Blueprint for Phasing and Assembling the Genomes of Heterozygous Polyploids: Application to the Octoploid Genome of Strawberry

The challenge of allelic diversity for assembling haplotypes is exemplified in polyploid genomes containing homoeologous chromosomes of identical ancestry, and significant homologous variation within their ancestral subgenomes. Cultivated strawberry (Fragaria x ananassa) and its wild progenitors are outbred octoploids (2n = 8x = 56) in which up to eight homologous and homoeologous alleles are preserved. This introduces significant risk of haplotype collapse, switching, and chimeric fusions during assembly. Using third generation HiFi sequences from PacBio, we assembled the genome of the day-neutral octoploid F. x ananassa hybrid 'Royal Royce' from the University of California. Our goal was to produce subgenome- and haplotype-resolved assemblies of all 56 chromosomes, accurately reconstructing the parental haploid chromosome complements. Previous work has demonstrated that partitioning sequences by parental phase supports direct assembly of haplotypes in heterozygous diploid species. We leveraged the accuracy of HiFi sequence data with pedigree-informed sequencing to partition long read sequences by phase, and reduce the downstream risk of subgenomic chimeras during assembly. We were able to utilize an octoploid strawberry recombination breakpoint map containing 3.6 M variants to identify and break chimeric junctions, and perform scaffolding of the phase-1 and phase-2 octoploid assemblies. The N50 contiguity of the phase-1 and phase-2 assemblies prior to scaffolding and gap-filling was 11 Mb. The final haploid assembly represented seven of 28 chromosomes in a single contiguous sequence, and averaged fewer than three gaps per pseudomolecule. Additionally, we re-annotated the octoploid genome to produce a custom F. x ananassa repeat library and improved set of gene models based on IsoSeq transcript data and an expansive RNA-seq expression atlas. Here we present 'FaRR1', a gold-standard reference genome of F. x ananassa cultivar 'Royal Royce' to assist future genomic research and molecular breeding of allo-octoploid strawberry.

Validation of a New Contactless and Continuous Respiratory Rate Monitoring Device Based on Ultra-Wideband Radar Technology

Respiratory rate (RR) is typically the first vital sign to change when a patient decompensates. Despite this, RR is often monitored infrequently and inaccurately. The Circadia Contactless Breathing Monitor™ (model C100) is a novel device that uses ultra-wideband radar to monitor RR continuously and un-obtrusively. Performance of the Circadia Monitor was assessed by direct comparison to manually scored reference data. Data were collected across a range of clinical and non-clinical settings, considering a broad range of user characteristics and use cases, in a total of 50 subjects. Bland–Altman analysis showed high agreement with the gold standard reference for all study data, and agreement fell within the predefined acceptance criteria of ±5 breaths per minute (BrPM). The 95% limits of agreement were −3.0 to 1.3 BrPM for a nonprobability sample of subjects while awake, −2.3 to 1.7 BrPM for a clinical sample of subjects while asleep, and −1.2 to 0.7 BrPM for a sample of healthy subjects while asleep. Accuracy rate, using an error margin of ±2 BrPM, was found to be 90% or higher. Results demonstrate that the Circadia Monitor can effectively and efficiently be used for accurate spot measurements and continuous bedside monitoring of RR in low acuity settings, such as the nursing home or hospital ward, or for remote patient monitoring.

A rapid calprotectin test for the diagnosis of pleural effusion

In previous studies, measuring the levels of calprotectin in patients with pleural effusion (PE) was an exceptionally accurate way to predict malignancy. Here, we evaluated a rapid method for the measurement of calprotectin levels as a useful parameter in the diagnosis of malignant pleural effusion (MPE) in order to minimise invasive diagnostic tests. Calprotectin levels were measured with Quantum Blue® sCAL (QB®sCAL) and compared with the gold standard reference ELISA method. Calprotectin levels in patients with benign pleural effusion (BPE) were significantly higher (p < 0.0001) than for MPE patients. We measured the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and positive and negative likelihood ratios (LRs) for a cut-off value of ≤ 14,150 ng/mL; the diagnostic accuracy was 64%. The odds ratio for PE calprotectin levels was 10.938 (95% CI [4.133 − 28.947]). The diagnostic performance of calprotectin concentration was better for predicting MPE compared to other individual parameters. Comparison of two assays showed a slope of 1.084, an intercept of 329.7, and a Pearson correlation coefficient of 0.798. The Bland–Altman test showed a positive bias for the QB®sCAL method compared to ELISA fCAL®. Clinical concordance between both these methods was 88.5% with a Cohen kappa index of 0.76 (95% CI [0.68 − 0.84]). We concluded that QB®sCAL is a fast, reliable, and non-invasive diagnostic tool for diagnosing MPE and represents an alternative to ELISA that could be implemented in medical emergencies.

John Watson (1807–1863) and his Pioneer Work On the Surgery of Esophagus

Backround. John Watson (1807-1863) was the first surgeon in USA who performed and published his results on esophagotomy. Methods. His pioneer surgical work was a gold standard reference for the later surgeons. Results. Watson was a devoted surgeon and his innovative operating techniques secured him a place in the hall of fame of the history of medicine.

Validation of a search strategy for randomized clinical trials related to periodontitis

Background Systematic reviews, considered the gold standard for the assessment of scientific evidence, may present conflicting findings for the same clinical issue, and such dissent may be justified by the forms of elaboration of the electronic search strategy. This paper aims to validate a search strategy to identify randomized clinical trials related to periodontitis. A gold standard reference set was developed to validate the identified clinical trials using the relative recall method. The choice of periodontitis is due to the fact that this disease has a high prevalence among chronic non-communicable diseases, is considered the second most common oral disease in the world, is associated with several health problems, such as cardiovascular diseases and diabetes, and, principally, has not been investigated sufficiently to prevent possible damages resulting from it. Methods A validation study was developed in MEDLINE/PubMed. In Stage 1, a methodological filter recommended by the Cochrane Collaboration to identify randomized clinical trials was applied. Stage 2 identified articles related only to periodontitis (gold standard reference set) from among the articles retrieved using the eligibility criteria. In Stage 3, a search statement for the retrieval of periodontitis-related articles was elaborated by experts. Stage 4 defined the proposed search strategy comprising of the combination of the search statement developed with the aforementioned methodological filter and subsequent application in MEDLINE/PubMed. The obtained data were analyzed using the set of articles identified in Stage 2, as the gold standard reference set. The following performance values were calculated - sensitivity, specificity, accuracy, and number needed to read - with their respective 95% confidence interval (95%CI). Results The search strategy under evaluation compared to the gold-standard showed a sensitivity of 93.2% (95%CI, 83.8–97.3), specificity of 99.9% (95%CI 99.8–99.9), and a precision of 77.5% (95%CI, 66.48–85.63). In addition, the number needed to read was 1.3. Conclusion According to the proposed methodological approach, the search strategy under evaluation performed well in the identification of randomized clinical trials related to periodontitis.

Comparison of PCR and phenotypic methods for the detection of methicillin resistant Staphylococcus aureus

Background and Objectives: Resistance to methicillin in methicillin resistant strains of Staphylococcus aureus (MRSA) is due to the presence of mec-A gene ,which encodes a low affinity penicillin binding protein (PBP)-2a or PBP2. Accurate and rapid identification of MRSA in clinical specimens is essential for timely decision on effective treatment. The aim of the study was to compare three different methods for detection of MRSA namely cefoxitin disc diffusion, CHROM agar MRSA and VITEK-2 susceptibility with PCR which is the gold standard reference method and to find the antibiotic susceptibility pattern of these isolates by VITEK-2. Materials and Methods: A Total of 100 non-duplicate S. aureus isolates were collected from different clinical samples among both outpatient and inpatients. Detection of MRSA among these isolates was done by cefoxitin disc diffusion, VITEK-2, CHROM agar MRSA and PCR. Results: The sensitivity and specificity of cefoxitin disc diffusion and Vitek was found to be 97.2% and 100%, while that of CHROM agar was found to be 100% and 78.6%. The overall prevalence of MRSA in our study by PCR was 72%. Conclusion: Based on the findings in our study, isolates which show cefoxitin zone diameter < 22 mm can be reported as MRSA. However, those isolates which have a zone diameter between 22-24 mm, should ideally be confirmed by PCR.

Chest patch for continuous vital-sign monitoring: A clinical validation study during movement and controlled hypoxia. (Preprint)

BACKGROUND Early warning scores in general wards are commonly limited by intermittent manual measurements; these are recognised as being time consuming and for impacting monitoring frequency. Wearable devices may support healthcare staff, improve patient safety and promote early deterioration detection. However available ambulatory monitoring devices need to be tested and validated before clinical implementation. OBJECTIVE The objective of this study is to determine the agreement between a chest-worn patch (VitalPatch®) and gold standard reference device for heart rate (HR) and respiratory rate (RR) measurements during movement and during gradual de-saturation in a controlled environment. METHODS After both VitalPatch and gold standard device (Philips MX450) were placed, participants performed 7 different movements: At rest, Sit-to-Stand, Tapping, Rubbing, Drinking, Turning Pages, and Using a Tablet. In a controlled environment. Participants were then made hypoxic gradually down to 80% peripheral oxygen saturations. The primary outcome measures were the accuracy, defined as the mean absolute error (MAE) of the VitalPatch estimates when compared with their gold-standards. We defined these to be clinical acceptable if within 5 beats per minute (bpm) for HR and 3 respirations per minute (rpm) for RR. RESULTS We acquired complete datasets of 29 participants. In the movement phase, HR estimates were within the pre-specified limits for all movements. For RR, estimates were also inside the acceptable range, with the exception of the Sit-to-Stand and Turning Pages movements, showing a MAE (95% CI) of 3.05 (2.48, 3.58) rpm and 3.45 (2.71, 4.11) rpm, respectively. For the hypoxia phase, these were always within the limits with an overall MAE for HR and RR of 0.72 (0.66, 0.78) bpm and 1.89 (1.75, 2.03) rpm, respectively. There were no significant differences in the VitalPatch performance across a range of oxygen desaturations. CONCLUSIONS The VitalPatch was highly accurate throughout movement tests except for its RR estimation during two movements. This device was reliable throughout the hypoxia stages, with no significant accuracy differences in normoxia (≥ 90%), mild (89.9 - 85%) and severe hypoxia (< 85%). CLINICALTRIAL ISRCTN61535692

Diagnostic accuracy of an in-house Scrub Typhus enzyme linked immunoassay for the detection of IgM and IgG antibodies in Laos

Scrub typhus is a major cause of morbidity and mortality in Southeast Asia. Diagnosis of scrub typhus is difficult due to a lack of accessible validated diagnostic tools. Despite its objectivity, the diagnostic accuracy of ELISA tests is influenced by methodological and patient factors. This study aims to evaluate the performance of a novel in-house ELISA developed in the Mahidol Oxford Tropical Medicine Research Unit (MORU) for anti-scrub typhus group IgM and IgG compared to the “gold standard” reference IFA and PCR, and to determine whether the in-house ELISA can be used as a seroepidemiological screening tool and/or stand-alone test for scrub typhus. A total of 1,976 admission and 1,438 participant follow-up sera collected in the Lao PDR (Laos) were tested with ELISA for IgM and IgG. Samples with an ELISA OD≥0.50 were tested with IFA for IgM and/or IgG. A strong positive relationship was present between ELISA ODs and IFA titers for admission IgM (r2: 0.70, p <0.005) and IgG (r2: 0.76, p<0.005), and for follow-up IgM and IgG (both r2: 0.76, p<0.005) samples. The best compromise between sensitivity and specificity for the ELISA OD cut-off is likely to be between 0.8–1.0 for IgM antibodies and 1.2–1.8 for IgG antibodies. These results demonstrate that the diagnostic accuracy of the MORU in-house scrub typhus group ELISA is comparable to that of IFA, with similar results as reported for the commonly used InBios Scrub Typhus Detect ELISA, validating the use of the in-house ELISA. The optimal ELISA cut-off would depend on the use of the test, and the desired sensitivity and specificity. Further studies are required to authenticate the use of these cut-offs in other endemic regions. This in-house ELISA has the potential to replace the imperfect IFA, which could ultimately reduce the burden of scrub typhus by improving the rate of scrub typhus diagnoses in endemic low-resource areas.

Pre-Collaborative Validation of an Amperometric Immunosensor for Salmonella

The method of Salmonella detection recommended is cultural, but it is laborious, presents a high consumption of material, and requires about five days for presumptive results. Immunosensor is an alternative tool that has shown promising and rapid results, although many devices have their performance evaluated only under buffering conditions and few achieve the validation stage. The objective was to perform a pre-collaborative validation of an electrochemical immunosensor assembled on screen-printed electrodes for the detection of Salmonella sp. in milk. The antibodies were immobilized by cysteamine self-assembled monolayer. The sandwich-type amperometric immunosensor was evaluated for contaminated raw and whole UHT milk and compared to performance with a gold standard reference method (BAM) according to AOAC recommendations for a single laboratory. A binary response (positive/negative) of the immunosensor was used based on a cut off established from current electric obtained for the absence of the pathogen. There was no significant difference for the results of the biosensor and the reference method, in the absence and the levels from 101 to 103 CFU mL−1 of Salmonella Typhimurium for the two types of milk. This result indicates the efficiency of the biosensor in detecting the pathogen into a complex matrix.