Sample Collection Research Articles

Clinical and immunophenotype correlating with response to immunotherapy in paediatric patients with primary liver carcinoma. A case series

Paediatric hepatocellular carcinomas (HCC) traditionally arise in the context of a normal structural and functional liver and carry a dismal prognosis. While chemotherapy is the frontline standard, there is emerging interest in the study of immunotherapies for paediatric patients with relapsed/refractory disease. There is limited data to support whether immunotherapies will be of utility in this patient population. Six paediatric patients (median age:16 years, range: 12-17at the time of treatment) with advanced hepatocellular neosplams, either conventional hepatocellular or fibrolamellar carcinoma, were treated with immunotherapy. Patients were consented to institutional genomic profiling and biobanking protocols. Baseline samples and serial tissue samples, when available, were evaluated for somatic mutation rate, actionable gene mutations, and pan-immune bulk RNA expression profiling. Results were correlated with clinical course. Three patients responded to checkpoint inhibition: one achieved a complete, durable response and the other two, prolonged stable disease. Three additional patients progressed. Diagnostic tissue from the complete responder demonstrated a higher relative mutational burden and robust immune infiltrate. Pre-treatment samples from the three responders demonstrated decreased expression of genes associated with T-cell dysfunction. A subset of patients with primary paediatric hepatocellular tumours will respond to immunotherapy. Immunotherapies are currently under prospective study for relapsed/refractory liver tumours in paediatric patients. Results from this report support the prospective collection of serial serum and tissue samples which may further identify genomic and immunophenotypic patterns predictive of response. This work was supported by Philanthropic funds (Pan Mass Challenge, Team Angus and Team Perspective).

Sampling technique to pool genetic materials of microorganism communities in blue-swimming crab processing plant industry

The crab and seafood processing industry must fulfill standard requirements for sanitation, hygiene, and good manufacturing methods to ensure the safety of the products and free from foodborne bacteria. However, equipment and processing unit surfaces are challenging to clean optimally, which can cause persistent bacteria to emerge. Eliminating persistent bacteria is the latest challenge in the fish processing industry for optimal disinfection, preventing cross-contamination, and controlling foodborne outbreaks. Microbiological testing in industry has been limited to selective culture-media techniques; thus, a rapid, sensitive, accurate, and routine applicable analytical method is urgently needed. The significant reduction in the costs of high-throughput sequencing technologies supports the possibility of routine applications in the industry. This study aimed to determine the profile of the microbial community on the surface of the production room and blue-swimming crab processing unit equipment using short-read metagenomic techniques. The analysis included the stages of sampling, bacterial incubation, bacterial DNA isolation, sequencing, and bioinformatics analysis. The first important step to increase the possibility of routine adoption in the seafood industry is to reduce the cost, complexity, and time required to complete the analysis. Therefore, in this protocol, we generate a scalable, flexible, cost-effective, and auditable workflow.• Collection of bacterial samples by swabbing the surface of the equipment using a sterile cotton swab and sterile cloth, which is easy to apply and follow in the blue-swimming crab processing plant industry.• Effective and efficient sample-pooling is an important step in identifying bacterial communities by metagenomic analysis.

Coarsened Mixtures of Hierarchical Skew Normal Kernels for Flow and Mass Cytometry Analyses

Cytometry is the standard multi-parameter assay for measuring single cell phenotype and functionality. It is commonly used for quantifying the relative frequencies of cell subsets in blood and disaggregated tissues. A typical analysis of cytometry data involves cell classification—that is, the identification of cell subgroups in the sample—and comparisons of the cell subgroups across samples or conditions. While modern experiments often necessitate the collection and processing of samples in multiple batches, analysis of cytometry data across batches is challenging because differences across samples may occur due to either true biological variation or technical reasons such as antibody lot effects or instrument optics across batches. Thus a critical step in comparative analyses of multi-sample cytometry data—yet missing in existing automated methods for analyzing such data—is cross-sample calibration, whose goal is to align corresponding cell subsets across multiple samples in the presence of technical variations, so that biological variations can be meaningfully compared. We introduce a Bayesian nonparametric hierarchical modeling approach for accomplishing both calibration and cell classification simultaneously in a unified probabilistic manner. Three important features of our method make it particularly effective for analyzing multi-sample cytometry data: a nonparametric mixture avoids prespecifying the number of cell clusters; a hierarchical skew normal kernel that allows flexibility in the shapes of the cell subsets and cross-sample variation in their locations; and finally the “coarsening” strategy makes inference robust to departures from the model not captured by the skew normal kernels. We demonstrate the merits of our approach in simulated examples and carry out a case study in the analysis of a multi-sample cytometry data set. We provide an R package for our method.

Relative Cost and Infectious Days Averted Associated With Rapid Gonorrhea and Chlamydia Testing Among Men Who Have Sex With Men.

Standard-of-care nucleic acid amplification tests (routine NAATs) for Neisseria gonorrhoeae (GC) and Chlamydia trachomatis (CT) can take several days to result and therefore delay treatment. Rapid point-of-care GC/CT NAAT (rapid NAAT) could reduce the time to treatment and therefore onward transmission. This study evaluated the incremental cost per infectious day averted and overall cost of implementation associated with rapid compared with routine NAAT. Prospective sexually transmitted infection (STI) treatment data from men who have sex with men and transgender women in San Diego who received rapid NAAT between November 2018 and February 2021 were evaluated. Historical time from testing to treatment for routine NAAT was abstracted from the literature. Costs per test for rapid and routine NAAT were calculated using a micro-costing approach. The incremental cost per infectious day averted comparing rapid to routine NAAT and the costs of rapid GC/CT NAAT implementation in San Diego Public Health STI clinics were calculated. Overall, 2333 individuals underwent rapid NAAT with a median time from sample collection to treatment of 2 days compared with 7 to 14 days for routine NAAT equating to a reduction of 5 to 12 days. The cost of rapid and routine GC/CT NAAT was $57.86 and $18.38 per test, respectively, with a cost-effectiveness of between $2.43 and $5.82 per infectious day averted. The incremental cost of rapid NAAT improved when at least 2000 tests were performed annually. Although rapid GC/CT NAAT is more expensive than routine testing, the reduction of infectious days between testing and treatment may reduce transmission and provide improved STI treatment services to patients.

Prevalence and antibiotic susceptibility patterns of Escherichia coli and Staphylococcus aureus isolated from bioaerosols of local markets located in Agulu, Anambara State

There is a need for improved understanding of aerosol-acquired disease due to the increasing environmental load of antibiotic resistant bacteria. Multidrug-resistant Escherichia coli and Staphylococcus aureus are examples of bacteria for which the role of exposure to aerosolized organisms in disease transmission should be more closely evaluated globally. This study is aimed at evaluating the prevalence and antibiogram of S. aureus and E. coli present in market bioaerosols, located in Nwagu-Agulu, Anambra Nigeria. Impaction sample collection method was used to collect bioaerosol samples within the market using nutrient agar as the collection media, A combination of the cultural and microscopic features as well as selected biochemical tests were employed to identify the isolates. The Kirby-Bauer disk diffusion method was used to determine antibiotic resistance to selected commonly used antibiotics. A total of 76 S. aureus and 24 E. coli were isolated. Both organisms have been implicated in community-acquired infections. The most common bacteria isolated were Staphylococcus aureus. Varying levels of resistance were observed by these isolates to selected commonly use antibiotics. There is high prevalence of S. aureus and E. coli within Nwagu market Agulu, Anambra State, Eastern region of Nigeria. The organisms have very high rates of resistance to antibiotics (MDR) as reported by other workers. The government as well as Health professionals must play an important role in the prevention of community acquired infections via regular environmental monitoring of open markets bioaerosol and establishing a common permissible limits or concentration for bioaerosols within the markets.

Efficient Use of Biological Data in the Web 3.0 Era by Applying Nonfungible Token Technology.

CryptoKitties, a trendy game on Ethereum that is an open-source public blockchain platform with a smart contract function, brought nonfungible tokens (NFTs) into the public eye in 2017. NFTs are popular because of their nonfungible properties and their unique and irreplaceable nature in the real world. The embryonic form of NFTs can be traced back to a P2P network protocol improved based on Bitcoin in 2012 that can realize decentralized digital asset transactions. NFTs have recently gained much attention and have shown an unprecedented explosive growth trend. Herein, the concept of digital asset NFTs is introduced into the medical and health field to conduct a subversive discussion on biobank operations. By converting biomedical data into NFTs, the collection and circulation of samples can be accelerated, and the transformation of resources can be promoted. In conclusion, the biobank can achieve sustainable development through "decentralization."

Gut microbiome and symptoms in females with irritable bowel syndrome: a cross-sectional analysis

Abstract Irritable bowel syndrome (IBS), a disorder of gut-brain interaction, is associated with abdominal pain and stool frequency/character alterations that are linked to changes in microbiome composition. We tested whether taxa differentially abundant between females with IBS vs healthy control females (HC) are associated with daily gastrointestinal and psychological symptom severity. Participants (age 18-50 year) completed a 3-day food record and collected a stool sample during the follicular phase. They also completed a 28-day diary rating symptom intensity; analysis focused on the three days after the stool sample collection. 16S rRNA gene sequencing was used for bacterial identification. Taxon abundance was compared between IBS and HC using zero-inflated quantile analysis (ZINQ). We found that females with IBS (n = 67) had greater Bacteroides abundance (q = 0.003) and lower odds of Bifidobacterium presence (q = 0.036) compared to HC (n = 46) after adjusting for age, race, body mass index, fibre intake, and hormonal contraception use. Intestimonas, Oscillibacter, and Phascolarctobacterium were more often present and Christensenellaceae R-7 group, Collinsella, Coprococcus 2, Moryella, Prevotella 9, Ruminococcaceae UCG-002, Ruminococcaceae UCG-005, and Ruminococcaceae UCG-014 were less commonly present in IBS compared to HC. Despite multiple taxon differences in IBS vs HC, we found no significant associations between taxon presence or abundance and average daily symptom severity within the IBS group. This may indicate the need to account for interactions between microbiome, dietary intake, metabolites, and host factors.

Production of redox-active-reactive filter material from charcoal

The surficial and textural features of a pyrogenic carbonaceous material (charcoal) was modulated with acidic Piranha solution (i.e., 1:3 30% H2O2 to H2SO4) to produce a redox-active adsorbent for the simultaneous adsorption and reduction of Cr(VI) in groundwater sample. The optimal modification time of 60 s is required to transform the raw charcoal to an efficacious redox-active adsorbent (MCh). The MCh surface was imbued with electron donor moieties that is required for the direct conversion of the adsorbed Cr(VI) to Cr(III). The rate parameters for Cr(VI) adsorption by the MCh were initial concentration dependent. The GW system variables had minimal influence on the performance of the MCh, which showed that the MCh is capable of effective performance within broad process variables. The direct conversion of Cr(VI) to Cr(III) on the MCh surface was affirmed. The treatment of Cr(VI) contaminated GW in a microcosm real-life system was highly effective over the period of study (40 hours). The residual Cr(VI) concentration in the hourly sample collection ranged between undetectable (i.e.,<0.01), at the inception, to 1.77 mg/L at the 40th hour. The pH values of the treated water were not vitiated, and the EC (μs cm− 1) values continually reduced over time.

Volatile Organic Compounds (VOCs) in Heritage Environments and Their Analysis: A Review

In the recent years, there has been an increased interest in indoor air quality in heritage environments, specifically in relation to volatile organic compounds (VOCs). These could originate from objects, furnishings, visitors and staff, as well as from olfactory exhibitions. This interest led to a number of studies investigating the “typical” emissions for diverse materials and their impact on the surrounding environment. The analysis of volatile compounds emitted by objects helps in the characterization of the material composition, its conservation history or its degradation processes. This contribution reviews how volatiles are emitted from objects and the commonly used sampling techniques for heritage science applications. A variety of methods are available, from bulk air sample collection to preconcentration using samplers. The commonly studied object types contributing to indoor VOCs are discussed. These include emissions from heritage objects, conservation products, furnishing materials and display cases. Furthermore, olfactory exhibitions are discussed in terms of indoor air quality. Finally, the findings are compared with the current guidelines on indoor volatile concentrations.

Ultra-diluted complex additive in the diet reveals benefits in the intestinal tract of nursery-phase piglets.

This study aimed to assess an ultra-diluted (UD) complex, as a replacement for an antimicrobial growth promoter in diets, on growth performance, intestinal health, and inflammatory response of nursery piglets. The experiment lasted 37 days and involved 126 animals weaned at 21 ± 1.3 d, with an initial body weight of 5.62 ± 1.16kg. Piglets were assigned to six dietary treatments in a randomized block design with seven replicates and three piglets per pen as experimental unit. The treatments were: positive control (PC)- basal diet + 120mg/kg of chlorohydroxyquinoline; negative control (NC)- basal diet without additives; and NC containing 4.5; 6.0; 7.5 or 9.0kg of UD additive/ton diet. Performance data were calculated, and daily diarrhea was observed. Blood samples were collected for hematological analysis. At the end of the experiment, one animal per pen was slaughtered for organ weighing, pH, and the collection of intestinal samples for histopathology. Feces and cecal contents were collected for microbiological and antibiogram analyses. There was no difference in the performance between the treatments. Throughout the study, UD levels were equal to those of PC for diarrhea occurrence. Higher levels of UD complex led to higher total leukocyte counts. The 4.5 treatment showed a reduction in total and thermotolerant Enterobacteriaceae populations in piglet feces and an increase in lactic acid bacteria compared to PC. All treatments resulted in fewer duodenal histopathological alterations than those in the NC group. The use of UD additives, especially at 4.5kg/ton, is a good alternative to chlorohydroxyquinoline in piglet diets.

Molecular characterization of Escherichia coli virulence markers in neonatal and postweaning piglets from major pig-producing districts of Uganda.

Piggery production is highly constrained by diseases, with diarrhoea in piglets being a major cause of economic losses to smallholder farmers in Uganda. Enterotoxigenic Escherichia coli (ETEC) is thought to be one of the major etiologies of this diarrhoea. A cross-sectional study was carried out in two high pig-producing districts of Uganda with the aim of determining the significance of piglet diarrhoea and the pathogenic determinants of causative E. coli. A total of 40 households with piglets were visited in each district for a questionnaire survey and faecal sample collection. The questionnaire-based data collected included; demographic data and pig management practices. E. coli were isolated from diarrheic (43) and non-diarrheic (172) piglets and were subjected to antimicrobial susceptibility testing against nine commonly used antimicrobial agents. The E. coli isolates were further screened for the presence of 11 enterotoxin and fimbrial virulence gene markers using multiplex polymerase chain reaction. Data entry, cleaning, verification and descriptive statistics were performed using Microsoft Excel. Statistical analysis to determine any association between the presence of virulence markers and diarrhea in piglets was done using SPSS software (Version 23), with a p value of less than 0.05 taken as a statistically significant association. Escherichia coli were recovered from 81.4% (175/215) of the faecal samples. All the isolates were resistant to erythromycin, and most showed high resistance to tetracycline (71%), ampicillin (49%), and trimethoprim sulfamethoxazole (45%). More than half of the isolates (58.3%) carried at least one of the 11 virulence gene markers tested. EAST1 was the most prevalent virulence marker detected (35.4%), followed by STb (14.8%). Expression of more than one virulence gene marker was observed in 6.2% of the isolates, with the EAST1/STa combination being the most prevalent. Three adhesins; F17 (0.6%), F18 (6.3%) and AIDA-I (0.6%) were detected, with F18 being the most encountered. There was a statistically significant association between the occurrence of piglet diarrhoea and the presence of the AIDA-1 (p value = 0.037) or EAST1 (p value = 0.011) gene marker among the isolates. The level of antimicrobial resistance among E. coli isolates expressing virulence markers were high in the sampled districts. The study established a significant association between presence of EAST1 and AIDA-I virulence markers and piglet diarrhea. Further studies should be carried out to elucidate the main adhesins borne by these organisms in Uganda and the actual role played by EAST1 in the pathogenesis of the infection since most isolates expressed this gene.

Production and Chemical Characterization of Silage from Saccharine Sorghum Fertirrigated with Sugarcane Vinasse.

Distillery vinasse is one of the promising bio-fertilizers, as it contains significant amounts of essential chemical elements, allied with sorghum that is widely used in the diet of ruminant animals and has been considered as an alternative to the production of other cereals or forages. This study aimed to evaluate saccharin sorghum silage from fertilization with vinasse. The research was conducted using the BRS-511, CR-1339, and CR-1342 geno-types. The silage was held for 170 days after sowing, with experimental design in blocks with triple factorial (genotypes x fertilization x inoculation) totaling 54 installments. At 95 days, the silos were opened for sample collection and analysis bromatological analysis. The results indicate the primary source of variation was genotype, characterizing them with different potentials in productivity and better results for BRS-511, CR-1339, and CR-1342. The bromatological results indicate good quality for CR-1339 and CR-1342 hybrids, however, better digestability for BRS-511. There was no observable difference between the factors of fertilization. The inoculation additive assists in the reduction of lignin appears to be high. PCA analysis showed differences between cultivars (BRS-511, CR-1339, and CR-1342) and fertili-zation. However, the PCAs showed the genotypes show similar results with conventional ferti-lization and sugarcane vinasse. The study reflected the possibility of producing sweet sorghum silage with soil sugarcane vinasse fertilization as fertilizer.

Isolation of Aspergillus sp from some Decomposing Fruits and Vegetables from Banana Rhyzosphere Soil for Citric Acid Production

Aims: The general objective of this study is to isolate certain strains capable of producing citric acid. Place and Duration of Study: Sample collection was carried out on two types of organic matter. Sampling was done from February to April 2021 in 3 municipalities in Abidjan with 10 samples per site. Methodology: To carry out this work, isolation and purification of Aspergillus sp strains were carried out. Macroscopic and microscopic identifications of the mold isolates were carried out. The search for aflatoxin-producing molds was carried out. The analysis ended with a screening of molds capable of synthesizing citric acid. Results: Aspergillus niger, Aspergillus sp1 and Aspergillus sp2 showed no fluorescence, while Aspergillus flavus, Aspergillus candidus and Aspergillus fumigatus showed fluorescent spots, indicating the presence of aflatoxin. Conclusion: Aspergillus isolates capable of producing citric acid were isolated during this study. Aspergillus niger, Aspergillus sp1 and Aspergillus sp2 do not produce aflatoxin and have the capacity to synthesize citric acid.

Assessment of the Bacteriological Properties of Indoor Dust in some Classrooms of Delta State University of Science and Technology, Ozoro

The bacteriological properties of indoor dust in some classrooms of the Delta State University of Science, Ozoro was examined with a view to identify any possible source of pollution of classroom indoor air quality; measurement of microbial load of classroom dust sample and to compare findings of selected locations as a means to determine high air polluted areas. This was done by the collection of dust samples from classrooms table and chair surfaces. It was observed that the amount of bacteria available in classrooms differ based on activities in and around the classrooms. Bacteria isolates indicated the presence of pathogen associated with disease such as Bacillus sp; Staphylococcus sp; Micrococcus sp. of indoor air pollution exist and this is identified as open dump practice birthing more microbial cells which are transported through air and poor sanitation practice around and within the classrooms. Contamination of sample was observed for dust at different locations with the bacteria count seen to be higher at sample C (1.8x102cfu/m3) and lower in sample D (0.2x102cfu/m3). Staphylococcus sp dominated all the locations that were analyzed while Micrococcus sp. only occurred in site C. This is because poor environmental sanitation is observed at certain classrooms in addition to shattered classroom windows given an ease of access of dust flow indoor. It can be concluded that only a minimal sources of indoor air pollution exist and hence only a small amount of bacteria is present. In spite of this, prevalence of such practices is likely to promote the growth of more microbial cells.

Sampling efficiency and nucleic acid stability during long-term sampling with different bioaerosol samplers

Aerosol microbiome studies have received increased attention as technological advancements have made it possible to dive deeper into the microbial diversity. To enhance biomass collection for metagenomic sequencing, long-term sampling is a common strategy. While the impact of prolonged sampling times on microorganisms’ culturability and viability is well-established, its effect on nucleic acid stability remains less understood but is essential to ensure representative sample collection. This study evaluated four air samplers (SKC BioSampler, SASS3100, Coriolis μ, BioSpot-VIVAS 300-P) against a reference sampler (isopore membrane filters) to identify nucleic acid stability during long-term sampling. Physical sampling efficiencies determined with a fluorescent tracer for three particle sizes (0.8, 1, and 3 μm), revealed high efficiencies (> 80% relative to reference) for BioSampler, SASS3100, and BioSpot-VIVAS for all particle sizes, and for Coriolis with 3 μm particles. Coriolis exhibited lower efficiency for 0.8 μm (7%) and 1 μm (50%) particles. During 2-h sampling with MS2 and Pantoea agglomerans, liquid-based collection with Coriolis and BioSampler showed a decrease in nucleic acid yields for all test conditions. BioSpot-VIVAS displayed reduced sampling efficiency for P. agglomerans compared to MS2 and the other air samplers, while filter-based collection with SASS3100 and isopore membrane filters, showed indications of DNA degradation for 1 μm particles of P. agglomerans after long-term sampling. These findings show that long-term air sampling affects nucleic acid stability in both liquid- and filter-based collection methods. These results highlight bias produced by bioaerosol collection and should be considered when selecting an air sampler and interpreting aerosol microbiome data.

Effects of a manganese complex with lysine and glutamic acid on growth performance, manganese deposition and emission, antioxidant capacity and metacarpal strength in weaned piglets.

The present study sought to assess the effects of manganese complexes with lysine and glutamic acid (Mn-LG) as manganese (Mn) sources on growth performance, trace element deposition, antioxidant capacity, and metacarpal strength in weaned piglets. The study involved 288 healthy Duroc × Landrace × Yorkshire piglets that were weaned at 25-28 days of age and weighed 8.66±0.96kg. These piglets were randomly divided into six groups: a control group (Mn-LG-0, receiving a basal diet without Mn supplementation), a Mn sulfate group (basal diet supplemented with 40 mg·kg-1 diet of Mn, Mn-S-40 group), and four Mn-LG groups (Mn-LG-20, Mn-LG-40, Mn-LG-60, Mn-LG-80, supplemented with 20, 40, 60, and 80 mg·kg-1 Mn from Mn-LG in the basal diet). Grouping began at weaning as the 0th day of the experiment. The corn-soybean-based basal diet during the early (d 0-14) and late (d 15-42) phases of the experiment contained 20.88 and 30.12 mg·kg-1 Mn, respectively. Blood samples were collected on days 14 and 42, and pigs were sacrificed for sample collection on day 42. The results indicated no significant differences in average daily gain, average daily feed intake, or feed-to-gain ratio among the groups (P > 0.05). The diarrhea rates of all Mn-LG groups and the Mn-S-40 group were significantly lower in the 0-14 d and during the entire experimental period than in the Mn-LG-0 group (P < 0.001). The Mn-LG-40 group exhibited a significant increase in liver Mn concentration and serum Mn superoxide dismutase (Mn-SOD) activity on d 42 (P < 0.01), as well as a significant decrease in fecal Mn concentration (P < 0.05), compared to those of the Mn-S-40 group. Significant differences (P < 0.05) were detected in the serum, liver, and fecal Mn concentrations, as well as in the serum and liver Mn-SOD activity, across the different Mn-LG groups. The serum and fecal Mn concentrations and serum Mn-SOD activity increased linearly or quadratically (P < 0.01) with increasing Mn-LG supplementation. No significant differences (P > 0.05) were found in kidney, heart, or metacarpal bone Mn concentrations or in bone strength indices. In summary, compared with the Mn-LG-0 diet, dietary supplementation with Mn-LG enhanced serum Mn deposition and Mn-SOD activity and decreased the incidence of diarrhea. Additionally, the fecal Mn concentration was lower in the Mn-LG group than in the inorganic group at equivalent dosages.

Serotype epidemiology and antibiotic resistance of pneumococcal isolates colonizing infants in Botswana (2016-2019).

In 2012, Botswana introduced 13-valent pneumococcal conjugate vaccine (PCV-13) to its childhood immunization program in a 3+0 schedule, achieving coverage rates of above 90% by 2014. In other settings, PCV introduction has been followed by an increase in carriage or disease caused by non-vaccine serotypes, including some serotypes with a high prevalence of antibiotic resistance. We characterized the serotype epidemiology and antibiotic resistance of pneumococcal isolates cultured from nasopharyngeal samples collected from infants (≤12 months) in southeastern Botswana between 2016 and 2019. Capsular serotyping was performed using the Quellung reaction. E-tests were used to determine minimum inhibitory concentrations for common antibiotics. We cultured 264 pneumococcal isolates from samples collected from 150 infants. At the time of sample collection, 81% of infants had received at least one dose of PCV-13 and 53% had completed the three-dose series. PCV-13 serotypes accounted for 27% of isolates, with the most prevalent vaccine serotypes being 19F (n = 20, 8%), 19A (n = 16, 6%), and 6A (n = 10, 4%). The most frequently identified non-vaccine serotypes were 23B (n = 29, 11%), 21 (n = 12, 5%), and 16F (n = 11, 4%). Only three (1%) pneumococcal isolates were resistant to amoxicillin; however, we observed an increasing prevalence of penicillin resistance using the meningitis breakpoint (2016: 41%, 2019: 71%; Cochran-Armitage test for trend, p = 0.0003) and non-susceptibility to trimethoprim-sulfamethoxazole (2016: 55%, 2019: 79%; p = 0.04). Three (1%) isolates were multi-drug resistant. PCV-13 serotypes accounted for a substantial proportion of isolates colonizing infants in Botswana during a four-year period starting four years after vaccine introduction. A low prevalence of amoxicillin resistance supports its continued use as the first-line agent for non-meningeal pneumococcal infections. The observed increase in penicillin resistance at the meningitis breakpoint and the low prevalence of resistance to ceftriaxone supports use of third-generation cephalosporins for empirical treatment of suspected bacterial meningitis.

The impact of kit, environment, and sampling contamination on the observed microbiome of bovine milk.

In low-microbial biomass samples such as bovine milk, contaminants can outnumber endogenous bacteria. Because of this, milk microbiome research suffers from a critical knowledge gap, namely, does non-mastitis bovine milk contain a native microbiome? In this study, we sampled external and internal mammary epithelia and stripped and cisternal milk and used numerous negative controls, including air and sampling controls and extraction and library preparation blanks, to identify the potential sources of contamination. Two algorithms were used to mathematically remove contaminants and track the potential movement of microbes among samples. Results suggest that the majority (i.e., >75%) of sequence data generated from bovine milk and mammary epithelium samples represents contaminating DNA. Contaminants in milk samples were primarily sourced from DNA extraction kits and the internal and external skin of the teat, while teat canal and apex samples were mainly contaminated during the sampling process. After decontamination, the milk microbiome displayed a more dispersed, less diverse, and compositionally distinct bacterial profile compared with epithelial samples. Similar microbial compositions were observed between cisternal and stripped milk samples, as well as between teat apex and canal samples. Staphylococcus and Acinetobacter were the predominant genera detected in milk sample sequences, and bacterial culture showed growth of Staphylococcus and Corynebacterium spp. in 50% (7/14) of stripped milk samples and growth of Staphylococcus spp. in 7% (1/14) of cisternal milk samples. Our study suggests that microbiome data generated from milk samples obtained from clinically healthy bovine udders may be heavily biased by contaminants that enter the sample during sample collection and processing workflows.IMPORTANCEObtaining a non-contaminated sample of bovine milk is challenging due to the nature of the sampling environment and the route by which milk is typically extracted from the mammary gland. Furthermore, the very low bacterial biomass of bovine milk exacerbates the impacts of contaminant sequences in downstream analyses, which can lead to severe biases. Our finding showed that bovine milk contains very low bacterial biomass and each contamination event (including sampling procedure and DNA extraction process) introduces bacteria and/or DNA fragments that easily outnumber the native bacterial cells. This finding has important implications for our ability to draw robust conclusions from milk microbiome data, especially if the data have not been subjected to rigorous decontamination procedures. Based on these findings, we strongly urge researchers to include numerous negative controls into their sampling and sample processing workflows and to utilize several complementary methods for identifying potential contaminants within the resulting sequence data. These measures will improve the accuracy, reliability, reproducibility, and interpretability of milk microbiome data and research.

Clinical determinants of blood biomarkers of atrial cardiomyopathy: results from the ISOLATION AF ablation cohort study

Abstract Background In patients with atrial fibrillation (AF), significant advances have been made in identifying biomarkers representing diverse pathophysiological pathways, such as inflammation, myocyte injury, collagen deposition, and fatty-fibrotic infiltration. Data on clinical determinants of biomarkers in patients with AF, however, are lacking. Objective Our primary aim was to explore the relationships between clinical determinants and biomarker levels in AF patients scheduled for catheter ablation. Methods A cross-sectional analysis was conducted in AF patients undergoing catheter ablation included in the prospective multicenter ISOLATION study between July 2020 and May 2022. Clinical characteristics, routine tests and blood samples were collected before ablation. Blood samples were analyzed for known and novel cardiovascular biomarkers, including Bone morphogenetic protein 10 (BMP10), Angiopoietin-2 (Ang-2), Fibroblast growth factor 23 (FGF-23), Dickkopf-related protein 3 (DKK-3), Endocan (ESM-1), Insulin-like growth factor-binding protein 7 (IGFBP-7), B-type natriuretic peptides (proBNP, NTproBNP), High sensitive Troponin-T (hsTnT)&amp;lt; Growth differentiation factor 15 (GDF-15), interleukin 6 (IL-6), Fatty acid binding protein 3 (FABP3), Cancer antigen-125 (Ca-125) (Table 1). For each biomarker we trained a lasso regression model to identify the most important clinical determinants out of all the 60 available determinants (including anthropometry, ECG, comorbidities). The strongest associations are summarized in figure 1. Results Blood samples of 508 patients were analyzed. The mean age was 63 ±9 years, and 31.1% were female. 354 patients (69.7%) had paroxysmal AF and 154 patients (30.3%) had persistent AF. Heart failure was reported in 77 patients (15.2%). 355 (72.3%) patients were in sinus rhythm during sample collection. The following clinical determinants of biomarker levels were frequently identified as important: rhythm during blood phlebotomy, age, heart failure, decreased kidney function and female sex (Figure 1). Most notably, rhythm at the time of sampling was strongly associated with various biomarker levels. Female sex was positively associated with BMP10 and FGF23 (atrial fibrosis), but negatively associated with hsTNT (cardiac injury). Conclusion In patients with AF scheduled for catheter ablation, we identified several clinical determinants of biomarker levels with potential implications for disease mechanisms. In women, pro-fibrotic biomarkers were frequently higher, while male patients more often showed signs of myocardial injury. Importantly, rhythm during blood draw is a very strong determinant of many biomarkers and should be taken into account in future biomarker studies.Figure 1.Table 1.

Is blood blood? Comparing quantitation of endogenous steroids and luteinizing hormone in concurrently collected venous serum and Tasso+ SST capillary serum samples.

The monitoring of endogenous steroids in urine has been an important component of the Athlete Biological Passport (ABP) for the last decade. Recently, the quantitation of endogenous steroids in blood has been incorporated into the ABP to increase sensitivity in circumstances where the excretion of urinary ABP biomarkers is low. Current ABP guidelines mandate the use of venous blood draws for blood steroid sample collections, however, recent efforts have focused on investigating the use of less invasive sample collection methods, such as capillary blood collected from the upper arm. The focus of this study was to compare the analytical results of venous and capillary blood collected weekly from 20 individuals, 10 males and 10 females, over six weeks. The two primary biomarkers of the blood steroid ABP module, testosterone (T) and the testosterone/androstenedione (T/A4) ratio, were compared, as well as luteinizing hormone (LH) and the T/LH ratio in male participants, two biomarkers known to be responsive to T use. All biomarkers showed excellent agreement between venous and capillary blood. Longitudinal stability between sample types within individuals was also comparable for all biomarkers. Finally, storage of simultaneously collected capillary samples at room temperature and frozen conditions was compared with evaluate the potential impact of non-cold chain shipping conditions. Most biomarkers showed excellent agreement between frozen and room temperature storage conditions. These results indicate capillary blood collections represent a promising alternative to venous blood collections for the blood steroid module of the ABP.