Is oncolytic adenoviral-mediated immunotherapy through p53-overexpression the solution to refractory pancreatic ductal adenocarcinoma?

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with poor prognosis. PDAC has a dense, desmoplastic stroma and immunosuppressive microenvironment, which impedes current treatment options. Immunotherapy delivered via oncolytic virotherapy is one potential solution to these barriers. Immune checkpoint inhibitors may facilitate immunogenic cell death by improving immune cell infiltration in cancer cells. PD-1 blockade shows better clinical outcomes for certain cancers. The addition of p53 to stimulate cell cycle arrest remains a novel field of research. The evaluated article by Araki et al. explores the efficacy of PD-1 blockade with oncolytic adenovirus platforms on immunogenic cell death and the possibility of combining PD-1 blockade and p53-activation. In vitroanalysis showed increased cell death in multiple cell lines infected with AdV mediating p53 expression. The underlying process may attribute to apoptosis and autophagy, with evidence of increased immunogenic cell death.In vivomodels demonstrated improved efficacy of p53-expressing AdV, particularly with the addition of PD-1 blockade which appears to be related to CD8+ cell infiltration.

CUREs for high-level Galectin-3 expression.

Galectins are a large and diverse protein family defined by the presence of a carbohydrate recognition domain (CRD) that binds ß-galactosides. They play important roles in early development, tissue regeneration, immune homeostasis, pathogen recognition, and cancer. In many cases, studies that examine galectin biology and the effect of manipulating galectins are aided by, or require the ability to express and purify, specific members of the galectin family. In many cases, E. coli is employed as a heterologous expression system, and galectin expression is induced with isopropyl ß-galactoside (IPTG). Here, we show that galectin-3 recognizes IPTG with micromolar affinity and that as IPTG induces expression, newly synthesized galectin can bind and sequester cytosolic IPTG, potentially repressing further expression. To circumvent this putative inhibitory feedback loop, we utilized an autoinduction protocol that lacks IPTG, leading to significantly increased yields of galectin-3. Much of this work was done within the context of a course-based undergraduate research experience, indicating the ease and reproducibility of the resulting expression and purification protocols.

Machine learning designs new GCGR/GLP-1R dual agonists with enhanced biological potency.

Several peptide dual agonists of the human glucagon receptor (GCGR) and the glucagon-like peptide-1 receptor (GLP-1R) are in development for the treatment of type 2 diabetes, obesity and their associated complications. Candidates must have high potency at both receptors, but it is unclear whether the limited experimental data available can be used to train models that accurately predict the activity at both receptors of new peptide variants. Here we use peptide sequence data labelled with in vitro potency at human GCGR and GLP-1R to train several models, including a deep multi-task neural-network model using multiple loss optimization. Model-guided sequence optimization was used to design three groups of peptide variants, with distinct ranges of predicted dual activity. We found that three of the model-designed sequences are potent dual agonists with superior biological activity. With our designs we were able to achieve up to sevenfold potency improvement at both receptors simultaneously compared to the best dual-agonist in the training set.

AmiA and AliA peptide ligands, found in Klebsiella pneumoniae, are imported into pneumococci and alter the transcriptome.

Klebsiella pneumoniae releases the peptides AKTIKITQTR and FNEMQPIVDRQ, which bind the pneumococcal proteins AmiA and AliA respectively, two substrate-binding proteins of the ABC transporter Ami-AliA/AliB oligopeptide permease. Exposure to these peptides alters pneumococcal phenotypes such as growth. Using a mutant in which a permease domain of the transporter was disrupted, by growth analysis and epifluorescence microscopy, we confirmed peptide uptake via the Ami permease and intracellular location in the pneumococcus. By RNA-sequencing we found that the peptides modulated expression of genes involved in metabolism, as pathways affected were mostly associated with energy or synthesis and transport of amino acids. Both peptides downregulated expression of genes involved in branched-chain amino acid metabolism and the Ami permease; and upregulated fatty acid biosynthesis genes but differed in their regulation of genes involved in purine and pyrimidine biosynthesis. The transcriptomic changes are consistent with growth suppression by peptide treatment. The peptides inhibited growth of pneumococcal isolates of serotypes 3, 8, 9N, 12F and 19A, currently prevalent in Switzerland, and caused no detectable toxic effect to primary human airway epithelial cells. We conclude that pneumococci take up K. pneumoniae peptides from the environment via binding and transport through the Ami permease. This changes gene expression resulting in altered phenotypes, particularly reduced growth.

Estimation of diagnostic sensitivity and specificity of abattoir registrations and bulk tank milk ELISA as herd-level tests for Fasciola hepatica using Bayesian latent class modelling.

The common liver fluke, Fasciola hepatica, is a trematode parasite found worldwide, typically with a focal distribution due to its requirement for suitable climatic and environmental conditions to complete its lifecycle. Bovine fasciolosis causes suboptimal production and economic losses, including liver condemnation at slaughter. The lack of reliable diagnostic methods is a disadvantage to the increasing demand for surveillance and control. The aim of this study was to evaluate the diagnostic accuracy of bulk tank milk (BTM) antibody testing and aggregated abattoir registrations (AAR) of liver fluke as herd-level tests for F. hepatica infection using Bayesian latent class models. Data from the abattoirs in 2019-2021 and BTM, sampled in the winter of 2020/2021, were collected from 437 herds on the southwest coast of Norway. The BTM samples were analysed with the SVANOVIR® F. hepatica-Ab ELISA test, with results given as an optical density ratio (ODR), and later dichotomized using the recommended cut-off value from the test manufacturer (ODR ≥0.3). Based on the BTM ELISA test, 47.8% of the herds tested positive. The AAR test was defined as the herd-level proportion of female slaughtered animals registered with liver fluke infection during the study period. For this test, three cut-offs were used (a proportion of 0.05, 0.1 and 0.2). The herds were split into two subpopulations ("Coastal" and "Inland"), which were expected to differ in true prevalence of F. hepatica infection based on climate-related and geographical factors. The diagnostic accuracies of both tests were estimated using Bayesian latent class models with minimally informative priors. Post-hoc analysis revealed that the maximum sum of sensitivity (Se) and specificity (Sp) of the tests was achieved with a herd-level proportion of ≥0.1 registered with liver fluke as the AAR test. Using this cut-off, the median estimate for the diagnostic accuracy of the BTM ELISA was 90.4% (84.0-96.2 95% Posterior Credible Interval (PCI)) for Se and 95.3% (90.6-100% PCI) for Sp, while the median estimate of Se for AAR was 87.5% (81.4-93.1% PCI) and the median estimate of Sp for AAR was 91.0% (85.2-96.5% PCI). The cut-off evaluation of the SVANOVIR® F. hepatica-Ab ELISA test for BTM confirmed the manufacturer's recommended cut-off of ODR ≥0.3 to denote positive and negative herds. This study suggests that AAR and BTM ELISA test can be used as herd-level tools to monitor liver fluke infection, so that appropriate interventions against infection can be implemented as necessary.

Children and young people's body mass index measures derived from routine data sources: A national data linkage study in Wales.

BACKGROUND: Routine monitoring of Body Mass Index (BMI) in general practice, and via national surveillance programmes, is essential for the identification, prevention, and management of unhealthy childhood weight. We examined and compared the presence and representativeness of children and young people's (CYPs) BMI recorded in two routinely collected administrative datasets: general practice electronic health records (GP-BMI) and the Child Measurement Programme for Wales (CMP-BMI), which measures height and weight in 4-5-year-old school children. We also assessed the feasibility of combining GP-BMI and CMP-BMI data for longitudinal analyses. METHODS: We accessed de-identified population-level GP-BMI data for calendar years 2011 to 2019 for 246,817 CYP, and CMP-BMI measures for 222,772 CYP, held within the Secure Anonymised Information Linkage Databank. We examined the proportion of CYP in Wales with at least one GP-BMI record, its distribution by child socio-demographic characteristics, and trends over time. We compared GP-BMI and CMP-BMI distributions. We quantified the proportion of children with a CMP-BMI measure and a follow-up GP-BMI recorded at an older age and explored the representativeness of these measures. RESULTS: We identified a GP-BMI record in 246,817 (41%) CYP, present in a higher proportion of females (54.2%), infants (20.7%) and adolescents. There was no difference in the deprivation profile of those with a GP-BMI measurement. 31,521 CYP with a CMP-BMI had at least one follow-up GP-BMI; those with a CMP-BMI considered underweight or very overweight were 87% and 70% more likely to have at least one follow-up GP-BMI record respectively compared to those with a healthy weight, as were males and CYP living in the most deprived areas of Wales. CONCLUSIONS: Records of childhood weight status extracted from general practice are not representative of the population and are biased with respect to weight status. Linkage of information from the national programme to GP records has the potential to enhance discussions around healthy weight at the point of care but does not provide a representative estimate of population level weight trajectories, essential to provide insights into factors determining a healthy weight gain across the early life course. A second CMP measurement is required in Wales.

A Novel Macrophage Subpopulation Conveys Increased Genetic Risk of Coronary Artery Disease.

BACKGROUND: Coronary artery disease (CAD), the leading cause of death worldwide, is influenced by both environmental and genetic factors. Although over 250 genetic risk loci have been identified through genome-wide association studies, the specific causal variants and their regulatory mechanisms are still largely unknown, particularly in disease-relevant cell types like macrophages. METHODS: We utilized single-cell RNA-seq and single-cell multiomics approaches in primary human monocyte-derived macrophages to explore the transcriptional regulatory network involved in a critical pathogenic event of coronary atherosclerosis-the formation of lipid-laden foam cells. The relative genetic contribution to CAD was assessed by partitioning disease heritability across different macrophage subpopulations. Meta-analysis of single-cell RNA-seq data sets from 38 human atherosclerotic samples was conducted to provide high-resolution cross-referencing to macrophage subpopulations in vivo. RESULTS: We identified 18 782 cis-regulatory elements by jointly profiling the gene expression and chromatin accessibility of >5000 macrophages. Integration with CAD genome-wide association study data prioritized 121 CAD-related genetic variants and 56 candidate causal genes. We showed that CAD heritability was not uniformly distributed and was particularly enriched in the gene programs of a novel CD52-hi lipid-handling macrophage subpopulation. These CD52-hi macrophages displayed significantly less lipoprotein accumulation and were also found in human atherosclerotic plaques. We investigated the cis-regulatory effect of a risk variant rs10488763 on FDX1, implicating the recruitment of AP-1 and C/EBP-ß in the causal mechanisms at this locus. CONCLUSIONS: Our results provide genetic evidence of the divergent roles of macrophage subsets in atherogenesis and highlight lipid-handling macrophages as a key subpopulation through which genetic variants operate to influence disease. These findings provide an unbiased framework for functional fine-mapping of genome-wide association study results using single-cell multiomics and offer new insights into the genotype-environment interactions underlying atherosclerotic disease.

Klebsiella pneumoniae peptide hijacks a Streptococcus pneumoniae permease to subvert pneumococcal growth and colonization.

Treatment of pneumococcal infections is limited by antibiotic resistance and exacerbation of disease by bacterial lysis releasing pneumolysin toxin and other inflammatory factors. We identified a previously uncharacterized peptide in the Klebsiella pneumoniae secretome, which enters Streptococcus pneumoniae via its AmiA-AliA/AliB permease. Subsequent downregulation of genes for amino acid biosynthesis and peptide uptake was associated with reduction of pneumococcal growth in defined medium and human cerebrospinal fluid, irregular cell shape, decreased chain length and decreased genetic transformation. The bacteriostatic effect was specific to S. pneumoniae and Streptococcus pseudopneumoniae with no effect on Streptococcus mitis, Haemophilus influenzae, Staphylococcus aureus or K. pneumoniae. Peptide sequence and length were crucial to growth suppression. The peptide reduced pneumococcal adherence to primary human airway epithelial cell cultures and colonization of rat nasopharynx, without toxicity. We identified a peptide with potential as a therapeutic for pneumococcal diseases suppressing growth of multiple clinical isolates, including antibiotic resistant strains, while avoiding bacterial lysis and dysbiosis.

Towards an actionable One Health approach.

BACKGROUND: Despite the increasing focus on strengthening One Health capacity building on global level, challenges remain in devising and implementing real-world interventions particularly in the Asia-Pacific region. Recognizing these gaps, the One Health Action Commission (OHAC) was established as an academic community for One Health action with an emphasis on research agenda setting to identify actions for highest impact. MAIN TEXT: This viewpoint describes the agenda of, and motivation for, the recently formed OHAC. Recognizing the urgent need for evidence to support the formulation of necessary action plans, OHAC advocates the adoption of both bottom-up and top-down approaches to identify the current gaps in combating zoonoses, antimicrobial resistance, addressing food safety, and to enhance capacity building for context-sensitive One Health implementation. CONCLUSIONS: By promoting broader engagement and connection of multidisciplinary stakeholders, OHAC envisions a collaborative global platform for the generation of innovative One Health knowledge, distilled practical experience and actionable policy advice, guided by strong ethical principles of One Health.

Shame, guilt, and drinking motives as mediators between child maltreatment and problematic alcohol use in college students.

Objective: Drinking for emotion regulation may be a concern for college students who have experienced childhood maltreatment, due to high levels of shame and guilt. The present cross-sectional survey study tested how trait shame-proneness, trait guilt-proneness, and trauma-related guilt are differently related to drinking motives and how these pathways mediate the links between maltreatment severity and alcohol outcomes. Participants: Undergraduate student drinkers (n = 464; M age = 19.50, SD = 2.20) from a midsized midwestern University. Methods: Participants completed an online survey inquiring about demographics, childhood maltreatment, shame, guilt, drinking motives, alcohol use, and alcohol-related consequences. Results: There were several significant serial indirect effects of maltreatment on alcohol consumption and related consequences, through trauma-related guilt, shame-proneness, guilt-proneness, drinking-to-cope, and drinking for mood enhancement. Conclusions: On college campuses, to address problematic drinking among childhood maltreatment survivors, interventions may target maladaptive feelings of shame and guilt stemming from trauma exposure.

Characterization of hOAT4 and mOat5 as functional orthologs for renal anion uptake and efflux transport.

Organic anions (OA) are compounds including drugs or toxicants that are negatively charged at physiological pH and are typically transported by Organic Anion Transporters (OATs). Human OAT4 (SLC22A11) is expressed in the apical membrane of renal proximal tubules. Although there is no rodent ortholog of hOAT4, rodents express Oat5 (Slc22a19), an anion exchanger that is also localized to the apical membrane of renal proximal tubule cells. The purpose of this study was to determine the functional similarity between mouse Oat5 and human OAT4. Chinese hamster ovary (CHO) cells expressing SLC22A11 or Slc22a19 were used to assess the transport characteristics of radiolabeled ochratoxin (OTA). We determined the kinetics of OTA transport; the resulting Kt and Jmax values were very similar for both hOAT4 and mOat5: Kt 3.9 and 7.2 µM, respectively, & Jmax 4.4 and 3.9 pmol/cm2, respectively. For the profile of OTA inhibition by OAs, IC50 values were determined for several clinically important drugs and toxicants. The resulting IC50 values ranged from 9 µM for indomethacin to ~600 µM for the diuretic hydrochlorothiazide. We measured the efflux of OTA from preloaded cells; both hOAT4 and mOat5 supported the efflux of OTA. These data support the hypothesis that OAT4 and Oat5 are functional orthologs and share selectivity for OTA both for reabsorption and secretion. Significance Statement This study compares the selectivity profile between human OAT4 and mouse Oat5. Our data revealed a similar selectivity profile for OTA reabsorption and secretion by these two transporters, thereby supporting the hypothesis that hOAT4 and mOat5, while not genetic orthologs, behave as functional orthologs for both uptake and efflux. These data will be instrumental in selecting an appropriate animal model when studying the renal disposition of anionic drugs and toxicants.

Perceived worsening of obsessive-compulsive disorder symptoms after childbirth in women and men: An understudied phenomenon.

The aim of this study was to examine worsening of OCD symptoms after childbirth in individuals seeking assessment or treatment of OCD. The postpartum period may make parents biologically and psychologically vulnerable to OCD symptoms. Participants included 222 parents with OCD who completed surveys through a self-help website. Most women and almost half of men with self-reported OCD reported an increase in OCD symptoms following childbirth. Retrospective report of perceived worsening of OCD symptoms after childbirth was associated with more aggressive obsessions for both men and women, in comparison to individuals whose OCD symptoms did not worsen around childbirth. Women whose OCD symptoms worsened after childbirth reported more impairment in social functioning than individuals whose symptoms did not worsen. These results highlight the need to develop a better understanding of aggressive obsessions in parents, and improve education about prevalence, content, assessment, and intervention for aggression-focused intrusive thoughts.

Children of trauma survivors: Influences of parental posttraumatic stress and child-perceived parenting.

BACKGROUND: Research has established a negative association between parental posttraumatic stress symptoms (PTSS), including subthreshold symptoms, and child physical and behavioral health outcomes. Such intergenerational transmission of risk has multiple possible mechanisms, including lack of positive parenting, increased negative parenting, shared environmental and contextual risks, and potential biological components such as shared genetics or even transmission of epigenetic risk. METHOD: This study examined 93 parent-child dyads (n = 171 participants total) from a mixed Urban-Suburban US metropolitan area to investigate the relations between parental PTSS and child-perceived parenting and child PTSS. We sought to examine interactions between parental PTSS and parenting on child PTSS. RESULTS: We found an association between parent and child PTSS, consistent with prior literature showing increased risk for children of trauma survivors. Interestingly, we found effects of positive parenting on diminished child PTSS symptoms only in parents without PTSS, whereas the effect of positive parenting on buffering child symptoms was absent in parents with PTSS. LIMITATIONS: The present findings are tempered by the use of self-report data to assess parent and child PTSS, which is not as reliable as clinician assessment of symptoms. Further, the use of survey data limits what is known about the extent of trauma exposure in parents and children, and different measures were used to assess PTSS in parents and kids, which limits comparability of these reported symptoms. DISCUSSION: Limitations notwithstanding, findings suggest joint attention paid to parenting practices and to a parent's recovery, even from subthreshold symptoms of PTSS, as two different but important ways to support trauma survivor parents in their efforts to most optimally parent and protect their children from intergenerational risk.

Programming sp3 Quantum Defects along Carbon Nanotubes with Halogenated DNA.

Atomic defect color centers in solid-state systems hold immense potential to advance various quantum technologies. However, the fabrication of high-quality, densely packed defects presents a significant challenge. Herein we introduce a DNA-programmable photochemical approach for creating organic color-center quantum defects on semiconducting single-walled carbon nanotubes (SWCNTs). Key to this precision defect chemistry is the strategic substitution of thymine with halogenated uracil in DNA strands that are orderly wrapped around the nanotube. Photochemical activation of the reactive uracil initiates the formation of sp3 defects along the nanotube as deep exciton traps, with a pronounced photoluminescence shift from the nanotube band gap emission (by 191 meV for (6,5)-SWCNTs). Furthermore, by altering the DNA spacers, we achieve systematic control over the defect placements along the nanotube. This method, bridging advanced molecular chemistry with quantum materials science, marks a crucial step in crafting quantum defects for critical applications in quantum information science, imaging, and sensing.

Considerable variation in current coronoid height and fracture measurement techniques: a systematic review.

BACKGROUND: Coronoid fractures usually occur in the presence of a significant osseoligamentous injury to the elbow. Fracture size and location correlate with degree of instability and many authors have attempted to analyze the effect of fracture variation on decision making and outcome. There remains no standardized technique for measuring coronoid height or fracture size. The aim of this study was to appraise the literature regarding techniques for coronoid height measurement in order to understand variation. METHODS: Preferred Reporting Items of Systematic Reviews and Meta-Analyses guidelines were followed. A search was performed to identify studies with either a description of coronoid height, fracture size, or bone loss using the terms (Coronoid) AND (Measurement) OR (Size) OR (Height). Articles were shortlisted by screening for topic relevance based on title, abstract and, if required, full-text review. Exclusion criteria were non-English articles, those on nonhuman species or parts other than the ulna coronoid process, and studies that included patients with pre-existing elbow pathology. Shortlisted articles were grouped based on study type, imaging modality, measurement technique, and measurement parameter as well as its location along the coronoid. RESULTS: Thirty out of the initially identified 494 articles met the inclusion criteria. Twenty-one articles were clinical studies, 8 were cadaveric studies, and 1 combined patients as well as cadavers. A variety of imaging modalities (plain radiographs, 2-dimensional computed tomography [CT], 3-dimensional CT, magnetic resonance imaging or a combination of these) were used with CT scan (either 2-dimensional images or 3-dimensional reconstructions or both) being the most common modality used by 21 studies. Measurement technique also varied from uniplanar linear measurements in 15 studies to multiplanar area and volumetric measurements in 6 studies to techniques describing various angles and indices as an indirect measure of coronoid height in 8 studies. Across the 30 shortlisted studies, 19 different measurement techniques were identified. Fifteen studies measured normal coronoid height while the other 15 measured intact coronoid and/or fracture fragment height. The location of this measurement was also variable between studies with measurements at the apex of the coronoid in 24/30 (80%) of studies. Measurement accuracy was assessed by only 1 study. A total of 12/30 (40%) studies reported on the interobserver and intraobserver reliability of their measurement technique. CONCLUSION: The systemic review demonstrated considerable variability between studies that report coronoid height or fracture size measurements. This variability makes comparison of coronoid height or fracture measurements and recommendations based on these between studies unreliable. There is need for development of a consistent, easy to use, and reproducible technique for coronoid height and bone loss.

Glycine supplementation can partially restore oxidative stress-associated glutathione deficiency in ageing cats.

Intracellular levels of glutathione, the major mammalian antioxidant, are reported to decline with age in several species. To understand whether ageing affects circulating glutathione levels in cats, blood was sampled from two age groups, < 3 years and > 9 years. Further, to determine whether dietary supplementation with glutathione precursor glycine (GLY) affects glutathione concentrations in senior cats (> 8 years), a series of free GLY inclusion level dry diets were fed. Subsequently, a 16-week GLY feeding study was conducted in senior cats (> 7 years), measuring glutathione, and markers of oxidative stress. Whole blood and erythrocyte total, oxidised and reduced glutathione levels were significantly decreased in senior cats, compared with their younger counterparts (P ≤ 0·02). The inclusion level study identified 1·5 % free GLY for the subsequent dry diet feeding study. Significant increases in erythrocyte total and reduced glutathione were observed between senior cats fed supplemented and control diets at 4 weeks (P ≤ 0·03; maximum difference of 1·23 µM). Oxidative stress markers were also significantly different between groups at 8 (P = 0·004; difference of 0·68 nG/ml in 8-hydroxy-2'-deoxyguanosine) and 12 weeks (P ≤ 0·049; maximum difference of 0·62 nG/mG Cr in F2-isoprostane PGF2α). Senior cats have lower circulating glutathione levels compared with younger cats. Feeding senior cats a complete and balanced dry diet supplemented with 1·5 % free GLY for 12 weeks elevated initial erythrocyte glutathione and altered markers of oxidative stress. Dietary supplementation with free GLY provides a potential opportunity to restore age-associated reduction in glutathione in cats.

The importance of estimating the burden of disease from foodborne transmission of Trypanosoma cruzi.

Chagas disease (ChD), caused by infection with the flagellated protozoan, Trypanosoma cruzi, has a complicated transmission cycle with many infection routes. These include vector-borne (via the triatomine (reduviid bug) vector defecating into a skin abrasion, usually following a blood meal), transplacental transmission, blood transfusion, organ transplant, laboratory accident, and foodborne transmission. Foodborne transmission may occur due to ingestion of meat or blood from infected animals or from ingestion of other foods (often fruit juice) contaminated by infected vectors or secretions from reservoir hosts. Despite the high disease burden associated with ChD, it was omitted from the original World Health Organization estimates of foodborne disease burden that were published in 2015. As these estimates are currently being updated, this review presents arguments for including ChD in new estimates of the global burden of foodborne disease. Preliminary calculations suggest a burden of at least 137,000 Disability Adjusted Life Years, but this does not take into account the greater symptom severity associated with foodborne transmission. Thus, we also provide information regarding the greater health burden in endemic areas associated with foodborne infection compared with vector-borne infection, with higher mortality and more severe symptoms. We therefore suggest that it is insufficient to use source attribution alone to determine the foodborne proportion of current burden estimates, as this may underestimate the higher disability and mortality associated with the foodborne infection route.

Preliminary investigations of parasite contamination of water sources in Armenia.

The intestinal protozoan parasites, Cryptosporidium and Giardia, are known to have a global distribution, infecting and causing disease in a range of hosts, including people, livestock, pets, and wildlife. However, data from some regions is very sparse. In Armenia, in the Caucasus region of West Asia, only scanty data are available, with just a few surveys on Cryptosporidium infections in livestock, and no available data on human infections or environmental contamination. As part of implementation of water analysis methods for these parasites in Armenia, 24 raw water samples and two sediment samples were analysed for these parasites using a range of approaches, including modified Ziehl-Neelsen, Lugol stain, immunofluorescent antibody test (IFAT), qPCR and, on sediment samples, immunomagnetic separation and IFAT. Results suggest substantial contamination of raw water sources and indicate the need for further targeted studies using appropriate methods and collecting data on host infections in catchment areas.