Association between asymptomatic infections and linear growth in 18-24-month-old Malawian children.

Inadequate diet and frequent symptomatic infections are considered major causes of growth stunting in low-income countries, but interventions targeting these risk factors have achieved limited success. Asymptomatic infections can restrict growth, but little is known about their role in global stunting prevalence. We investigated factors related to length-for-age Z-score (LAZ) at 24 months by constructing an interconnected network of various infections, biomarkers of inflammation (as assessed by alpha-1-acid glycoprotein [AGP]), and growth (insulin-like growth factor 1 [IGF-1] and collagen X biomarker [CXM]) at 18 months, as well as other children, maternal, and household level factors. Among 604 children, there was a continuous decline in mean LAZ and increased mean length deficit from birth to 24 months. At 18 months of age, the percentage of asymptomatic children who carried each pathogen was: 84.5% enterovirus, 15.5% parechovirus, 7.7% norovirus, 4.6% rhinovirus, 0.6% rotavirus, 69.6% Campylobacter, 53.8% Giardia lamblia, 11.9% malaria parasites, 10.2% Shigella, and 2.7% Cryptosporidium. The mean plasma IGF-1 concentration was 12.5 ng/ml and 68% of the children had systemic inflammation (plasma AGP concentration >1 g/L). Shigella infection was associated with lower LAZ at 24 months through both direct and indirect pathways, whereas enterovirus, norovirus, Campylobacter, Cryptosporidium, and malaria infections were associated with lower LAZ at 24 months indirectly, predominantly through increased systemic inflammation and reduced plasma IGF-1 and CXM concentration at 18 months.

High-molecular-weight organic matter in the particles of comet 67P/Churyumov-Gerasimenko.

The presence of solid carbonaceous matter in cometary dust was established by the detection of elements such as carbon, hydrogen, oxygen and nitrogen in particles from comet 1P/Halley. Such matter is generally thought to have originated in the interstellar medium, but it might have formed in the solar nebula-the cloud of gas and dust that was left over after the Sun formed. This solid carbonaceous material cannot be observed from Earth, so it has eluded unambiguous characterization. Many gaseous organic molecules, however, have been observed; they come mostly from the sublimation of ices at the surface or in the subsurface of cometary nuclei. These ices could have been formed from material inherited from the interstellar medium that suffered little processing in the solar nebula. Here we report the in situ detection of solid organic matter in the dust particles emitted by comet 67P/Churyumov-Gerasimenko; the carbon in this organic material is bound in very large macromolecular compounds, analogous to the insoluble organic matter found in the carbonaceous chondrite meteorites. The organic matter in meteorites might have formed in the interstellar medium and/or the solar nebula, but was almost certainly modified in the meteorites' parent bodies. We conclude that the observed cometary carbonaceous solid matter could have the same origin as the meteoritic insoluble organic matter, but suffered less modification before and/or after being incorporated into the comet.

Associations between Gut Microbiota and Intestinal Inflammation, Permeability and Damage in Young Malawian Children.

BACKGROUND: Environmental enteric dysfunction (EED) is common in low- and middle-income countries and associated with childhood undernutrition. The composition of gut microbiota has been implicated in the pathogenesis of EED. Our aim was to assess the associations between gut microbiota and EED biomarkers in rural Malawian children. We hypothesized that there would be an inverse association between microbiota maturity and diversity and fecal concentrations of EED biomarkers. METHODS: We used data from fecal samples collected at 6, 18 and 30 months from 611 children who were followed up during a nutrition intervention trial. The primary time point for analysis was 18 months. Microbiota data were obtained through 16S rRNA sequencing and variables included microbiota maturity and diversity, phylogenetic dissimilarity and relative abundances of individual taxa. EED biomarkers included calprotectin (marker of inflammation), alpha-1 antitrypsin (intestinal permeability) and REG1B (intestinal damage). RESULTS: There was an inverse association between microbiota maturity and diversity and fecal concentrations of all 3 EED biomarkers at 18 months (p≤0.001). The results were similar at 30 months, while at 6 months inverse associations were found only with calprotectin and alpha-1 antitrypsin concentrations. At 18 months, EED biomarkers were not associated with phylogenetic dissimilarity, but at 6 and 30 months several associations were observed. Individual taxa predicting EED biomarker concentrations at 18 months included several Bifidobacterium and Enterobacteriaceae taxa as well as potentially displaced oral taxa. CONCLUSIONS: Our findings support the hypothesis of an inverse association between microbiota maturity and diversity and EED in rural Malawian children.

Chronic childhood undernutrition is an important public health concern that affects about 150 million children, mostly in low- and middle-income countries. Undernutrition is caused by insufficient nutrient intake and frequent infections, but there are also other underlying factors. One of these is a condition called environmental enteric dysfunction (EED), which is characterized by intestinal inflammation and damage without apparent clinical symptoms. EED is thought to be caused by the ingestion of pathogenic bacteria that leads to changes in the intestine such as increased permeability and decreased absorptive capacity. This might make the intestinal wall vulnerable to bacterial invasion and reduce the absorption of nutrients. Besides potentially pathogenic bacteria, there are many commensal bacteria in the gastrointestinal tract that have beneficial functions and that interact with the immune system. The aim of our study was to assess the associations between all these bacteria, that is the intestinal microbiota and biomarkers of EED. We used data from fecal samples collected from young children participating in a nutrition intervention trial in rural Malawi. Our findings support an inverse association between the diversity and maturity of the intestinal microbiota and biomarkers of EED. Additionally, we identified the differences at the level of individual bacterial taxa (groups of bacteria defined by genetic similarity) between participants with different levels of EED biomarkers. Due to the type of study, we cannot determine whether the observed associations represent a causal relationship between the intestinal microbiota and EED. This as well as the exact mechanisms behind these associations should be assessed in further studies.

Faecal regenerating 1B protein concentration is not associated with child growth in rural Malawi.

AIM: This study was designed to determine whether faecal regenerating 1B protein (REG1B) concentration is associated with physical growth among 6-30-month-old children in rural Malawi. METHODS: This was a secondary analysis from a randomised controlled trial in rural Malawi in which we followed-up 790 live-born infants from birth to 30 months of age. We collected anthropometric data at the age of 6, 12, 18, 24 and 30 months. We measured faecal REG1B concentration by enzyme-linked immunosorbent assay (ELISA) technique using stool samples collected at 6, 18 and 30 months of age. We assessed the association between faecal REG1B concentration and children's physical growth using linear regression and longitudinal data analysis. RESULTS: Of 790 live-born infants enrolled, 694 (87%) with at least one faecal REG1B concentration measurement were included in the analysis. Faecal REG1B concentration was not associated with the children's concurrent length-for-age z-score (LAZ), weight-for-age z-score (WAZ), weight-for-length z-score (WLZ) and mid-upper arm circumference-for-age z-score (MUACZ) at any time point (P > 0.05), nor with a change in their anthropometric indices in the subsequent 6-month period (P > 0.05). CONCLUSIONS: Faecal REG1B concentration is not associated with LAZ, WAZ, WLZ and MUACZ among 6-30-month-old infants and children in rural Malawi.

Pouch failures following restorative proctocolectomy in ulcerative colitis.

PURPOSE: Restorative proctocolectomy (RPC) is the most common operation in ulcerative colitis. Nevertheless, permanent ileostomy will sometimes be unavoidable. The aim was to evaluate the reasons for pouch failure and early morbidity after pouch excision. METHODS: The number and the reasons for pouch failures were analysed in patients undergoing RPC 1985-2016. RESULTS: Out of 491 RPC patients, 53 experienced pouch failure (10 women, 43 men); 52 out of 53 underwent pouch excision. The cumulative risk for excision at 5, 10 and 20 years was 5.6, 9.4 and 15.5%, respectively. The reasons for failure included septic events such as fistula in 12 (23%), chronic pouchitis in 11 (21%) and leakage in 8 (15%) patients. Functional reasons for pouch failure were recorded as poor function in 16 (30%), incontinence in 12 (23%) and stricture in 12 (23%) patients. Multiple causes for pouch failure were recorded for individual patients. Seven cases of Crohn's disease were found among the failure cases: two before pouch excision and five after. Altogether, 15 Crohn's disease diagnoses were set in the RPC cohort, giving a percentage of 47% of pouch failure in this disorder. A complication occurred in 23 (44%) patients within 30 days after surgery; 16 were mild (Clavien-Dindo grades I-II). CONCLUSIONS: Eleven percent of RPC patients suffered pouch failure: more men than women. The reasons were multiple. Crohn's disease created a risk of failure, but a half of these patients maintained the pouch. Morbidity after pouch excision was moderate, but in most cases slight.

Presence of Giardia lamblia in stools of six- to 18-month old asymptomatic Malawians is associated with children's growth failure.

AIM: Despite high pathogen burden and malnutrition in low-income settings, knowledge on relationship between asymptomatic viral or parasitic infections, nutrition and growth is insufficient. We studied these relationships in a cohort of six-month-old Malawian infants. METHODS: As part of a nutrient supplementation trial for 12 months, we documented disease symptoms of 840 participant daily and anthropometric measurements every three months. Stool specimens were collected every six months and analysed for Giardia lamblia, Cryptosporidium species and enterovirus, rotavirus, norovirus, parechovirus and rhinovirus using polymerase chain reaction (PCR). The prevalence of the microbes was compared to the children's linear growth and the dietary. RESULTS: The prevalence of the microbes was similar in every intervention group. All age groups combined, children negative for G. lamblia had a mean standard deviation (SD) of -0.01 (0.49) change in length-for-age Z-score (LAZ), compared to -0.12 (0.045) among G. lamblia positive children (difference -0.10, 95% CI -0.21 to -0.00, p = 0.047). The LAZ change difference was also statistically significant (p = 0.042) at age of 18-21 months but not at the other time points. CONCLUSION: Asymptomatic G. lamblia infection was mainly associated with growth reduction in certain three-month periods. The result refers to the chronic nature of G. lamblia infection.

Eukaryote specific folds: Part of the whole.

The origin of eukaryotes is one of the central transitions in the history of life; without eukaryotes there would be no complex multicellular life. The most accepted scenarios suggest the endosymbiosis of a mitochondrial ancestor with a complex archaeon, even though the details regarding the host and the triggering factors are still being discussed. Accordingly, phylogenetic analyses have demonstrated archaeal affiliations with key informational systems, while metabolic genes are often related to bacteria, mostly to the mitochondrial ancestor. Despite of this, there exists a large number of protein families and folds found only in eukaryotes. In this study, we have analyzed structural superfamilies and folds that probably appeared during eukaryogenesis. These folds typically represent relatively small binding domains of larger multidomain proteins. They are commonly involved in biological processes that are particularly complex in eukaryotes, such as signaling, trafficking/cytoskeleton, ubiquitination, transcription and RNA processing, but according to recent studies, these processes also have prokaryotic roots. Thus the folds originating from an eukaryotic stem seem to represent accessory parts that have contributed in the expansion of several prokaryotic processes to a new level of complexity. This might have taken place as a co-evolutionary process where increasing complexity and fold innovations have supported each other.

Lipid-based Nutrient Supplements Do Not Affect Gut Bifidobacterium Microbiota in Malawian Infants: A Randomized Trial.

OBJECTIVES: The aim of the study was to assess the effect of nutritional supplementation with lipid-based nutrient supplements (LNS) and corn-soy blend flour on Bifidobacterium and Staphylococcus aureus gut microbiota composition in Malawian infants. In addition, the microbiota changes over time were characterized in the study infants. METHODS: Healthy 6-month-old Malawian infants were randomly assigned to 1 of 4 intervention schemes for a 6-month period. Infants in the control group were not provided with any supplementary food. Infants in other 3 groups received either micronutrient-fortified corn-soy blend, micronutrient-fortified LNS with milk protein base, or micronutrient-fortified LNS with soy protein base between 6 and 12 months of age. Fecal bifidobacteria and S aureus gut microbiota at 6 and 12 months of age were analyzed by quantitative real-time polymerase chain reaction method. RESULTS: There was no difference in change in bacterial prevalence or counts between the intervention groups during the 6-month study period. When looking at the total study population, higher counts of total bacteria (P = 0.028), Bifidobacterium genus (P = 0.027), B catenulatum (P = 0.031), and lower counts of B infantis (P < 0.001), B lactis (P < 0.001), B longum (P < 0.001), and S aureus (P < 0.001) were detected in the children's stools at 12 months rather than at 6 months of age. CONCLUSIONS: The dietary supplementation did not have an effect on the Bifidobacterium and S aureus microbiota composition of the study infants. The fecal bifidobacterial diversity of the infants, however, changed toward a more adult-like microbiota profile within the observed time.

Pressurized Martian-Like Pure CO2 Atmosphere Supports Strong Growth of Cyanobacteria, and Causes Significant Changes in their Metabolism.

Surviving of crews during future missions to Mars will depend on reliable and adequate supplies of essential life support materials, i.e. oxygen, food, clean water, and fuel. The most economical and sustainable (and in long term, the only viable) way to provide these supplies on Martian bases is via bio-regenerative systems, by using local resources to drive oxygenic photosynthesis. Selected cyanobacteria, grown in adequately protective containment could serve as pioneer species to produce life sustaining substrates for higher organisms. The very high (95.3 %) CO2 content in Martian atmosphere would provide an abundant carbon source for photo-assimilation, but nitrogen would be a strongly limiting substrate for bio-assimilation in this environment, and would need to be supplemented by nitrogen fertilizing. The very high supply of carbon, with rate-limiting supply of nitrogen strongly affects the growth and the metabolic pathways of the photosynthetic organisms. Here we show that modified, Martian-like atmospheric composition (nearly 100 % CO2) under various low pressure conditions (starting from 50 mbar to maintain liquid water, up to 200 mbars) supports strong cellular growth. Under high CO2 / low N2 ratio the filamentous cyanobacteria produce significant amount of H2 during light due to differentiation of high amount of heterocysts.

Anal incontinence: long-term alterations in the incidence and healthcare usage.

OBJECTIVE: The aim of the study was to determine the alterations in the occurrence of incontinence and how subjects suffering from incontinence seek and receive healthcare services over a 10-year follow-up period. METHODS: Postal questionnaires (Wexner Incontinence Score, Fecal Incontinence Quality of Life Scale, a questionnaire to investigate the management of fecal incontinence and the frequency of urinary incontinence) were sent to subjects who had reported anal incontinence in our population-based study in 2003. For each incontinent person (n = 155) from the 2003 series, we identified two control subjects (n = 310) who did not suffer from incontinence. RESULTS: Of the initially incontinent, 47 (58%) had remained incontinent after a follow up of 10 years. Almost 80% of the incontinent subjects in 2012 were female. Of the 152 initially continent, 12 (7.9%) had developed symptoms, all of whom were females. Urinary incontinence was present in approximately 60% of incontinent subjects. The majority (57.8%) of the subjects still incontinent in 2012 felt that they needed help for the complaint, but only 30.9% had received any, and only 7.4% received any benefit. The most common treatment was medication. The subjective incontinence impaired the quality of life. CONCLUSION: Incontinence is a chronic long-lasting disorder. The current management of anal incontinence is not satisfactory. The primary healthcare system should be more aware of the nature of this condition to find and offer treatment for the patients.

Simultaneous biomass concentration and subsequent quantitation of multiple infectious disease agents and antimicrobial resistance genes from community wastewater.

Wastewater-based surveillance (WBS) of infectious disease agents is increasingly seen as a reliable source of population health data. To date, wastewater-based surveillance efforts have largely focused on individual pathogens. However, given that wastewater contains a broad range of pathogens circulating in the population, a more comprehensive approach could enhance its usability. We focused on the simultaneous detection of SARS-CoV-2, sapovirus, Campylobacter jejuni, Campylobacter coli, Salmonella spp., pathogenic Escherichia coli, Cryptosporidium spp., Giardia spp. and antimicrobial resistance genes (ARGs) of clinical relevance. To achieve this goal, biomass concentration and nucleic acid extraction methods were optimized, and samples were analyzed by using a set of (RT)-qPCR and (HT)-qPCR methods. We determined the prevalence and the spatial and temporal trends of the targeted pathogens and collected novel information on ARGs in Finnish wastewater. In addition, the use of different wastewater concentrates, namely the ultrafiltered concentrate of the supernatant and the centrifuged pellet, and the effect of freezing and thawing wastewater prior to sample processing were investigated with the indicator microbe crAssphage. Freeze-thawing of wastewater decreased the gene copy count of crAssphage in comparison to analyzing fresh samples (p < 0.001). Campylobacters were most abundant in two of the four studied summer months (30 % detection rate) and in wastewaters from regions with intensive animal farming. Salmonella, however, was detected in 40 % of the samples without any clear seasonal trends, and the highest gene copy numbers were recorded from the largest wastewater treatment plants. Beta-lactamase resistance genes that have commonly been detected in bacteria isolated from humans in Finland, namely blaCTX-M, blaOXA48, blaNDM, and blaKPC, were also frequently detected in wastewaters (100, 98, 98, and 70 % detection rates, respectively). These results confirm the reliability of using wastewater in public health surveillance and demonstrate the possibility to simultaneously perform WBS of multiple pathogens.

Comparison of Different Reverse Transcriptase-Polymerase Chain Reaction-Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study.

BACKGROUND: Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required. OBJECTIVE: This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings. METHODS: We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively. RESULTS: Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater. CONCLUSIONS: As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.

Parastomal Hernia Prevention Using Funnel-Shaped Intra-Abdominal Mesh Compared to No Mesh: The Chimney Randomized Clinical Trial.

Importance: Prophylactic placement of a mesh has been suggested to prevent parastomal hernia. Evidence to support this practice is contradictory. Objective: To determine whether funnel-shaped permanent synthetic parastomal mesh is effective and safe in parastomal hernia prevention. Design, Setting, and Participants: The Chimney Trial was a randomized single-blinded multicenter trial conducted in 4 hospitals in Finland and 1 in Sweden from February 2019 and September 2021. Of 439 patients with rectal adenocarcinoma undergoing either laparoscopic or robotic-assisted abdominoperineal resection or the Hartmann procedure, 143 were enrolled in the trial, 135 received their allocated intervention, and 121 were analyzed at 12-month follow-up. Data were analyzed from December 2023 to May 2024. Intervention: In the intervention group, a permanent colostomy was created with a funnel-shaped intraperitoneal mesh and compared to a control group with a stoma without the mesh. Main Outcome and Measure: The primary end point was the incidence of computed tomography (CT)-confirmed parastomal hernia 12 months after surgery. Results: There were 68 patients (mean [SD] age, 68.7 [11.6] years; 36 [53% male and 32 [47%] female) who received the intended allocation in the mesh group and 67 (mean [SD] age, 66.4 [11.7] years; 48 [72%] male and 19 [28%] female) who received the intended allocation in the control group. CT scans were available for 58 patients in the mesh group and 59 patients in the control group at the 12-month follow-up. CT scans confirmed parastomal hernia in 6 of 58 patients (10%) in the mesh group compared to 22 of 59 patients (37%) in the control group (difference, 27%; 95% CI, 12-41; P < .001). Clinical parastomal hernia as a secondary outcome was recorded in 1 of 60 patients (2%) in the mesh group compared to 27 of 61 (43%) in the control group (difference, 41%; 95% CI, 29-55; P < .001). The number of patients with Clavien-Dindo class II ileus was 23 (35%) in the mesh group compared to 11 (17%) in the control group (difference, 18%; 95% CI, 3-32; P = .006). Only slight differences between the groups were detected in other stoma-related complications, readmissions, operative time, surgical site infections, reoperations, and quality of life. Conclusions and Relevance: In this study, funnel-shaped parastomal mesh prevented a significant number of parastomal hernias without predisposing patients to mesh- or stoma-related complications during 12-month follow-up. The results of this study suggest the funnel-shaped mesh is a feasible option to prevent parastomal hernia. Trial Registration: ClinicalTrials.gov Identifier: NCT03799939.

Wastewater-based surveillance is an efficient monitoring tool for tracking influenza A in the community.

Around the world, influenza A virus has caused severe pandemics, and the risk of future pandemics remains high. Currently, influenza A virus surveillance is based on the clinical diagnosis and reporting of disease cases. In this study, we apply wastewater-based surveillance to monitor the amount of the influenza A virus RNA at the population level. We report the influenza A virus RNA levels in 10 wastewater treatment plant catchment areas covering 40 % of the Finnish population. Altogether, 251 monthly composite influent wastewater samples (collected between February 2021 and February 2023) were analysed from supernatant fraction using influenza A virus specific RT-qPCR method. During the study period, an influenza A virus epidemic occurred in three waves in Finland. This study shows that the influenza A virus RNA can be detected from the supernatant fraction of 24 h composite influent wastewater samples. The influenza A virus RNA gene copy number in wastewater correlated with the number of confirmed disease cases in the Finnish National Infectious Diseases Register. The median Kendall's τ correlation strength was 0.636 (min= 0.486 and max=0.804) and it was statistically significant in all 10 WTTPs. Wastewater-based surveillance of the influenza A virus RNA is an independent from individual testing method and cost-efficiently reflects the circulation of the virus in the entire population. Thus, wastewater monitoring complements the available, but often too sparse, information from individual testing and improves health care and public health preparedness for influenza A virus pandemics.

Clinically relevant sequence types of carbapenemase-producing Escherichia coli and Klebsiella pneumoniae detected in Finnish wastewater in 2021-2022.

BACKGROUND: Antimicrobial resistance (AMR) is a critical threat to human health. Escherichia coli and Klebsiella pneumoniae are clinically the most important species associated with AMR and are the most common carbapenemase-producing (CP) Enterobacterales detected in human specimens in Finland. Wastewater surveillance has emerged as a potential approach for population-level surveillance of AMR, as wastewater could offer a reflection from a larger population with one sample and minimal recognized ethical issues. In this study, we investigated the potential of wastewater surveillance to detect CP E. coli and K. pneumoniae strains similar to those detected in human specimens. METHODS: Altogether, 89 composite samples of untreated community wastewater were collected from 10 wastewater treatment plants across Finland in 2021-2022. CP E. coli and K. pneumoniae were isolated using selective culture media and identified using MALDI-TOF MS. Antimicrobial susceptibility testing was performed using disk diffusion test and broth microdilution method, and a subset of isolates was characterized using whole-genome sequencing. RESULTS: CP E. coli was detected in 26 (29.2%) and K. pneumoniae in 25 (28.1%) samples. Among E. coli, the most common sequence type (ST) was ST410 (n = 7/26, 26.9%), while ST359 (n = 4/25, 16.0%) predominated among K. pneumoniae. Globally successful STs were detected in both E. coli (ST410, ST1284, ST167, and ST405) and K. pneumoniae (ST512, ST101, and ST307). K. pneumoniae carbapenemases (KPC) were the most common carbapenemases in both E. coli (n = 11/26, 42.3%) and K. pneumoniae (n = 13/25, 52.0%), yet also other carbapenemases, such as blaNDM-5, blaOXA-48, and blaOXA-181, were detected. We detected isolates harboring similar ST and enzyme type combinations previously linked to clusters in Finland, such as E. coli ST410 with blaKPC-2 and K. pneumoniae ST512 with blaKPC-3. CONCLUSIONS: Our study highlights the presence of clinically relevant strains of CP E. coli and K. pneumoniae in community wastewater. The results indicate that wastewater surveillance could serve as a monitoring tool for CP Enterobacterales. However, the specificity and sensitivity of the methods should be improved, and technologies, like advanced sequencing methods, should be utilized to distinguish data with public health relevance, harness the full potential of wastewater surveillance, and implement the data in public health surveillance.

Detection of SARS-COV-2 variants and their proportions in wastewater samples using next-generation sequencing in Finland.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants may have different characteristics, e.g., in transmission, mortality, and the effectiveness of vaccines, indicating the importance of variant detection at the population level. Wastewater-based surveillance of SARS-CoV-2 RNA fragments has been shown to be an effective way to monitor the COVID-19 pandemic at the population level. Wastewater is a complex sample matrix affected by environmental factors and PCR inhibitors, causing insufficient coverage in sequencing, for example. Subsequently, results where part of the genome does not have sufficient coverage are not uncommon. To identify variants and their proportions in wastewater over time, we utilized next-generation sequencing with the ARTIC Network's primer set and bioinformatics pipeline to evaluate the presence of variants in partial genome data. Based on the wastewater data from November 2021 to February 2022, the Delta variant was dominant until mid-December in Helsinki, Finland's capital, and thereafter in late December 2022 Omicron became the most common variant. At the same time, the Omicron variant of SARS-CoV-2 outcompeted the previous Delta variant in Finland in new COVID-19 cases. The SARS-CoV-2 variant findings from wastewater are in agreement with the variant information obtained from the patient samples when visually comparing trends in the sewerage network area. This indicates that the sequencing of wastewater is an effective way to monitor temporal and spatial trends of SARS-CoV-2 variants at the population level.

Developing wastewater-based surveillance schemes for multiple pathogens: The WastPan project in Finland.

Wastewater comprises multiple pathogens and offers a potential for wastewater-based surveillance (WBS) to track the prevalence of communicable diseases. The Finnish WastPan project aimed to establish wastewater-based pandemic preparedness for multiple pathogens (viruses, bacteria, parasites, fungi), including antimicrobial resistance (AMR). This article outlines WastPan's experiences in this project, including the criteria for target selection, sampling locations, frequency, analysis methods, and results communication. Target selection relied on epidemiological and microbiological evidence and practical feasibility. Within the WastPan framework, wastewater samples were collected between 2021 and 2023 from 10 wastewater treatment plants (WWTPs) covering 40 % of Finland's population. WWTP selection was validated for reported cases of Extended Spectrum Beta-lactamase-producing bacterial pathogens (Escherichia coli and Klebsiella pneumoniae) from the National Infectious Disease Register. The workflow included 24-h composite influent samples, with one fraction for culture-based analysis (bacteria and fungi) and the rest of the sample was reserved for molecular analysis (viruses, bacteria, antibiotic resistance genes, and parasites). The reproducibility of the monitoring workflow was assessed for SARS-CoV-2 through inter-laboratory comparisons using the N2 and N1 assays. Identical protocols were applied to same-day samples, yielding similar positivity trends in the two laboratories, but the N2 assay achieved a significantly higher detection rate (Laboratory 1: 91.5 %; Laboratory 2: 87.4 %) than the N1 assay (76.6 %) monitored only in Laboratory 2 (McNemar, p < 0.001 Lab 1, = 0.006 Lab 2). This result indicates that the selection of monitoring primers and assays may impact monitoring sensitivity in WBS. Overall, the current study recommends that the selection of sampling frequencies and population coverage of the monitoring should be based on pathogen-specific epidemiological characteristics. For example, pathogens that are stable over time may need less frequent annual sampling, while those that are occurring across regions may require reduced sample coverage. Here, WastPan successfully piloted WBS for monitoring multiple pathogens, highlighting the significance of one-litre community composite wastewater samples for assessing community health. The infrastructure established for COVID-19 WBS is valuable for monitoring various pathogens. The prioritization of the monitoring targets optimizes resource utilization. In the future legislative support in target selection, coverage determination, and sustained funding for WBS is recomended.

Organ-specific alterations in tobacco transcriptome caused by the PVX-derived P25 silencing suppressor transgene.

BACKGROUND: RNA silencing affects a broad range of regulatory processes in all eukaryotes ranging from chromatin structure maintenance to transcriptional and translational regulation and longevity of the mRNAs. Particularly in plants, it functions as the major defense mechanism against viruses. To counter-act this defense, plant viruses produce suppressors of RNA silencing (Viral suppressors of RNA silencing, VSRSs), which are essential for viruses to invade their specific host plants. Interactions of these VSRSs with the hosts' silencing pathways, and their direct and indirect interference with different cellular regulatory networks constitute one of the main lines of the molecular virus-host interactions. Here we have used a microarray approach to study the effects of the Potato virus X Potexvirus (PVX)-specific P25 VSRS protein on the transcript profile of tobacco plants, when expressed as a transgene in these plants. RESULTS: The expression of the PVX-specific P25 silencing suppressor in transgenic tobacco plants caused significant up-regulation of 1350 transcripts, but down-regulation of only five transcripts in the leaves, and up- and down-regulation of 51 and 13 transcripts, respectively, in the flowers of these plants, as compared to the wild type control plants. Most of the changes occurred in the transcripts related to biotic and abiotic stresses, transcription regulation, signaling, metabolic pathways and cell wall modifications, and many of them appeared to be induced through up-regulation of the signaling pathways regulated by ethylene, jasmonic acid and salicylic acid. Correlations of these alterations with the protein profile and related biological functions were analyzed. Surprisingly, they did not cause significant alterations in the protein profile, and caused only very mild alteration in the phenotype of the P25-expressing transgenic plants. CONCLUSION: Expression of the PVX-specific P25 VSRS protein causes major alterations in the transcriptome of the leaves of transgenic tobacco plants, but very little of any effects in the young flowers of the same plants. The fairly stable protein profile in the leaves and lack of any major changes in the plant phenotype indicate that the complicated interplay and interactions between different regulatory levels are able to maintain homeostasis in the plants.

Seven-year follow-up after anterior sphincter reconstruction for faecal incontinence.

PURPOSE: The aim was to evaluate the effects of anterior sphincter repair on faecal incontinence and quality of life. PATIENTS AND METHODS: Fifty-six patients who underwent anterior anal sphincteroplasty between January 2003 and December 2005 were asked to complete questionnaires containing the Wexner Incontinence Score (a score of 0 corresponds to full continence and 20 to total incontinence) and Faecal Incontinence Quality of Life Scale preoperatively, in May 2006 (mean follow-up time, 22.8 months) and in August 2011 (mean follow-up time, 89.3 months). Thirty-nine (69.6 %) patients completed the questionnaires before the operation and in 2006, and 36 (64.3 %) in 2011. RESULTS: The overall severity of faecal incontinence improved in 26 patients (67 %), and quality of life improved in 2006 as a whole, but after a longer follow-up (in 2011), the severity of faecal incontinence was about the same as preoperatively (median, 12.0 months) in all the patients. Among younger patients (≤50 years), the situation was better, but older patients (>50 years) had an even worse situation than before the operation. In the group of younger patients, the preoperative median of the overall incontinence score was 10.5, and in 2011, it was 9.0, while in the group of older patients, the corresponding numbers were 13.0 and 15.0. In 2011, quality of life was still better than preoperatively as a whole, but the results had deteriorated from those in 2006. CONCLUSIONS: Initially, both overall faecal incontinence and quality of life improved, but younger patients achieved a greater benefit. However, the results deteriorated with a longer follow-up. Operative management should be considered preferably in relatively young subjects as their results are better.

Genomic epidemiology of nosocomial carbapenemase-producing Citrobacter freundii in sewerage systems in the Helsinki metropolitan area, Finland.

Multi-drug resistance is emerging in Citrobacter freundii, which is the third most common carbapenemase-producing (CP) Enterobacteriaceae in humans in Finland due to recent outbreaks. The objective of this study was to determine if wastewater surveillance (WWS) could detect CP C. freundii strains causing infections in humans. Selective culturing was used to isolate CP C. freundii from the hospital environment, hospital wastewater, and untreated municipal wastewater in Helsinki, Finland, between 2019 and 2022. Species were identified using MALDI-TOF, and presumptive CP C. freundii isolates were subjected to antimicrobial susceptibility testing and further characterized by whole genome sequencing. A genomic comparison was conducted to compare isolates collected from the hospital environment, untreated municipal wastewater, and a selection of isolates from human specimens from two hospitals in the same city. We also examined the persistence of CP C. freundii in the hospital environment and the impact of our attempts to eradicate it. Overall, 27 blaKPC - 2-carrying C. freundii were detected in the hospital environment (ST18; n = 23 and ST8; n = 4), while 13 blaKPC - 2-carrying C. freundii (ST8) and five blaVIM - 1-carrying (ST421) C. freundii were identified in untreated municipal wastewater. CP C. freundii was not identified in hospital wastewater. We found three clusters (cluster distance threshold ≤ 10 allelic difference) after comparing the recovered isolates and a selection of isolates from human specimens. The first cluster consisted of ST18 isolates from the hospital environment (n = 23) and human specimens (n = 4), the second consisted of ST8 isolates from the hospital environment (n = 4), untreated municipal wastewater (n = 6), and human specimens (n = 2), and the third consisted of ST421 isolates from the untreated municipal wastewater (n = 5). Our results support previous studies suggesting that the hospital environment could act as a source of transmission of CP C. freundii in clinical settings. Furthermore, the eradication of CP Enterobacteriaceae from the hospital environment is challenging. Our findings also showed that CP C. freundii is persistent throughout the sewerage system and demonstrate the potential of WWS for detecting CP C. freundii.