Small Bowel Transit Scintigraphy in Children With Pediatric Intestinal Pseudo-Obstruction.

INTRODUCTION: Objective evidence of small intestinal dysmotility is a key criterion for the diagnosis of pediatric intestinal pseudo-obstruction (PIPO). Small bowel scintigraphy (SBS) allows for objective measurement of small bowel transit (SBT), but limited data are available in children. We aimed to evaluate the utility of SBS in children suspected of gastrointestinal dysmotility. METHODS: Patients undergoing gastric emptying studies for suspected foregut dysmotility, including PIPO, from 2016 to 2022 at 2 tertiary children's hospitals were recruited to an extended protocol of gastric emptying studies to allow for assessment of SBT. PIPO was classified based on antroduodenal manometry (ADM). SBT was compared between PIPO and non-PIPO patients. Scintigraphic parameters were assessed and correlated against ADM scores. RESULTS: Fifty-nine patients (16 PIPO and 43 non-PIPO diagnoses) were included. SBS was performed with liquid and solid meals in 40 and 26 patients, respectively. As compared to the non-PIPO group, PIPO patients had a significantly lower median percentage of colonic filling at 6 hours, with both liquid (48% vs 83%) and solid tests (5% vs 65%). SBT in PIPO patients with myopathic involvement was significantly slower than in patients with neuropathic PIPO, both for liquid and solid meal. A significant correlation was found between solid SBT and ADM scores (r = -0.638, P = 0.036). DISCUSSION: SBS provides a practically feasible assessment of small intestinal motility. It shows a potential utility to help diagnose and characterize PIPO. SBS seems most discriminative in PIPO patients with myopathic involvement. Studies in a larger pediatric population and across different ages are required.

Draft Genome Assemblies of Phage AP50c-Resistant Derivatives of Bacillus anthracis Sterne Strain 7702 Lacking Plasmid pXO2.

Mutants of the attenuated Bacillus anthracis (Sterne) strain 7702 that are resistant to phage AP50c have been previously described. Here, we report the draft genome assemblies of the parent strain, several phage-resistant derivatives, and mutants of genes in the pathways for synthesis and assembly of the S-layer.

Optimization of Oxford Nanopore Technology Sequencing Workflow for Detection of Amplicons in Real Time Using ONT-DART Tool.

An optimized, well-tested and validated targeted genomic sequencing-based high-throughput assay is currently not available ready for routine biodefense and biosurveillance applications. Earlier, we addressed this gap by developing and establishing baseline comparisons of a multiplex end-point Polymerase Chain Reaction (PCR) assay followed by Oxford Nanopore Technology (ONT) based amplicon sequencing to real time PCR and customized data processing. Here, we expand upon this effort by identifying the optimal ONT library preparation method for integration into a novel software platform ONT-DART (ONT-Detection of Amplicons in Real-Time). ONT-DART is a dockerized, real-time, amplicon-sequence analysis workflow that is used to reproducibly process and filter read data to support actionable amplicon detection calls based on alignment metrics, within sample statistics, and no-template control data. This analysis pipeline was used to compare four ONT library preparation protocols using R9 and Flongle (FL) flow cells. The two 4-Primer methods tested required the shortest preparation times (5.5 and 6.5 h) for 48 libraries but provided lower fidelity data. The Native Barcoding and Ligation methods required longer preparation times of 8 and 12 h, respectively, and resulted in higher overall data quality. On average, data derived from R9 flow cells produced true positive calls for target organisms more than twice as fast as the lower throughput FL flow cells. These results suggest that utilizing the R9 flowcell with an ONT Native Barcoding amplicon library method in combination with ONT-DART platform analytics provides the best sequencing-based alternative to current PCR-based biodetection methods.

Impact of SARS-CoV-2 Mutations on PCR Assay Sequence Alignment.

Real-time reverse transcription polymerase chain reaction (RT-PCR) assays are the most widely used molecular tests for the detection of SARS-CoV-2 and diagnosis of COVID-19 in clinical samples. PCR assays target unique genomic RNA regions to identify SARS-CoV-2 with high sensitivity and specificity. In general, assay development incorporates the whole genome sequences available at design time to be inclusive of all target species and exclusive of near neighbors. However, rapid accumulation of mutations in viral genomes during sustained growth in the population can result in signature erosion and assay failures, creating situational blind spots during a pandemic. In this study, we analyzed the signatures of 43 PCR assays distributed across the genome against over 1.6 million SARS-CoV-2 sequences. We present evidence of significant signature erosion emerging in just two assays due to mutations, while adequate sequence identity was preserved in the other 41 assays. Failure of more than one assay against a given variant sequence was rare and mostly occurred in the two assays noted to have signature erosion. Assays tended to be designed in regions with statistically higher mutations rates. in silico analyses over time can provide insights into mutation trends and alert users to the emergence of novel variants that are present in the population at low proportions before they become dominant. Such routine assessment can also potentially highlight false negatives in test samples that may be indicative of mutations having functional consequences in the form of vaccine and therapeutic failures. This study highlights the importance of whole genome sequencing and expanded real-time monitoring of diagnostic PCR assays during a pandemic.

Halo-A Universal Fluorescence Reader Based Threat Agent Detection Platform-A Proof of Concept Study Using SARS-CoV-2 Assays.

Polymerase chain reaction (PCR) remains the gold standard in disease diagnostics due to its extreme sensitivity and specificity. However, PCR tests are expensive and complex, require skilled personnel and specialized equipment to conduct the tests, and have long turnaround times. On the other hand, lateral flow immunoassay-based antigen tests are rapid, relatively inexpensive, and can be performed by untrained personnel at the point of care or even in the home. However, rapid antigen tests are less sensitive than PCR since they lack the inherent target amplification of PCR. It has been argued that rapid antigen tests are better indicators of infection in public health decision-making processes to test, trace, and isolate infected people to curtail further transmission. Hence, there is a critical need to increase the sensitivity of rapid antigen tests and create innovative solutions to achieve that goal. Herein, we report the development of a low-cost diagnostic platform, enabling rapid detection of SARS-CoV-2 under field or at-home conditions. This platform (Halo™) is a small, highly accurate, consumer-friendly diagnostic reader paired with fluorescently labeled lateral flow assays and custom software for collection and reporting of results. The focus of this study is to compare the analytical performance of HaloTM against comparable tests that use either colloidal gold nanoparticles or fluorescence-based reporters in simulated nasal matrix and not in clinical samples. Live virus data has demonstrated limit of detection performance of 1.9 TCID50/test in simulated nasal matrix for the delta variant, suggesting that single-assay detection of asymptomatic SARS-CoV-2 infections may be feasible. Performance of the system against all tested SARS CoV-2 virus variants showed comparable sensitivities indicating mutations in SARS-CoV-2 variants do not negatively impact the assay.

Development, testing and validation of a SARS-CoV-2 multiplex panel for detection of the five major variants of concern on a portable PCR platform.

Many SARS-CoV-2 variants have emerged during the course of the COVID-19 pandemic. These variants have acquired mutations conferring phenotypes such as increased transmissibility or virulence, or causing diagnostic, therapeutic, or immune escape. Detection of Alpha and the majority of Omicron sublineages by PCR relied on the so-called S gene target failure due to the deletion of six nucleotides coding for amino acids 69-70 in the spike (S) protein. Detection of hallmark mutations in other variants present in samples relied on whole genome sequencing. However, whole genome sequencing as a diagnostic tool is still in its infancy due to geographic inequities in sequencing capabilities, higher cost compared to other molecular assays, longer turnaround time from sample to result, and technical challenges associated with producing complete genome sequences from samples that have low viral load and/or high background. Hence, there is a need for rapid genotyping assays. In order to rapidly generate information on the presence of a variant in a given sample, we have created a panel of four triplex RT-qPCR assays targeting 12 mutations to detect and differentiate all five variants of concern: Alpha, Beta, Gamma, Delta, and Omicron. We also developed an expanded pentaplex assay that can reliably distinguish among the major sublineages (BA.1-BA.5) of Omicron. In silico, analytical and clinical testing of the variant panel indicate that the assays exhibit high sensitivity and specificity. This panel can help fulfill the need for rapid identification of variants in samples, leading to quick decision making with respect to public health measures, as well as treatment options for individuals. Compared to sequencing, these genotyping PCR assays allow much faster turn-around time from sample to results-just a couple hours instead of days or weeks.

Blind Spot: A Braille Patterned Novel Multiplex Lateral Flow Immunoassay Sensor Array for the Detection of Biothreat Agents.

Lateral flow immunoassays (LFIs) are simple, point-of-care diagnostic devices used for detecting biological agents or other analytes of interest in a sample. LFIs are predominantly singleplex assays, interrogating one target analyte at a time. There is a need for multiplex LFI devices, e.g., a syndromic panel to differentiate pathogens causing diseases exhibiting similar symptoms. Multiplex LFI devices would be especially valuable in instances where sample quantity is limiting and reducing assay time and costs is critical. There are limitations to the design parameters and performance characteristics of a multiplex LFI assay with many horizontal test lines due to constraints in capillary flow dynamics. To address some of the performance issues, we have developed a spot array multiplex LFI using Braille format (hence called Blind Spot) and a sensor, MACAW (Modular Automated Colorimetric Analyses Widget), that can analyze and interpret the results. As a proof of concept, we created a multiplex toxin panel, for detecting three toxins, using two letter codes for each. The results indicated that the six-plex, triple toxin assay performs as well as singleplex assays. The sensor-based calls are better compared to human interpretation in discriminating and interpreting ambiguous test results correctly especially at lower antigen concentrations and from strips with blemishes.

Estimation of prevalence of transthyretin (ATTR) cardiac amyloidosis in an Australian subpopulation using bone scans with echocardiography and clinical correlation.

BACKGROUND: Bone scans differentiate transthyretin (ATTR) cardiac amyloidosis from light chain amyloidosis and other causes of increased left ventricular (LV) wall thickness. We examined the prevalence and implications of cardiac uptake in the general population. METHODS: Patients were included based on having undertaken a bone scan for non-cardiac indications using Technetium 99m hydroxymethylene diphosphonate (HMDP) or Technetium 99m methylene diphosphonate (MDP). Blinded image review was undertaken. Positive was defined as cardiac uptake ≥ rib AND heart/whole body ratio (H/WB) > 0.0388. Echocardiography and clinical records were reviewed. RESULTS: 6918 patients were included. 15/3472 HMDP scans were positive (14 males, 1 female): none in individuals aged < 65; 1.44% in males and 0.17% in females ≥ 65; 6.15% in males and 1.69% in females ≥ 85. Only 1/3446 MDP scans were positive. All HMDP positive patients had increased septal wall thickness on echocardiography. H/WB correlated positively with LV mass, and negatively with LV ejection fraction. No individual had an explanation other than ATTR for their positive scan. CONCLUSION: In this Australian subpopulation, the prevalence of positive bone scans consistent with cardiac ATTR is 0% in individuals aged < 65. Prevalence increased with age, reaching 6.15% in men ≥ 85. The amount of HMDP uptake correlated with echocardiographic features of more advanced cardiac involvement. MDP does not appear useful in ATTR.

Complete Genome Sequence of Francisella tularensis Live Vaccine Strain NR-28537 (BEI Master Cell Bank).

The genome of Francisella tularensis live vaccine strain NR-28537 was sequenced by a hybrid approach utilizing an Oxford Nanopore Technologies R9 flow cell and an Illumina MiSeq platform. De novo assembly of the resulting long and short reads produced a single-contig whole-genome sequence.

Draft Whole-Genome Sequences of Ciprofloxacin-Resistant Derivatives of a Bacillus anthracis ΔANR Strain Lacking pXO1 and pXO2 Plasmids.

Mutants of an attenuated Bacillus anthracis (ΔANR) strain conferring increasing levels of ciprofloxacin resistance have been described. Here, we report the draft genome sequences of the parent strain (ΔANR pXO1-, pXO2-) and its derivatives conferring low (step 1; 0.5 µg/ml), medium (step 2; 8 to 16 µg/ml), and high (step 3; 32 to 64 µg/ml) levels of ciprofloxacin resistance.

Comparison of the performance of an amplicon sequencing assay based on Oxford Nanopore technology to real-time PCR assays for detecting bacterial biodefense pathogens.

BACKGROUND: The state-of-the-art in nucleic acid based biodetection continues to be polymerase chain reaction (PCR), and many real-time PCR assays targeting biodefense pathogens for biosurveillance are in widespread use. These assays are predominantly singleplex; i.e. one assay tests for the presence of one target, found in a single organism, one sample at a time. Due to the intrinsic limitations of such tests, there exists a critical need for high-throughput multiplex assays to reduce the time and cost incurred when screening multiple targets, in multiple pathogens, and in multiple samples. Such assays allow users to make an actionable call while maximizing the utility of the small volumes of test samples. Unfortunately, current multiplex real-time PCR assays are limited in the number of targets that can be probed simultaneously due to the availability of fluorescence channels in real-time PCR instruments. RESULTS: To address this gap, we developed a pipeline in which the amplicons produced by a 14-plex end-point PCR assay using spiked samples were subsequently sequenced using Nanopore technology. We used bar codes to sequence multiple samples simultaneously, leading to the generation and subsequent analysis of sequence data resulting from a short sequencing run time (< 10 min). We compared the limits of detection (LoD) of real-time PCR assays to Oxford Nanopore Technologies (ONT)-based amplicon sequencing and estimated the sample-to-answer time needed for this approach. Overall, LoDs determined from the first 10 min of sequencing data were at least one to two orders of magnitude lower than real-time PCR. Given enough time, the amplicon sequencing approach is approximately 100 times more sensitive than real-time PCR, with detection of amplicon specific reads even at the lowest tested spiking concentration (around 2.5-50 Colony Forming Units (CFU)/ml). CONCLUSIONS: Based on these results, we propose amplicon sequencing assay as a viable alternative to replace the current real-time PCR based singleplex assays for higher throughput biodefense applications. We note, however, that targeted amplicon specific reads were not detectable even at the highest tested spike concentrations (2.5 X 104-5.0 X105 CFU/ml) without an initial amplification step, indicating that PCR is still necessary when utilizing this protocol.

Avirulent Bacillus anthracis Strain with Molecular Assay Targets as Surrogate for Irradiation-Inactivated Virulent Spores.

The revelation in May 2015 of the shipment of γ irradiation-inactivated wild-type Bacillus anthracis spore preparations containing a small number of live spores raised concern about the safety and security of these materials. The finding also raised doubts about the validity of the protocols and procedures used to prepare them. Such inactivated reference materials were used as positive controls in assays to detect suspected B. anthracis in samples because live agent cannot be shipped for use in field settings, in improvement of currently deployed detection methods or development of new methods, or for quality assurance and training activities. Hence, risk-mitigated B. anthracis strains are needed to fulfill these requirements. We constructed a genetically inactivated or attenuated strain containing relevant molecular assay targets and tested to compare assay performance using this strain to the historical data obtained using irradiation-inactivated virulent spores.

Evaluation of Signature Erosion in Ebola Virus Due to Genomic Drift and Its Impact on the Performance of Diagnostic Assays.

Genome sequence analyses of the 2014 Ebola Virus (EBOV) isolates revealed a potential problem with the diagnostic assays currently in use; i.e., drifting genomic profiles of the virus may affect the sensitivity or even produce false-negative results. We evaluated signature erosion in ebolavirus molecular assays using an in silico approach and found frequent potential false-negative and false-positive results. We further empirically evaluated many EBOV assays, under real time PCR conditions using EBOV Kikwit (1995) and Makona (2014) RNA templates. These results revealed differences in performance between assays but were comparable between the old and new EBOV templates. Using a whole genome approach and a novel algorithm, termed BioVelocity, we identified new signatures that are unique to each of EBOV, Sudan virus (SUDV), and Reston virus (RESTV). Interestingly, many of the current assay signatures do not fall within these regions, indicating a potential drawback in the past assay design strategies. The new signatures identified in this study may be evaluated with real-time reverse transcription PCR (rRT-PCR) assay development and validation. In addition, we discuss regulatory implications and timely availability to impact a rapidly evolving outbreak using existing but perhaps less than optimal assays versus redesign these assays for addressing genomic changes.