Distinct T cell signatures are associated with Staphylococcus aureus skin infection in pediatric atopic dermatitis.

Atopic dermatitis (AD) is an inflammatory skin condition with a childhood prevalence of up to 25%. Microbial dysbiosis is characteristic of AD, with Staphylococcus aureus the most frequent pathogen associated with disease flares and increasingly implicated in disease pathogenesis. Therapeutics to mitigate the effects of S. aureus have had limited efficacy and S. aureus-associated temporal disease flares are synonymous with AD. An alternative approach is an anti-S. aureus vaccine, tailored to AD. Experimental vaccines have highlighted the importance of T cells in conferring protective anti-S. aureus responses; however, correlates of T cell immunity against S. aureus in AD have not been identified. We identify a systemic and cutaneous immunological signature associated with S. aureus skin infection (ADS.aureus) in a pediatric AD cohort, using a combined Bayesian multinomial analysis. ADS.aureus was most highly associated with elevated cutaneous chemokines IP10 and TARC, which preferentially direct Th1 and Th2 cells to skin. Systemic CD4+ and CD8+ T cells, except for Th2 cells, were suppressed in ADS.aureus, particularly circulating Th1, memory IL-10+ T cells, and skin-homing memory Th17 cells. Systemic γδ T cell expansion in ADS.aureus was also observed. This study suggests that augmentation of protective T cell subsets is a potential therapeutic strategy in the management of S. aureus in AD.

Computable phenotype for real-world, data-driven retrospective identification of relapse in ANCA-associated vasculitis.

OBJECTIVE: ANCA-associated vasculitis (AAV) is a relapsing-remitting disease, resulting in incremental tissue injury. The gold-standard relapse definition (Birmingham Vasculitis Activity Score, BVAS>0) is often missing or inaccurate in registry settings, leading to errors in ascertainment of this key outcome. We sought to create a computable phenotype (CP) to automate retrospective identification of relapse using real-world data in the research setting. METHODS: We studied 536 patients with AAV and >6 months follow-up recruited to the Rare Kidney Disease registry (a national longitudinal, multicentre cohort study). We followed five steps: (1) independent encounter adjudication using primary medical records to assign the ground truth, (2) selection of data elements (DEs), (3) CP development using multilevel regression modelling, (4) internal validation and (5) development of additional models to handle missingness. Cut-points were determined by maximising the F1-score. We developed a web application for CP implementation, which outputs an individualised probability of relapse. RESULTS: Development and validation datasets comprised 1209 and 377 encounters, respectively. After classifying encounters with diagnostic histopathology as relapse, we identified five key DEs; DE1: change in ANCA level, DE2: suggestive blood/urine tests, DE3: suggestive imaging, DE4: immunosuppression status, DE5: immunosuppression change. F1-score, sensitivity and specificity were 0.85 (95% CI 0.77 to 0.92), 0.89 (95% CI 0.80 to 0.99) and 0.96 (95% CI 0.93 to 0.99), respectively. Where DE5 was missing, DE2 plus either DE1/DE3 were required to match the accuracy of BVAS. CONCLUSIONS: This CP accurately quantifies the individualised probability of relapse in AAV retrospectively, using objective, readily accessible registry data. This framework could be leveraged for other outcomes and relapsing diseases.

Data quality and patient characteristics in European ANCA-associated vasculitis registries: data retrieval by federated querying.

OBJECTIVES: This study aims to describe the data structure and harmonisation process, explore data quality and define characteristics, treatment, and outcomes of patients across six federated antineutrophil cytoplasmic antibody-associated vasculitis (AAV) registries. METHODS: Through creation of the vasculitis-specific Findable, Accessible, Interoperable, Reusable, VASCulitis ontology, we harmonised the registries and enabled semantic interoperability. We assessed data quality across the domains of uniqueness, consistency, completeness and correctness. Aggregated data were retrieved using the semantic query language SPARQL Protocol and Resource Description Framework Query Language (SPARQL) and outcome rates were assessed through random effects meta-analysis. RESULTS: A total of 5282 cases of AAV were identified. Uniqueness and data-type consistency were 100% across all assessed variables. Completeness and correctness varied from 49%-100% to 60%-100%, respectively. There were 2754 (52.1%) cases classified as granulomatosis with polyangiitis (GPA), 1580 (29.9%) as microscopic polyangiitis and 937 (17.7%) as eosinophilic GPA. The pattern of organ involvement included: lung in 3281 (65.1%), ear-nose-throat in 2860 (56.7%) and kidney in 2534 (50.2%). Intravenous cyclophosphamide was used as remission induction therapy in 982 (50.7%), rituximab in 505 (17.7%) and pulsed intravenous glucocorticoid use was highly variable (11%-91%). Overall mortality and incidence rates of end-stage kidney disease were 28.8 (95% CI 19.7 to 42.2) and 24.8 (95% CI 19.7 to 31.1) per 1000 patient-years, respectively. CONCLUSIONS: In the largest reported AAV cohort-study, we federated patient registries using semantic web technologies and highlighted concerns about data quality. The comparison of patient characteristics, treatment and outcomes was hampered by heterogeneous recruitment settings.

A Comparison of Relative-Efficacy Estimate(S) Derived From Both Matching-Adjusted Indirect Comparisons and Standard Anchored Indirect Treatment Comparisons: A Review of Matching-Adjusted Indirect Comparisons.

OBJECTIVES: We present an empirical comparison of relative-efficacy estimate(s) from matching-adjusted indirect comparisons (MAICs) with estimates from corresponding standard anchored indirect treatment comparisons. METHODS: A total of 80 comparisons were identified from 17 publications through a systematic rapid review. A standardized metric that used reported relative treatment efficacy estimates and their associated uncertainty was used to compare the methods across different treatment indications and outcome measures. RESULTS: On aggregate, MAICs presented for connected networks tended to report a more favorable relative-efficacy estimate for the treatment for which individual-level patient data were available relative to the reported indirect treatment comparison estimate. CONCLUSIONS: Although we recognize the importance of MAIC and other population adjustment methods in certain situations, we recommend that results from these analyses are interpreted with caution. Researchers and analysts should carefully consider if MAICs are appropriate where presented and whether MAICs would have added value where omitted.

Extending Beyond Bagust and Beale: Fully Parametric Piecewise Exponential Models for Extrapolation of Survival Outcomes in Health Technology Assessment.

OBJECTIVES: When extrapolating time-to-event data the Bagust and Beale (B&B) approach uses the Kaplan-Meier survival function until a manually chosen time point, after which a constant hazard is assumed. This study demonstrates an objective statistical approach to estimate this time point. METHODS: We estimate piecewise exponential models (PEMs), whereby the hazard function is partitioned into segments each with constant hazards. The boundaries of these segments are known as change points. Our approach determines the location and number of change points in PEMs from which the hazard in the final segment is used to model long-term survival. We reviewed previous applications of the B&B approach in National Institute for Health and Care Excellence Technology Appraisals (TAs) completed between July 2011 and June 2017. The time points after which constant hazards were assumed were compared between PEMs and the B&B approaches. When further survival data were published following the original TA, we compared these updated estimates to predicted survival from the PEM and other parametric models adjusted for general population mortality. RESULTS: Six of the 59 TAs in this review considered the B&B approach. There was general agreement between the location of time points identified through the PEM and the B&B approaches. In 2 of the identified TAs the best fitting model to the data was a no-change-point model. Of the 3 TAs for which further survival data became available, PEM provided the closest prediction for survival outcomes in 2 TAs. CONCLUSIONS: PEMs are useful for survival extrapolation when a long-term constant hazard trend for the disease is clinically plausible.

Direct Incorporation of Expert Opinion into Parametric Survival Models to Inform Survival Extrapolation.

BACKGROUND: In decision modeling with time-to-event data, there are a variety of parametric models that can be used to extrapolate the survival function. Each model implies a different hazard function, and in situations in which there is moderate censoring, this can result in quite different survival projections. External information such as expert opinion on long-term survival can more accurately characterize the uncertainty in these extrapolations. OBJECTIVE: We present a general and easily implementable approach to incorporate various types of expert opinions into parametric survival models, focusing on opinions about survival at various landmark time points. METHODS: Expert opinion is incorporated into parametric survival models using Bayesian and frequentist approaches. In the Bayesian method, expert opinion is included through a loss function and in the frequentist approach by penalizing the likelihood function, although in both cases the core approach is the same. The issue of aggregating multiple expert opinions is also considered. RESULTS: We apply this method to data from a leukemia trial and use previously elicited expert opinion on survival probabilities for that particular trial population at years 4 and 5 to inform our analysis. We take a robust approach to modeling expert opinion by using pooled distributions and fit a broad class of parametric models to the data. We also assess statistical goodness of fit of the models to both the observed data and expert opinion. CONCLUSIONS: Expert opinions can be implemented in a straightforward manner using this novel approach; however, more work is required on the correct elicitation of these quantities. HIGHLIGHTS: Presentation of a novel and open-source method to incorporate expert opinion into decision modeling.Extends upon earlier work in that expert opinion can be incorporated into a wide range of parametric models.Provides methodological guidance for directly including expert opinion in decision modeling, which is a research focus area in NICE TSD 21.1.

The association between ambient UVB dose and ANCA-associated vasculitis relapse and onset.

BACKGROUND: The aetiology of ANCA-associated vasculitis (AAV) and triggers of relapse are poorly understood. Vitamin D (vitD) is an important immunomodulator, potentially responsible for the observed latitudinal differences between granulomatous and non-granulomatous AAV phenotypes. A narrow ultraviolet B spectrum induces vitD synthesis (vitD-UVB) via the skin. We hypothesised that prolonged periods of low ambient UVB (and by extension vitD deficiency) are associated with the granulomatous form of the disease and an increased risk of AAV relapse. METHODS: Patients with AAV recruited to the Irish Rare Kidney Disease (RKD) (n = 439) and UKIVAS (n = 1961) registries were studied. Exposure variables comprised latitude and measures of ambient vitD-UVB, including cumulative weighted UVB dose (CW-D-UVB), a well-validated vitD proxy. An n-of-1 study design was used to examine the relapse risk using only the RKD dataset. Multi-level models and logistic regression were used to examine the effect of predictors on AAV relapse risk, phenotype and serotype. RESULTS: Residential latitude was positively correlated (OR 1.41, 95% CI 1.14-1.74, p = 0.002) and average vitD-UVB negatively correlated (0.82, 0.70-0.99, p = 0.04) with relapse risk, with a stronger effect when restricting to winter measurements (0.71, 0.57-0.89, p = 0.002). However, these associations were not restricted to granulomatous phenotypes. We observed no clear relationship between latitude, vitD-UVB or CW-D-UVB and AAV phenotype or serotype. CONCLUSION: Our findings suggest that low winter ambient UVB and prolonged vitD status contribute to AAV relapse risk across all phenotypes. However, the development of a granulomatous phenotype does not appear to be directly vitD-mediated. Further research is needed to determine whether sufficient vitD status would reduce relapse propensity in AAV.

Severe COVID-19 is characterised by inflammation and immature myeloid cells early in disease progression.

SARS-CoV-2 infection causes a wide spectrum of disease severity. Identifying the immunological characteristics of severe disease and the risk factors for their development are important in the management of COVID-19. This study aimed to identify and rank clinical and immunological features associated with progression to severe COVID-19 in order to investigate an immunological signature of severe disease. One hundred and eight patients with positive SARS-CoV-2 PCR were recruited. Routine clinical and laboratory markers were measured, as well as myeloid and lymphoid whole-blood immunophenotyping and measurement of the pro-inflammatory cytokines IL-6 and soluble CD25. All analysis was carried out in a routine hospital diagnostic laboratory. Univariate analysis demonstrated that severe disease was most strongly associated with elevated CRP and IL-6, loss of DLA-DR expression on monocytes and CD10 expression on neutrophils. Unbiased machine learning demonstrated that these four features were strongly associated with severe disease, with an average prediction score for severe disease of 0.925. These results demonstrate that these four markers could be used to identify patients developing severe COVID-19 and allow timely delivery of therapeutics.

ANCA-associated vasculitis in Ireland: a multi-centre national cohort study.

Background: Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a rare multisystem autoimmune disease. There is a need for interoperable national registries to enable reporting of real-world long-term outcomes and their predictors in AAV. Methods: The Irish National Rare Kidney Disease (RKD) registry was founded in 2012. To date, 842 patients with various forms of vasculitis have been recruited across eight nephrology, rheumatology and immunology centres. We focus here on patient- and disease- characteristics, treatment and outcomes of the 397 prospectively recruited patients with AAV. Results: Median age was 64 years (IQR 55-73), 57.9% were male, 58.9% had microscopic polyangiitis and 85.9% had renal impairment. Cumulative one- and five-year patient survival was 94% and 77% respectively. Median follow-up was 33.5 months (IQR 10.7-52.7). After controlling for age, baseline renal dysfunction (p = 0.04) and the burden of adverse events (p <0.001) were independent predictors of death overall. End-stage-kidney-disease (ESKD) occurred in 73 (18.4%) patients; one- and five-year renal survival was 85% and 79% respectively. Baseline severity of renal insufficiency (p = 0.02), urine soluble CD163 (usCD163) (p = 0.002) and "sclerotic" Berden histological class (p = 0.001) were key determinants of ESKD risk. Conclusions: Long-term outcomes of Irish AAV patients are comparable to other reported series. Our results emphasise the need for personalisation of immunosuppression, to limit treatment toxicity, particularly in those with advanced age and renal insufficiency. Baseline usCD163 is a potential biomarker for ESKD prediction and should be validated in a large independent cohort.

Altered Cytokine Endotoxin Responses in Neonatal Encephalopathy Predict MRI Outcomes.

Background: Neonatal encephalopathy (NE) is associated with adverse neurodevelopmental outcome and is linked with systemic inflammation. Pro-inflammatory and anti-inflammatory cytokines are known to play a role in the pathology of NE by activating innate immune cells. Methods: Eighty-seven infants were enrolled including 53 infants with NE of whom 52 received therapeutic hypothermia (TH) and 34 term infant healthy controls (TC). Whole blood sampling was performed in the first 4 days of life, and a 14-spot ELISA Multiplex Cytokine Array was carried out on baseline samples or after stimulation with lipopolysaccharide (LPS) as an additional inflammatory stimulus. The cytokine medians were examined for differences between infants with NE and healthy TC; and then short-term outcomes of Sarnat stage, seizures, and MRI brain were examined within the NE group. The potential of LPS stimulation to predict abnormal MRI was explored using receiver operating characteristic (ROC) curves. Results: At baseline, infants with NE had significantly higher levels of erythropoietin (Epo), interleukin (IL)-6, and IL-1ra and significantly lower vascular endothelial growth factor (VEGF) than had controls. All cytokines were increased after LPS stimulation in infants with NE with an excessive Epo and IL-1ra response than in controls. Infants with NE had lower IL-8, IL-2, IL-6, tumor necrosis factor (TNF)-α, granulocyte-macrophage colony-stimulating factor (GM-CSF), VEGF, and interferon (IFN)-γ than controls had following LPS. GM-CSF and IFN-γ, IL-1ß, IL-1ra, and VEGF were higher on days 1-2 in NE infants with abnormal neuroimaging. GM-CSF, IFN-γ, and TNF-α levels with LPS stimulation were different upon stimulation between normal and abnormal neuroimaging. TNF-α is the only strong cytokine predictor both pre- and post-LPS stimulation of abnormal brain imaging. Conclusions: Altered cytokine responses are found in infants with NE vs. controls, and more significant differences are unmasked by the additional stimulus of LPS, which potentially improves the predictive power of these cytokines for the detection of abnormal MRIs. Infants with NE undergoing TH demonstrate both trained immunity and tolerance, and understanding these responses will facilitate adjunctive immunomodulatory treatments.

Comparisons of disease cluster patterns, prevalence and health factors in the USA, Canada, England and Ireland.

BACKGROUND: Identification of those who are most at risk of developing specific patterns of disease across different populations is required for directing public health policy. Here, we contrast prevalence and patterns of cross-national disease incidence, co-occurrence and related risk factors across population samples from the U.S., Canada, England and Ireland. METHODS: Participants (n = 62,111) were drawn from the US Health and Retirement Study (n = 10,858); the Canadian Longitudinal Study on Ageing (n = 36,647); the English Longitudinal Study of Ageing (n = 7938) and The Irish Longitudinal Study on Ageing (n = 6668). Self-reported lifetime prevalence of 10 medical conditions, predominant clusters of multimorbidity and their specific risk factors were compared across countries using latent class analysis. RESULTS: The U.S. had significantly higher prevalence of multimorbid disease patterns and nearly all diseases when compared to the three other countries, even after adjusting for age, sex, BMI, income, employment status, education, alcohol consumption and smoking history. For the U.S. the most at-risk group were younger on average compared to Canada, England and Ireland. Socioeconomic gradients for specific disease combinations were more pronounced for the U.S., Canada and England than they were for Ireland. The rates of obesity trends over the last 50 years align with the prevalence of eight of the 10 diseases examined. While patterns of disease clusters and the risk factors related to each of the disease clusters were similar, the probabilities of the diseases within each cluster differed across countries. CONCLUSIONS: This information can be used to better understand the complex nature of multimorbidity and identify appropriate prevention and management strategies for treating multimorbidity across countries.

A cohort study to investigate sex-specific differences in ANCA-associated glomerulonephritis outcomes.

Data surrounding sex-specific differences in ANCA-associated vasculitis glomerulonephritis (ANCA-GN) outcomes is sparse. We hypothesised that the previously observed increased risk of end-stage kidney disease (ESKD) in males is driven by sex-specific variation in immunosuppression dosing. Patients were recruited to the Irish Rare Kidney Disease Registry or followed by the Royal Free Hospital vasculitis team (2012-2020). Inclusion criteria: prior diagnosis of ANCA-GN (biopsy proven pauci-immune glomerulonephritis) and positive serology for anti-MPO or -PR3 antibodies. Renal and patient survival, stratified by sex and Berden histological class, was analysed. The cumulative- and starting dose/kilogram of induction agents and prednisolone, respectively, was compared between sexes. 332 patients were included. Median follow-up was time 40.2 months (IQR 17.3-69.2). 73 (22%) reached ESKD and 47 (14.2%) died. Overall 1- and 5-year renal survival was 82.2% and 76.7% in males and 87.1% and 82.0% in females, respectively (p 0.13). The hazard ratio for ESKD in males versus females, after adjustment for age, ANCA serology, baseline creatinine and histological class was 1.07 (95% CI 0.59-1.93). There was no difference between sexes in the dose/kilogram of any induction agent. We did not observe a strong impact of sex on renal outcome in ANCA-GN. Treatment intensity does not vary by sex.

Investigation of type I interferon responses in ANCA-associated vasculitis.

Type I interferon (IFN) dysregulation is a major contributory factor in the development of several autoimmune diseases, termed type I interferonopathies, and is thought to be the pathogenic link with chronic inflammation in these conditions. Anti-neutrophil cytoplasmic antibody (ANCA)-Associated Vasculitis (AAV) is an autoimmune disease characterised by necrotising inflammation of small blood vessels. The underlying biology of AAV is not well understood, however several studies have noted abnormalities in type I IFN responses. We hypothesised that type I IFN responses are systemically dysregulated in AAV, consistent with features of a type I interferonopathy. To investigate this, we measured the expression of seven interferon regulated genes (IRGs) (ISG15, SIGLEC1, STAT1, RSAD2, IFI27, IFI44L and IFIT1) in peripheral blood samples, as well as three type I IFN regulated proteins (CXCL10, MCP-1 and CCL19) in serum samples from AAV patients, healthy controls and disease controls. We found no difference in type I IFN regulated gene or protein expression between AAV patients and healthy controls. Furthermore, IRG and IFN regulated protein expression did not correlate with clinical measurements of disease activity in AAV patients. Thus, we conclude that systemic type I IFN responses are not key drivers of AAV pathogenesis and AAV should not be considered a type I interferonopathy.

Difficulties arising in reimbursement recommendations on new medicines due to inadequate reporting of population adjustment indirect comparison methods.

Indirect treatment comparisons are useful to estimate relative treatment effects when head-to-head studies are not conducted. Statisticians at the National Centre for Pharmacoeconomics Ireland (NCPE) and Scottish Medicines Consortium (SMC) assess the clinical and cost-effectiveness of new medicines as part of multidisciplinary teams. We describe some shared observations on areas where reporting of population-adjustment indirect comparison methods is causing uncertainty in our recommendations to decision-making committees when assessing reimbursement of medicines.

Incorporating single-arm evidence into a network meta-analysis using aggregate level matching: Assessing the impact.

Increasingly, single-armed evidence is included in health technology assessment submissions when companies are seeking reimbursement for new drugs. While it is recognized that randomized controlled trials provide a higher standard of evidence, these are not available for many new agents that have been granted licenses in recent years. Therefore, it is important to examine whether alternative strategies for assessing this evidence may be used. In this work, we examine approaches to incorporating single-armed evidence formally in the evaluation process. We consider matching aggregate level covariates to comparator arms or trials and including this evidence in a network meta-analysis. We consider two methods of matching: (i) we include the chosen matched arm in the data set itself as a comparator for the single-arm trial; (ii) we use the baseline odds of an event in a chosen matched trial to use as a plug-in estimator for the single-arm trial. We illustrate that the synthesis of evidence resulting from such a setup is sensitive to the between-study variability, formulation of the prior for the between-design effect, weight given to the single-arm evidence, and extent of the bias in single-armed evidence. We provide a flowchart for the process involved in such a synthesis and highlight additional sensitivity analyses that should be carried out. This work was motivated by a hepatitis C data set, where many agents have only been examined in single-arm studies. We present the results of our methods applied to this data set.

A Novel Calcium-Dependent Kinase Inhibitor, Bumped Kinase Inhibitor 1517, Cures Cryptosporidiosis in Immunosuppressed Mice.

Cryptosporidium is recognized as one of the main causes of childhood diarrhea worldwide. However, the current treatment for cryptosporidiosis is suboptimal. Calcium flux is essential for entry in apicomplexan parasites. Calcium-dependent protein kinases (CDPKs) are distinct from protein kinases of mammals, and the CDPK1 of the apicomplexan Cryptosporidium lack side chains that typically block a hydrophobic pocket in protein kinases. We exploited this to develop bumped kinase inhibitors (BKIs) that selectively target CDPK1. We have shown that several BKIs of Cryptosporidium CDPK1 potently reduce enzymatic activity and decrease parasite numbers when tested in vitro. In the present work, we studied the anticryptosporidial activity of BKI-1517, a novel BKI. The half maximal effective concentration for Cryptosporidium parvum in HCT-8 cells was determined to be approximately 50 nM. Silencing experiments of CDPK1 suggest that BKI-1517 acts on CDPK1 as its primary target. In a mouse model of chronic infection, 5 of 6 SCID/beige mice (83.3%) were cured after treatment with a single daily dose of 120 mg/kg BKI-1517. No side effects were observed. These data support advancing BKI-1517 as a lead compound for drug development for cryptosporidiosis.

Kato-Katz and Lumbreras rapid sedimentation test to evaluate helminth prevalence in the setting of a school-based deworming program.

The sensitivity of the Kato-Katz test is suboptimal for the evaluation of intestinal helminth prevalence. Moreover, during mass deworming, as helminth egg burden decreases, the sensitivity is likely to decrease. The Lumbreras rapid sedimentation (Lumbreras) is a low-cost non-quantitative test, but may provide useful information in low burden areas. We compared the prevalence of intestinal helminth infections assessed by the Kato-Katz and the Lumbreras rapid sedimentation test on 3 stool specimens from each of 1083 children. The sensitivities were compared using the McNemar paired test. Using the combined outcome of the 3 different stool tests as the standard, Kato-Katz had lower sensitivity than Lumbreras rapid sedimentation tests for Ascaris lumbricoides (85.1% vs. 95.1%, p = 0.03), Hymenolepis nana (77.7% vs. 97.9%, p < 0.01), Trichuris trichura (41.7% vs. 100%, p = 0.01), hookworm (0% vs. 100%, p = 0.01), and Strongyloides stercoralis (0% vs. 88%, p < 0.01). Kato-Katz demonstrated significantly lower sensitivity, missing most T. trichiura, hookworm, and S. stercoralis infections. The combination of Kato-Katz and Lumbreras rapid sedimentation tests enables the detection of more intestinal helminths infections in post-deworming low prevalence areas.

Fasciola hepatica Infection in an Indigenous Community of the Peruvian Jungle.

Fasciola hepatica is a zoonotic infection with a worldwide distribution. Autochthonous cases have not been reported in the Amazon region of Peru. Operculated eggs resembling F. hepatica were identified in the stools of five out of 215 subjects in a remote indigenous community of the Peruvian jungle. Polymerase chain reaction targeting Fasciola hepatica cytochrome oxidase subunit 1 (COI) gene and sequencing of the products confirmed Fasciola infection.

Recombinase polymerase amplification-based assay to diagnose Giardia in stool samples.

Giardia duodenalis is one of the most commonly identified parasites in stool samples. Although relatively easy to treat, giardiasis can be difficult to detect as it presents similar to other diarrheal diseases. Here, we present a recombinase polymerase amplification-based Giardia (RPAG) assay to detect the presence of Giardia in stool samples. The RPAG assay was characterized on the bench top using stool samples spiked with Giardia cysts where it showed a limit-of-detection nearly as low as the gold standard polymerase chain reaction assay. The RPAG assay was then tested in the highlands of Peru on 104 stool samples collected from the surrounding communities where it showed 73% sensitivity and 95% specificity against a polymerase chain reaction and microscopy composite gold standard. Further improvements in clinical sensitivity will be needed for the RPAG assay to have clinical relevance.