An evaluation of efficacy and acceptability of a novel manualised JuniorLEAP group programme for compulsive exercise, for children and adolescents with anorexia nervosa, within an inpatient setting.

PURPOSE: Compulsive exercise is a symptom and a maintenance factor of eating disorders, which increases the risk of relapse. It has been considered a target for treatment, particularly for anorexia nervosa (AN). This audit aims to review the efficacy and acceptability of a new seven-week JuniorLEAP group therapy programme, for children and adolescents with anorexia nervosa. JuniorLEAP was adapted by the authors and based on the Loughborough Eating Disorder Activity Programme (LEAP) for adults. METHODS: 32 children and adolescents with anorexia nervosa were allocated to the group in an in-patient setting using entry criteria. All children and adolescents completed seven weekly sessions of the JuniorLEAP programme, as well as pre- and post-treatment questionnaires, including the Eating Disorder Examination Questionnaire (EDE-Q) and the Compulsive Exercise Test (CET). The children and adolescents were also asked to provide qualitative responses about the acceptability of the group. A paired t test was conducted to review the efficacy of the JuniorLEAP programme. RESULTS: Significant changes in eating disorder psychopathology was observed, as measured by the EDE-Q, with total mean scores reducing from 3.53 to 2.77 (p = 0.001). Compulsive exercise attitudes were also observed to reduce, as measured by the CET, with total mean scores reducing from 15.39 to 10.90 (p ≤ 0.001). Furthermore, there was a significant reduction in all five subscales of the CET following completion of the group. Qualitative results also demonstrate the group to be acceptable to the patients. CONCLUSION: This study finds that a new manualised JuniorLEAP group therapy, specifically adapted for adolescents and children with AN, when used as an adjuvant with other therapies in a residential setting, significantly reduces their compulsive exercise, as measured by CET. The patients reported that the treatment was acceptable. Further research testing the new treatment in a randomised controlled trial is now needed, particularly to disentangle the impact of other aspects of standard treatment in reducing compulsive exercise. LEVEL OF EVIDENCE: II.

A novel 'practical body image' therapy for adolescent inpatients with anorexia nervosa: a randomised controlled trial.

PURPOSE: To determine the potential effectiveness of a novel 10-week manualised Practical Body Image therapy (PBI) with mirror exposure (ME), when used as an adjuvant to an intensive treatment package (TAU) in adolescent inpatients with Anorexia Nervosa (AN). To evaluate the effectiveness of ME in an adolescent population. METHODS: Using a randomised control design, 40 girls aged 11-17 years with AN were assigned to PBI with TAU (n = 20) and TAU alone (n = 20). Both groups completed self-report measures of body image at week 1 and week 10 of the study to measure the potential effectiveness of PBI. The PBI group completed measures at week 7 to evaluate the ME component. RESULTS: 31 participants completed the study; 16 TAU, 15 PBI. PBI participants had greater improvement in all outcomes than TAU participants. Medium effect sizes were seen for self-reported weight concern, body image avoidance in terms of clothing and body image anxiety. ME produced effect sizes in self-reported body image avoidance in terms of clothing and grooming that were greater than 0.40, n = 14. CONCLUSION: The findings demonstrate that PBI supports an intensive inpatient treatment package and addresses elements of negative body image. PBI was beneficial for addressing body image dissatisfaction with improvements in weight concerns, body image avoidance and physical appearance trait anxiety following the ME component. The magnitude of the effect sizes is comparable to previous studies. Positive qualitative feedback indicated the intervention was acceptable to users. PBI is a promising new adjuvant treatment for AN. EMB RATING: Level I: randomized controlled trial.

Taxon-rich phylogenomic analyses resolve the eukaryotic tree of life and reveal the power of subsampling by sites.

Most eukaryotic lineages are microbial, and many have only recently been sampled for phylogenetic studies or remain in the "dark area" of the tree of life where there are no molecular data. To assess relationships among eukaryotic lineages, we perform a taxon-rich phylogenomic analysis including 232 eukaryotes selected to maximize taxonomic diversity and up to 1554 genes chosen as vertically inherited based on their broad distribution among eukaryotes. We also include sequences from 486 bacteria and 84 archaea to assess the impact of endosymbiotic gene transfer (EGT) from plastids and to detect contamination. Overall, our analyses are consistent with other less taxon-rich estimates of the eukaryotic tree of life, and we recover strong support for five major clades: Amoebozoa, Excavata (without the genus Malawimonas), Opisthokonta, Archaeplastida, and SAR (Stramenopila, Alveolata, and Rhizaria). Our analyses also highlight the existence of "orphan" lineages, lineages that lack robust placement in the eukaryotic tree of life, and indicate the possibility of as yet undiscovered diversity. In analyses including bacteria and archaea, we find that approximately 10% of the 1554 genes, which we choose because they are found in four or five of the five major eukaryotic clades and hence may be more likely to be inherited vertically, appear to have been acquired from cyanobacteria through EGT in photosynthetic lineages. Removing these EGT genes places the green algae as sister to the glaucophytes instead of the red algae, suggesting that unknowingly including genes of plastid origin, and combining them with genes of nuclear origin, may mislead phylogenetic estimates. Finally, the large size of our data set allows comparative analyses of subsets of data; alignments built from randomly sampled sites provide greater support, particularly for deep relationships, than do equivalent-sized data sets built from randomly sampled genes.

Sapocribrum chincoteaguense n. gen. n. sp.: A Small, Scale-bearing Amoebozoan with Flabellinid Affinities.

The isolate American Type Culture Collection (ATCC)® 50979™ is a small amoebozoan whose actin gene was previously characterized, but did not allow a stable phylogenetic placement. This isolate was originally mis-identified upon deposition, and subsequently mis-illustrated in a recent publication. Here, we provide both a detailed morphological description as well as additional molecular analyses in order to clarify the isolate's phylogenetic relationships. The amoeba is minute (less than 5 µm), and presents the behavior of staying in a fixed location, while emitting one or two thin pseudopods. Transmission electron microscopy reveals that the cell is covered in a layer with embedded scales, giving the cell an armored appearance. Molecular phylogenetic analyses of data (actin, alpha- and beta-tubulin, elongation factor 2, and 14-3-3) from transcriptomes of this and four other isolates reveals that ATCC® 50979(™) is closely related to the recently described Squamamoeba japonica and in a novel, stable clade. Due to the unique nature of the scale covering, as well as other gross morphological characters and the molecular phylogenetic analyses, we formally describe the isolate as Sapocribrum chincoteaguense n. gen. n. sp.

Proposed Specifiers for Conduct Disorder (PSCD) self-report: Factor structure and validation in a community sample of Belgian youth.

The current study examined the psychometric properties of the Proposed Specifiers for Conduct Disorder (PSCD) in a sample of school-attending adolescent Belgian youth (N = 599; M age = 16.51 years, SD = 1.27). Given the recent interest in the PSCD-Short Version (PSCD-SV), this study focused on the 13-item variant of the PSCD. Study findings showed that the PSCD-SV had a hierarchical four-factor structure including the components of grandiose-manipulative (GM), callous-unemotional (CU), daring-impulsive (DI), and conduct disorder (CD). These interrelated factors were found to be internally consistent. The study also showed that the PSCD-SV total score was positively and significantly related to an alternate measure of psychopathy. Further, the study revealed the PSCD-SV was meaningfully related to the five-factor personality domains (i.e., extraversion, conscientiousness, agreeableness) as well as peer functioning and prosocial behavior. Bivariate correlations demonstrated that the dimensions differed in their associations with external correlates (e.g., peer functioning). Regression analyses showed that the GM, CU, and CD components of the PSCD-SV were uniquely associated to externalizing difficulties, whereas only the GM and CU components of the PSCD-SV were associated with low prosocial behaviors. These findings shed light on the conceptual and developmental models for the consideration of psychopathy and conduct problems. The use of the broader psychopathy condition as well as its underpinning dimensions may have important implications for assessment, treatment, and diagnostic manuals. The implications of the current study are further discussed.

Rigidity analysis of protein biological assemblies and periodic crystal structures.

BACKGROUND: We initiate in silico rigidity-theoretical studies of biological assemblies and small crystals for protein structures. The goal is to determine if, and how, the interactions among neighboring cells and subchains affect the flexibility of a molecule in its crystallized state. We use experimental X-ray crystallography data from the Protein Data Bank (PDB). The analysis relies on an effcient graph-based algorithm. Computational experiments were performed using new protein rigidity analysis tools available in the new release of our KINARI-Web server http://kinari.cs.umass.edu. RESULTS: We provide two types of results: on biological assemblies and on crystals. We found that when only isolated subchains are considered, structural and functional information may be missed. Indeed, the rigidity of biological assemblies is sometimes dependent on the count and placement of hydrogen bonds and other interactions among the individual subchains of the biological unit. Similarly, the rigidity of small crystals may be affected by the interactions between atoms belonging to different unit cells. CONCLUSION: The rigidity analysis of a single asymmetric unit may not accurately reflect the protein's behavior in the tightly packed crystal environment. Using our KINARI software, we demonstrated that additional functional and rigidity information can be gained by analyzing a protein's biological assembly and/or crystal structure. However, performing a larger scale study would be computationally expensive (due to the size of the molecules involved). Overcoming this limitation will require novel mathematical and computational extensions to our software.

Turning the crown upside down: gene tree parsimony roots the eukaryotic tree of life.

The first analyses of gene sequence data indicated that the eukaryotic tree of life consisted of a long stem of microbial groups "topped" by a crown-containing plants, animals, and fungi and their microbial relatives. Although more recent multigene concatenated analyses have refined the relationships among the many branches of eukaryotes, the root of the eukaryotic tree of life has remained elusive. Inferring the root of extant eukaryotes is challenging because of the age of the group (∼1.7-2.1 billion years old), tremendous heterogeneity in rates of evolution among lineages, and lack of obvious outgroups for many genes. Here, we reconstruct a rooted phylogeny of extant eukaryotes based on minimizing the number of duplications and losses among a collection of gene trees. This approach does not require outgroup sequences or assumptions of orthology among sequences. We also explore the impact of taxon and gene sampling and assess support for alternative hypotheses for the root. Using 20 gene trees from 84 diverse eukaryotic lineages, this approach recovers robust eukaryotic clades and reveals evidence for a eukaryotic root that lies between the Opisthokonta (animals, fungi and their microbial relatives) and all remaining eukaryotes.

Meloxicam in Combination with Mitoxantrone or Vinblastine as First-Line Treatment for Non-Resectable Urothelial Cell Carcinoma in Dogs.

Cyclooxygenase (COX) inhibitors have been demonstrated to have antitumour activity in canine urothelial cell carcinoma (UCC), given as a sole treatment or in combination with chemotherapy. The purpose of this retrospective multi-institutional study was to assess the efficacy of meloxicam in combination with mitoxantrone or vinblastine as a first-line treatment for non-resectable canine UCC. Gastrointestinal adverse effects (AEs) of these treatment combinations were also assessed. A total of 28 dogs met the inclusion criteria, 21/28 dogs received mitoxantrone and meloxicam, and 7/28 received vinblastine and meloxicam. Tumour response (TR) and AE were evaluated according to Veterinary Co-Operative Oncology Group (VCOG) criteria. The endpoint of the study was the time to tumour progression (TTP). The mitoxantrone-group induced 24% partial response and 62% stable disease, while the vinblastine-group induced 14% and 86%, respectively. Median TTP was 84 days (mitoxantrone and meloxicam, 70 days; and vinblastine and meloxicam, 178 days). The presence of metastatic disease significantly decreased TTP (p = 0.007). Gastrointestinal AEs were reported in 21.4% of the patients, with the most common being VCOG grade 1-2 diarrhoea. Meloxicam is a well-tolerated NSAID when combined with mitoxantrone or vinblastine as first-line treatment for non-resectable canine UCC.

Communication Interventions and Assessment of Drivers for Hendra Virus Vaccination Uptake.

Hendra virus disease (HeVD) is an emerging zoonosis in Australia, resulting from the transmission of Hendra virus (HeV) to horses from Pteropus bats. Vaccine uptake for horses is low despite the high case fatality rate of HeVD in both horses and people. We reviewed evidence-based communication interventions to promote and improve HeV vaccine uptake for horses by horse owners and conducted a preliminary evaluation of potential drivers for HeV vaccine uptake using the Behavioural and Social Drivers of Vaccination (BeSD) framework developed by the World Health Organization. Six records were eligible for review following a comprehensive search and review strategy of peer-reviewed literature, but evidence-based communication interventions to promote and improve HeV vaccine uptake for horses were lacking. An evaluation of potential drivers for HeV vaccine uptake using the BeSD framework indicated that horse owners' perceptions, beliefs, social processes, and practical issues are similar to those experienced by parents making decisions about childhood vaccines, although the overall motivation to vaccinate is lower amongst horse owners. Some aspects of HeV vaccine uptake are not accounted for in the BeSD framework (for example, alternative mitigation strategies such as covered feeding stations or the zoonotic risk of HeV). Overall, problems associated with HeV vaccine uptake appear well-documented. We, therefore, propose to move from a problems-focused to a solutions-focused approach to reduce the risk of HeV for humans and horses. Following our findings, we suggest that the BeSD framework could be modified and used to develop and evaluate communication interventions to promote and improve HeV vaccine uptake by horse owners, which could have a global application to promote vaccine uptake for other zoonotic diseases in animals, such as rabies.

Optimized strategy for real-time qPCR detection of Onchocerca volvulus DNA in pooled Simulium sp. blackfly vectors.

BACKGROUND: Onchocerca volvulus is a filarial parasite that is a major cause of dermatitis and blindness in endemic regions primarily in sub-Saharan Africa. Widespread efforts to control the disease caused by O. volvulus infection (onchocerciasis) began in 1974 and in recent years, following successful elimination of transmission in much of the Americas, the focus of efforts in Africa has moved from control to the more challenging goal of elimination of transmission in all endemic countries. Mass drug administration (MDA) with ivermectin has reached more than 150 million people and elimination of transmission has been confirmed in four South American countries, with at least two African countries having now stopped MDA as they approach verification of elimination. It is essential that accurate data for active transmission are used to assist in making the critical decision to stop MDA, since missing low levels of transmission and infection can lead to continued spread or recrudescence of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Current World Health Organization guidelines for MDA stopping decisions and post-treatment surveillance include screening pools of the Simulium blackfly vector for the presence of O. volvulus larvae using a PCR-ELISA-based molecular technique. In this study, we address the potential of an updated, practical, standardized molecular diagnostic tool with increased sensitivity and species-specificity by comparing several candidate qPCR assays. When paired with heat-stable reagents, a qPCR assay with a mitochondrial DNA target (OvND5) was found to be more sensitive and species-specific than an O150 qPCR, which targets a non-protein coding repetitive DNA sequence. The OvND5 assay detected 19/20 pools of 100 blackfly heads spiked with a single L3, compared to 16/20 for the O150 qPCR assay. CONCLUSIONS/SIGNIFICANCE: Given the improved sensitivity, species-specificity and resistance to PCR inhibitors, we identified OvND5 as the optimal target for field sample detection. All reagents for this assay can be shipped at room temperature with no loss of activity. The qPCR protocol we propose is also simpler, faster, and more cost-effective than the current end-point molecular assays.

Is the ASVAB ST composite score a reliable predictor of first-attempt graduation for the U.S. Army operating room specialist course?

The U.S. Army Operating Room Specialist (68D) Course provides first class medical technician training to U.S. Army enlisted soldiers of the Army Medical Command. With a failure rate of approximately 12% over a 2-year period, this study was commissioned to determine whether the Armed Services Vocational Aptitude Battery (ASVAB) skilled technical (ST) Score served as a reliable predictor for successful first-attempt completion of the 68D course. A sample size of 373 was analyzed via a multivariate binary logistic regression model with 6 distinct independent variables. This study found that the ASVAB ST score, gender, and rank were predictors to first-attempt successful completion of the 68D training program. Specifically, students with an ST score 10 points higher than their peers were 5 times more likely to graduate. In addition, females were 2.5 times more likely to succeed than males and Army Privates (E2) were 3.2 times more likely than Privates (El). Specialists, Corporals (E4), Sergeants (E5), and Staff Sergeants (E6) combined, were 34 times more likely to succeed than Els. Although further study may be warranted, increasing the minimum ST score requirement in the admission guidelines and/or specific preventive assistance for lower-ranked students may decrease the first-attempt failure rate.

Broadly sampled multigene analyses yield a well-resolved eukaryotic tree of life.

An accurate reconstruction of the eukaryotic tree of life is essential to identify the innovations underlying the diversity of microbial and macroscopic (e.g., plants and animals) eukaryotes. Previous work has divided eukaryotic diversity into a small number of high-level "supergroups," many of which receive strong support in phylogenomic analyses. However, the abundance of data in phylogenomic analyses can lead to highly supported but incorrect relationships due to systematic phylogenetic error. Furthermore, the paucity of major eukaryotic lineages (19 or fewer) included in these genomic studies may exaggerate systematic error and reduce power to evaluate hypotheses. Here, we use a taxon-rich strategy to assess eukaryotic relationships. We show that analyses emphasizing broad taxonomic sampling (up to 451 taxa representing 72 major lineages) combined with a moderate number of genes yield a well-resolved eukaryotic tree of life. The consistency across analyses with varying numbers of taxa (88-451) and levels of missing data (17-69%) supports the accuracy of the resulting topologies. The resulting stable topology emerges without the removal of rapidly evolving genes or taxa, a practice common to phylogenomic analyses. Several major groups are stable and strongly supported in these analyses (e.g., SAR, Rhizaria, Excavata), whereas the proposed supergroup "Chromalveolata" is rejected. Furthermore, extensive instability among photosynthetic lineages suggests the presence of systematic biases including endosymbiotic gene transfer from symbiont (nucleus or plastid) to host. Our analyses demonstrate that stable topologies of ancient evolutionary relationships can be achieved with broad taxonomic sampling and a moderate number of genes. Finally, taxon-rich analyses such as presented here provide a method for testing the accuracy of relationships that receive high bootstrap support (BS) in phylogenomic analyses and enable placement of the multitude of lineages that lack genome scale data.

Development of a novel real-time polymerase chain reaction assay for the sensitive detection of Schistosoma japonicum in human stool.

BACKGROUND: Elimination and control of Schistosoma japonicum, the most virulent of the schistosomiasis-causing blood flukes, requires the development of sensitive and specific diagnostic tools capable of providing an accurate measurement of the infection prevalence in endemic areas. Typically, detection of S. japonicum has occurred using the Kato-Katz technique, but this methodology, which requires skilled microscopists, has been shown to radically underestimate levels of infection. With the ever-improving capabilities of next-generation sequencing and bioinformatic analysis tools, identification of satellite sequences and other highly repetitive genomic elements for use as real-time PCR diagnostic targets is becoming increasingly common. Assays developed using these targets have the ability to improve the sensitivity and specificity of results for epidemiological studies that can in turn be used to inform mass drug administration and programmatic decision making. METHODOLOGY/PRINCIPAL FINDINGS: Utilizing Tandem Repeat Analyzer (TAREAN) and RepeatExplorer2, a cluster-based analysis of the S. japonicum genome was performed and a tandemly arranged genomic repeat, which we named SjTR1 (Schistosoma japonicum Tandem Repeat 1), was selected as the target for a real-time PCR diagnostic assay. Based on these analyses, a primer/probe set was designed and the assay was optimized. The resulting real-time PCR test was shown to reliably detect as little as 200 ag of S. japonicum genomic DNA and as little as 1 egg per gram of human stool. Based on these results, the index assay reported in this manuscript is more sensitive than previously published real-time PCR assays for the detection of S. japonicum. CONCLUSIONS/SIGNIFICANCE: The extremely sensitive and specific diagnostic assay described in this manuscript will facilitate the accurate detection of S. japonicum, particularly in regions with low levels of endemicity. This assay will be useful in providing data to inform programmatic decision makers, aiding disease control and elimination efforts.

Assessing outcomes of ear molding therapy by health care providers and convolutional neural network.

Ear molding therapy is a nonsurgical technique to correct certain congenital auricular deformities. While the advantages of nonsurgical treatments over otoplasty are well-described, few studies have assessed aesthetic outcomes. In this study, we compared assessments of outcomes of ear molding therapy for 283 ears by experienced healthcare providers and a previously developed deep learning CNN model. 2D photographs of ears were obtained as a standard of care in our onsite photography studio. Physician assistants (PAs) rated the photographs using a 5-point Likert scale ranging from 1(poor) to 5(excellent) and the CNN assessment was categorical, classifying each photo as either "normal" or "deformed". On average, the PAs classified 75.6% of photographs as good to excellent outcomes (scores 4 and 5). Similarly, the CNN classified 75.3% of the photographs as normal. The inter-rater agreement between the PAs ranged between 72 and 81%, while there was a 69.6% agreement between the machine model and the inter-rater majority agreement between at least two PAs (i.e., when at least two PAs gave a simultaneous score < 4 or ≥ 4). This study shows that noninvasive ear molding therapy has excellent outcomes in general. In addition, it indicates that with further training and validation, machine learning techniques, like CNN, have the capability to accurately mimic provider assessment while removing the subjectivity of human evaluation making it a robust tool for ear deformity identification and outcome evaluation.

Identification of new molecular markers for assembling the eukaryotic tree of life.

Six eukaryotic supergroups have been proposed based on both morphological and molecular data. However, some of these supergroups are contentious and the deep relationships among them are poorly resolved. This is due to a limited number of morphological characters and few molecular markers in current use. The lack of resolution in most multigene analyses, including phylogenomic analyses, necessitates a search for additional, appropriate molecular markers to enable targeted sampling of taxa in key phylogenetic positions. We evaluated the phylogenetic signal of 860 proteins obtained from the Clusters of Orthologous Groups of proteins (COGs) database. We report a total of 17 markers that resulted in well-resolved topologies that are congruent with well-established components of the eukaryotic tree. To establish their utility, we designed universal degenerate primers for six markers, some of which showed promising results in unicellular eukaryotes. Finally, we present phylogenetic informativeness profiles for seven selected markers, revealing that the markers contain phylogenetic signal that spans the whole tree including the deeper branches.

A description of a new "Amoebozoan" isolated from the American lobster, Homarus americanus.

Our knowledge of the diversity of amoeboid protists is rapidly expanding as new and old habitats are more fully explored. In 2003, while investigating the cause of an amoeboid disease afflicting lobsters on the East Coast, samples were examined for the presence of amoebae from the carapace washings of the American lobster, Homarus americanus. During this survey a unique community of gymnamoebae was discovered. Among the new taxa discovered was a small Thecamoeba-like organism with a single posteriorly directed pseudopodium. Although resembling Parvamoeba rugata, this amoeba displayed distinctive morphology from that isolate or any other amoebozoan. Phylogenetic analysis shows this amoeba is distantly related to the Thecamoebidae. In this paper we describe the unique morphology of a second species of Parvamoeba and discuss its phylogenetic position with respect to the "Amoebozoa."

Selection and exploitation of prevalent, tandemly repeated genomic targets for improved real-time PCR-based detection of Wuchereria bancrofti and Plasmodium falciparum in mosquitoes.

BACKGROUND: Optimization of polymerase chain reaction (PCR)-based diagnostics requires the careful selection of molecular targets that are both highly repetitive and pathogen-specific. Advances in both next-generation sequencing (NGS) technologies and bioinformatics-based analysis tools are facilitating this selection process, informing target choices and reducing labor. Once developed, such assays provide disease control and elimination programs with an additional set of tools capable of evaluating and monitoring intervention successes. The importance of such tools is heightened as intervention efforts approach their endpoints, as accurate and complete information is an essential component of the informed decision-making process. As global efforts for the control and elimination of both lymphatic filariasis and malaria continue to make significant gains, the benefits of diagnostics with improved analytical and clinical/field-based sensitivities and specificities will become increasingly apparent. METHODOLOGY/PRINCIPAL FINDINGS: Coupling Illumina-based NGS with informatics approaches, we have successfully identified the tandemly repeated elements in both the Wuchereria bancrofti and Plasmodium falciparum genomes of putatively greatest copy number. Utilizing these sequences as quantitative real-time PCR (qPCR)-based targets, we have developed assays capable of exploiting the most abundant tandem repeats for both organisms. For the detection of P. falciparum, analysis and development resulted in an assay with improved analytical and field-based sensitivity vs. an established ribosomal sequence-targeting assay. Surprisingly, analysis of the W. bancrofti genome predicted a ribosomal sequence to be the genome's most abundant tandem repeat. While resulting cycle quantification values comparing a qPCR assay targeting this ribosomal sequence and a commonly targeted repetitive DNA sequence from the literature supported our finding that this ribosomal sequence was the most prevalent tandemly repeated target in the W. bancrofti genome, the resulting assay did not significantly improve detection sensitivity in conjunction with field sample testing. CONCLUSIONS/SIGNIFICANCE: Examination of pathogen genomes facilitates the development of PCR-based diagnostics targeting the most abundant and specific genomic elements. While in some instances currently available tools may deliver equal or superior performance, systematic analysis of potential targets provides confidence that the selected assays represent the most advantageous options available and that informed assay selection is occurring in the context of a particular study's objectives.

Molecular detection of intestinal helminths and protozoa among young children in Dosso Region, Niger.

Eukaryotic parasites are significant contributors to childhood illness in Niger. While helminthiases have received national attention through mass deworming efforts, the epidemiology of intestinal protozoa in Niger remains underexamined. This study employed real-time PCR diagnostics to describe the prevalence of two schistosomes, four soil-transmitted helminths, and one protozoan parasite in Boboye Department, Dosso Region. Prevalence was assessed using bulk stool specimens collected from a population-based sample of 86 children residing in 9 communities. Anthropometric measurements were used to calculate child growth z-scores and stool consistency was graded. Helminths were absent from the study population, with the exception of a single Schistosoma haematobium infection (1/86; 1.2%). Giardia duodenalis was the only protozoa present, detected in 65% (56/86) of children. Prevalence of G. duodenalis peaked in 2-year-olds with 88% (15/17) positivity. The population was generally undernourished, though growth indices did not differ significantly between children with and without G. duodenalis infection.

Rectal Swabs as an Alternative Sample Collection Method to Bulk Stool for the Real-Time PCR Detection of Giardia duodenalis.

Though bulk stool remains the gold standard specimen type for enteropathogen diagnosis, rectal swabs may offer comparable sensitivity with greater ease of collection for select pathogens. This study sought to evaluate the validity and reproducibility of rectal swabs as a sample collection method for the molecular diagnosis of Giardia duodenalis. Paired rectal swab and bulk stool samples were collected from 86 children ages 0-4 years living in southwest Niger, with duplicate samples collected among a subset of 50 children. Infection was detected using a previously validated real-time PCR diagnostic targeting the small subunit ribosomal RNA gene. Giardia duodenalis was detected in 65.5% (55/84) of bulk stool samples and 44.0% (37/84) of swab samples. The kappa evaluating test agreement was 0.81 (95% CI: 0.54-1.00) among duplicate stool samples (N = 49) and 0.75 (95% CI: 0.47-1.00) among duplicate rectal swabs (N = 48). Diagnostic sensitivity was 93% (95% CI: 84-98) by bulk stool and 63% (95% CI: 49-75) by rectal swabs. When restricting to the lowest three quartiles of bulk stool quantitation cycle values (an indication of relatively high parasite load), sensitivity by rectal swabs increased to 78.0% (95% CI: 64-89, P < 0.0001). These findings suggest that rectal swabs provide less sensitive and reproducible results than bulk stool for the real-time PCR diagnosis of G. duodenalis. However, their fair sensitivity for higher parasite loads suggests that swabs may be a useful tool for detecting higher burden infections when stool collection is excessively expensive or logistically challenging.

Comparison of multi-parallel qPCR and double-slide Kato-Katz for detection of soil-transmitted helminth infection among children in rural Bangladesh.

There is growing interest in local elimination of soil-transmitted helminth (STH) infection in endemic settings. In such settings, highly sensitive diagnostics are needed to detect STH infection. We compared double-slide Kato-Katz, the most commonly used copromicroscopic detection method, to multi-parallel quantitative polymerase chain reaction (qPCR) in 2,799 stool samples from children aged 2-12 years in a setting in rural Bangladesh with predominantly low STH infection intensity. We estimated the sensitivity and specificity of each diagnostic using Bayesian latent class analysis. Compared to double-slide Kato-Katz, STH prevalence using qPCR was almost 3-fold higher for hookworm species and nearly 2-fold higher for Trichuris trichiura. Ascaris lumbricoides prevalence was lower using qPCR, and 26% of samples classified as A. lumbricoides positive by Kato-Katz were negative by qPCR. Amplicon sequencing of the 18S rDNA from 10 samples confirmed that A. lumbricoides was absent in samples classified as positive by Kato-Katz and negative by qPCR. The sensitivity of Kato-Katz was 49% for A. lumbricoides, 32% for hookworm, and 52% for T. trichiura; the sensitivity of qPCR was 79% for A. lumbricoides, 93% for hookworm, and 90% for T. trichiura. Specificity was ≥ 97% for both tests for all STH except for Kato-Katz for A. lumbricoides (specificity = 68%). There were moderate negative, monotonic correlations between qPCR cycle quantification values and eggs per gram quantified by Kato-Katz. While it is widely assumed that double-slide Kato-Katz has few false positives, our results indicate otherwise and highlight inherent limitations of the Kato-Katz technique. qPCR had higher sensitivity than Kato-Katz in this low intensity infection setting.