzed by the use of an [89Zr]-labeled anti-PD-1 antibody and measured as 89Zr tumor uptake. Results: Conventional [18F]-FDG-PET scans failed to detect the antitumor activity exerted by anti-PD-1 therapy. However, [89Zr]-anti-PD-1 uptake was substantially higher in mice that responded to PD-1 blockade. The analysis of tumor-infiltrating immune cell populations and interleukins demonstrated an increased anti-tumor effect elicited by activation of effector immune cells in PD-1-responder mice. Interestingly, a positive correlation between [89Zr]-anti-PD-1 uptake and the proportion of tumor-infiltrating lymphocytes (TILs) was found (Cor = 0.8; p = 0.001). Conclusion: Our data may support the clinical implementation of immuno-PET as a promising novel imaging tool to predict and assess the response of PD-1/PD-L1 inhibitors in patients with NSCLC.

Importance of breastfeeding and complementary feeding for management and prevention of childhood diarrhoea in low- and middle-income countries.

Background: Early and exclusive breastfeeding have been shown to protect young infants from all-cause and diarrhoea-related mortality. Ideally breastfeeding should be initiated within the first hour of birth. Despite efforts to increase rates of early and exclusive breastfeeding in low- and middle-income countries (LMICs), challenges with uptake remain. This analysis reviews trends in early and exclusive breastfeeding, and the impact of infant feeding interventions in reducing childhood diarrhoea. Methods: We conducted a detailed review of articles written in English between 1990 and 2020 on the impact and efficacy of breastfeeding and complementary feeding on diarrhoea in children aged 0-2 years in LMICs. Using data from 86 countries and all WHO global regions collected from the mid-1980s through 2018 obtained from publicly available Demographic Health Surveys, we assessed trends in five-year intervals of timing of breastfeeding initiation, exclusive breastfeeding, median and mean duration of exclusive breastfeeding, and complementary feeding. Results: The literature search identified ten articles that described variable rates of early initiation of breastfeeding from 20% in Pakistan to 76% in Egypt. An analysis of 288 DHS studies found that the proportion of women who reported initiating breastfeeding within an hour of birth increased from 32% in the early 1990s to 55% between 2016 and 2020. Exclusive breastfeeding increased from 20% in the late 1980s to 48% between 2016 and 2020 and the mean duration of exclusive breastfeeding of 2-to-4-month-old infants doubled. Early initiation of breastfeeding and exclusive breastfeeding was associated with reductions in diarrhoea prevalence in the South East Asian, Western Pacific, Eastern Mediterranean, and African regions. Eight studies evaluating the effectiveness of different maternal education interventions, health care worker training, and media campaigns demonstrated improvements in exclusive breastfeeding, and most resulted in reductions in the incidence or duration of diarrhoea. Conclusions: During the last two decades, early and exclusive breastfeeding have increased. Nevertheless, the uptake of this basic, low-cost intervention remains suboptimal across all global regions. Given the potential benefits the in reduction of diarrhoea and diarrhoea-associated mortality, interventions for improving the uptake of early and exclusive breastfeeding in different sociological contexts need to be designed, implemented, and evaluated.

Development of intranasal implantable devices for schizophrenia treatment.

In this work the preparation and characterisation of intranasal implants for the delivery of risperidone (RIS) is described. The aim of this work is to develop better therapies to treat chronic conditions affecting the brain such as schizophrenia. This type of systems combines the advantages of intranasal drug delivery with sustained drug release. The resulting implants were prepared using biodegradable materials, including poly(caprolactone) (PCL) and poly(lactic-co-glycolic acid) (PLGA). These polymers were combined with water-soluble compounds, such as poly(ethylene glycol) (PEG) 600, PEG 3000, and Tween® 80 using a solvent-casting method. The resulting implants contained RIS loadings ranging between 25 and 50 %. The obtained implants were characterised using a range of techniques including thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), attenuated total reflectance-Fourier transform infrared (ATR-FTIR), X-ray diffraction (XRD), and Scanning Electron Microscopy (SEM). Moreover, in vitro RIS release was evaluated showing that the addition of water-soluble compounds exhibited significant faster release profiles compared to pristine PCL and PLGA-based implants. Interestingly, PCL-based implants containing 25 % of RIS and PLGA-based implants loaded with 50 % of RIS showed sustained drug release profiles up to 90 days. The former showed faster release rates over the first 28 days but after this period PLGA implants presented higher release rates. The permeability of RIS released from the implants through a model membrane simulating nasal mucosa was subsequently evaluated showing desirable permeation rate of around 2 mg/day. Finally, following in vitro biocompatibility studies, PCL and PLGA-based implants showed acceptable biocompatibility. These results suggested that the resulting implants displayed potential of providing prolonged drug release for brain-targeting drugs.

Identification of microorganisms responsible for foam formation in mesophilic anaerobic digesters treating surplus activated sludge.

Foaming is a common operational problem in anaerobic digestion (AD) systems, where hydrophobic filamentous microorganisms are usually considered to be the major cause. However, little is known about the identity of foam-stabilising microorganisms in AD systems, and control measures are lacking. This study identified putative foam forming microorganisms in 13 full-scale mesophilic digesters located at 11 wastewater treatment plants in Denmark, using 16S rRNA gene amplicon sequencing with species-level resolution and fluorescence in situ hybridization (FISH) for visualization. A foaming potential aeration test was applied to classify the digester sludges according to their foaming propensity. A high foaming potential for sludges was linked to the abundance of species from the genus Candidatus Microthrix, immigrating with the feed stream (surplus activated sludge), but also to several novel phylotypes potentially growing in the digester. These species were classified to the genera Ca. Brevefilum (Ca. B. fermentans) and Tetrasphaera (midas_s_5), the families ST-12K33 (midas_s_22), and Rikenellaceae (midas_s_141), and the archaeal genus Methanospirillum (midas_s_2576). Application of FISH showed that these potential foam-forming organisms all had a filamentous morphology. Additionally, it was shown that concentrations of ammonium and total nitrogen correlated strongly to the presence of foam-formers. This study provided new insight into the identity of putative foam-forming microorganisms in mesophilic AD systems, allowing for the subsequent surveillance of their abundances and studies of their ecology. Such information will importantly inform the development of control measures for these problematic microorganisms.

Generation of Comprehensive Ecosystem-Specific Reference Databases with Species-Level Resolution by High-Throughput Full-Length 16S rRNA Gene Sequencing and Automated Taxonomy Assignment (AutoTax).

High-throughput 16S rRNA gene amplicon sequencing is an essential method for studying the diversity and dynamics of microbial communities. However, this method is presently hampered by the lack of high-identity reference sequences for many environmental microbes in the public 16S rRNA gene reference databases and by the absence of a systematic and comprehensive taxonomy for the uncultured majority. Here, we demonstrate how high-throughput synthetic long-read sequencing can be applied to create ecosystem-specific full-length 16S rRNA gene amplicon sequence variant (FL-ASV) resolved reference databases that include high-identity references (>98.7% identity) for nearly all abundant bacteria (>0.01% relative abundance) using Danish wastewater treatment systems and anaerobic digesters as an example. In addition, we introduce a novel sequence identity-based approach for automated taxonomy assignment (AutoTax) that provides a complete seven-rank taxonomy for all reference sequences, using the SILVA taxonomy as a backbone, with stable placeholder names for unclassified taxa. The FL-ASVs are perfectly suited for the evaluation of taxonomic resolution and bias associated with primers commonly used for amplicon sequencing, allowing researchers to choose those that are ideal for their ecosystem. Reference databases processed with AutoTax greatly improves the classification of short-read 16S rRNA ASVs at the genus- and species-level, compared with the commonly used universal reference databases. Importantly, the placeholder names provide a way to explore the unclassified environmental taxa at different taxonomic ranks, which in combination with in situ analyses can be used to uncover their ecological roles.

The morphology and metabolic potential of the Chloroflexi in full-scale activated sludge wastewater treatment plants.

Filamentous bacteria belonging to the phylum Chloroflexi have received considerable attention in wastewater treatment systems for their suggested role in the operational problem of impaired sludge settleability known as bulking. Their consistently high abundance in full-scale systems, even in the absence of bulking, indicates that they make a substantial contribution to the nutrient transformations during wastewater treatment. In this study, extensive 16S rRNA amplicon surveys of Danish wastewater treatment plants (WWTPs) with nutrient removal were screened to identify numerically important Chloroflexi genera. Fluorescence in situ hybridization probes were designed for their in situ characterization. All abundant Chloroflexi phylotypes were putatively identified as facultative anaerobic chemoorganotrophs involved in sugar fermentation. They were all filamentous but differed in their morphology and spatial arrangement. 'Candidatus Villigracilis' was predominantly located within the activated sludge flocs, where they possibly have structural importance, and their abundance was relatively stable. Conversely, the abundance of 'Candidatus Amarolinea' was highly dynamic, relative to other genera, sometimes reaching abundances in excess of 30% of the biovolume, suggesting their likely role in bulking episodes. This study gives an important insight into the role of Chloroflexi in WWTPs, thus contributing to the broader goal of understanding the ecology of these biotechnologically important systems.

Resolving the individual contribution of key microbial populations to enhanced biological phosphorus removal with Raman-FISH.

Enhanced biological phosphorus removal (EBPR) is a globally important biotechnological process and relies on the massive accumulation of phosphate within special microorganisms. Candidatus Accumulibacter conform to the classical physiology model for polyphosphate accumulating organisms and are widely believed to be the most important player for the process in full-scale EBPR systems. However, it was impossible till now to quantify the contribution of specific microbial clades to EBPR. In this study, we have developed a new tool to directly link the identity of microbial cells to the absolute quantification of intracellular poly-P and other polymers under in situ conditions, and applied it to eight full-scale EBPR plants. Besides Ca. Accumulibacter, members of the genus Tetrasphaera were found to be important microbes for P accumulation, and in six plants they were the most important. As these Tetrasphaera cells did not exhibit the classical phenotype of poly-P accumulating microbes, our entire understanding of the microbiology of the EBPR process has to be revised. Furthermore, our new single-cell approach can now also be applied to quantify storage polymer dynamics in individual populations in situ in other ecosystems and might become a valuable tool for many environmental microbiologists.

In situ visualisation of the abundant Chloroflexi populations in full-scale anaerobic digesters and the fate of immigrating species.

Anaerobic digestion is a key process for the conversion of waste organics to biogas for energy and is reliant on the synergistic activities of complex microbial communities. Members of the phylum Chloroflexi are often found to be abundant in these systems, yet little is known of their role, with most members yet to be cultured or identified. The aim of this study was to characterize the Chloroflexi communities present in full-scale anaerobic digesters receiving excess sludge from wastewater treatment plants. The core genus-level-phylotypes were identified from extensive 16S rRNA gene amplicon sequencing surveys of 19 full-scale systems over a 6 year period. The T78 and Leptolinea, and the RB349 and SJA-170, were found to be the most abundant genera of mesophilic and thermophilic digesters, respectively. With the exception of Leptolinea, these phylotypes are known only by their 16S rRNA gene sequence, and their morphology and metabolic potentials are not known. Fluorescence in situ hybridisation (FISH) probes were designed for these phylotypes, with their application revealing a similar thin filamentous morphology, indicating a possible role for these organisms in maintaining floc structure. The new FISH probes provide a useful tool for future efforts to characterize these organisms in situ. FISH also suggests that immigrating Chloroflexi species die off in the anaerobic digester environment and their high abundance in anaerobic digesters, observed with DNA based sequencing surveys, was quite possibly due to the persistence of their DNA after their death. This observation is important for the interpretation of popular DNA-based sequencing methods applied for the characterisation of communities with substantial immigration rates, such as anaerobic digesters.

Monitoring foaming potential in anaerobic digesters.

Foaming in anaerobic digestion (AD) systems for biogas generation can give serious operational problems. The cause of such foaming events is often unclear, and it is therefore not an easy task to predict and subsequently apply preventative measures. Methods for the measurement of the foaming potential of digester sludge are often implemented, but no standardized method is available. In this study, we investigated parameters influencing the foam formation during experimental aeration tests of full-scale digester sludge, including air flow, time, and total solids concentration, and proposed an optimized method for standard use. In a survey of 16 full-scale AD systems located at wastewater treatment plants in Denmark, all sludge samples were classified into three groups (non-foaming, pre-foaming, and actually foaming) according to their foam height/propensity and stability. Extensive surveillance of plants with the proposed classification system will enable the determination of cut-off values to help to identify foaming or pre-foaming sludge, and to associate these with operational conditions leading to foaming episodes.

Novel prosthecate bacteria from the candidate phylum Acetothermia.

Members of the candidate phylum Acetothermia are globally distributed and detected in various habitats. However, little is known about their physiology and ecological importance. In this study, an operational taxonomic unit belonging to Acetothermia was detected at high abundance in four full-scale anaerobic digesters by 16S rRNA gene amplicon sequencing. The first closed genome from this phylum was obtained by differential coverage binning of metagenomes and scaffolding with long nanopore reads. Genome annotation and metabolic reconstruction suggested an anaerobic chemoheterotrophic lifestyle in which the bacterium obtains energy and carbon via fermentation of peptides, amino acids, and simple sugars to acetate, formate, and hydrogen. The morphology was unusual and composed of a central rod-shaped cell with bipolar prosthecae as revealed by fluorescence in situ hybridization combined with confocal laser scanning microscopy, Raman microspectroscopy, and atomic force microscopy. We hypothesize that these prosthecae allow for increased nutrient uptake by greatly expanding the cell surface area, providing a competitive advantage under nutrient-limited conditions.

Paediatric malaria case-management with artemether-lumefantrine in Zambia: a repeat cross-sectional study.

BACKGROUND: Zambia was the first African country to change national antimalarial treatment policy to artemisinin-based combination therapy--artemether-lumefantrine. An evaluation during the early implementation phase revealed low readiness of health facilities and health workers to deliver artemether-lumefantrine, and worryingly suboptimal treatment practices. Improvements in the case-management of uncomplicated malaria two years after the initial evaluation and three years after the change of policy in Zambia are reported. METHODS: Data collected during the health facility surveys undertaken in 2004 and 2006 at all outpatient departments of government and mission facilities in four Zambian districts were analysed. The surveys were cross-sectional, using a range of quality of care assessment methods. The main outcome measures were changes in health facility and health worker readiness to deliver artemether-lumefantrine, and changes in case-management practices for children below five years of age presenting with uncomplicated malaria as defined by national guidelines. RESULTS: In 2004, 94 health facilities, 103 health workers and 944 consultations for children with uncomplicated malaria were evaluated. In 2006, 104 facilities, 135 health workers and 1125 consultations were evaluated using the same criteria of selection. Health facility and health worker readiness improved from 2004 to 2006: availability of artemether-lumefantrine from 51% (48/94) to 60% (62/104), presence of artemether-lumefantrine dosage wall charts from 20% (19/94) to 75% (78/104), possession of guidelines from 58% (60/103) to 92% (124/135), and provision of in-service training from 25% (26/103) to 41% (55/135). The proportions of children with uncomplicated malaria treated with artemether-lumefantrine also increased from 2004 to 2006: from 1% (6/527) to 27% (149/552) in children weighing 5 to 9 kg, and from 11% (42/394) to 42% (231/547) in children weighing 10 kg or more. In both weight groups and both years, 22% (441/2020) of children with uncomplicated malaria were not prescribed any antimalarial drug. CONCLUSION: Although significant improvements in malaria case-management have occurred over two years in Zambia, the quality of treatment provided at the point of care is not yet optimal. Strengthening weak health systems and improving the delivery of effective interventions should remain high priority in all countries implementing new treatment policies for malaria.

MiDAS 2.0: an ecosystem-specific taxonomy and online database for the organisms of wastewater treatment systems expanded for anaerobic digester groups.

Wastewater is increasingly viewed as a resource, with anaerobic digester technology being routinely implemented for biogas production. Characterising the microbial communities involved in wastewater treatment facilities and their anaerobic digesters is considered key to their optimal design and operation. Amplicon sequencing of the 16S rRNA gene allows high-throughput monitoring of these systems. The MiDAS field guide is a public resource providing amplicon sequencing protocols and an ecosystem-specific taxonomic database optimized for use with wastewater treatment facility samples. The curated taxonomy endeavours to provide a genus-level-classification for abundant phylotypes and the online field guide links this identity to published information regarding their ecology, function and distribution. This article describes the expansion of the database resources to cover the organisms of the anaerobic digester systems fed primary sludge and surplus activated sludge. The updated database includes descriptions of the abundant genus-level-taxa in influent wastewater, activated sludge and anaerobic digesters. Abundance information is also included to allow assessment of the role of emigration in the ecology of each phylotype. MiDAS is intended as a collaborative resource for the progression of research into the ecology of wastewater treatment, by providing a public repository for knowledge that is accessible to all interested in these biotechnologically important systems. Database URL: http://www.midasfieldguide.org.

Genomic insights into members of the candidate phylum Hyd24-12 common in mesophilic anaerobic digesters.

Members of the candidate phylum Hyd24-12 are globally distributed, but no genomic information or knowledge about their morphology, physiology or ecology is available. In this study, members of the Hyd24-12 lineage were shown to be present and abundant in full-scale mesophilic anaerobic digesters at Danish wastewater treatment facilities. In some samples, a member of the Hyd24-12 lineage was one of the most abundant genus-level bacterial taxa, accounting for up to 8% of the bacterial biomass. Three closely related and near-complete genomes were retrieved using metagenome sequencing of full-scale anaerobic digesters. Genome annotation and metabolic reconstruction showed that they are Gram-negative bacteria likely involved in acidogenesis, producing acetate and hydrogen from fermentation of sugars, and may play a role in the cycling of sulphur in the digesters. Fluorescence in situ hybridization revealed single rod-shaped cells dispersed within the flocs. The genomic information forms a foundation for a more detailed understanding of their role in anaerobic digestion and provides the first insight into a hitherto undescribed branch in the tree of life.