Confirmation of multi-parallel quantitative real-time PCR as the gold standard for detecting soil-transmitted helminths in stool

Due to its simplicity and cost-effectiveness, microscopy has seen extensive field-use as the diagnostic standard for the detection of soil-transmitted helminths (STH) in stool samples. However, the sensitivity of microscopy-based detection is inadequate in reduced-transmission settings where worm burden is oftentimes low. Equally problematic, eggs of closely related species oftentimes have indistinguishable morphologies, leading to species misidentification. In light of these shortcomings, the purpose of this study was to demonstrate multi-parallel quantitative real-time PCR (qPCR) as the new “gold standard” for STH detection. Accordingly, stool samples from non-endemic participants were spiked with limited numbers of eggs or larvae (1 to 40) of five different species of STH. DNA extracts were tested using two unique multi-parallel real-time PCR-based diagnostic methods. These methods employed different target sequences (ribosomal internal transcribed spacer, or highly repetitive non-coding regions), to evaluate the detection of DNA from as little as one egg per sample. There was a statistically significant kendall correlation between egg/larvae counts and qPCR from both methods for Trichuris trichiura (0.86 and 0.872 for NHM and Baylor assays) and a strong correlation (0.602 and 0.631 for NHM and Baylor assays, respectively) for Ascaris lumbricoides. Less strong but still significant was the Kendall Tau-b value for A. duodenale (0.408 for both) and for S. stercoralis (0.483 and 0.653, respectively). In addition, using field stool samples from rural Argentina both assays had fair to moderate kappa agreement (0.329-0.454), except for Strongyloides stercoralis (0.121) that both assays had slight agreement. In spite of the small cohort of samples, both qPCR assays, targeting of two independent genomic regions, provided reproducible results and we believe that, low cost multi-parallel quantitative real-time PCR-based diagnostics should supplant microscopy as the new gold standard for stool-based detection of soil transmitted helminths in public-health and community settings.

Laboratory and Field Evaluations of a Commercially Available Real-Time Loop-Mediated Isothermal Amplification Assay for the Detection of West Nile Virus in Mosquito Pools

ABSTRACT Although the specific cDNA amplification mechanisms of reverse-transcriptase polymerase chain reaction (RT-PCR) and RT loop-mediated isothermal amplification (RT-LAMP) are very different, both molecular assays serve as options to detect arboviral RNA in mosquito pools. Like RT-PCR, RT-LAMP uses a reverse transcription step to synthesize complementary DNA (cDNA) from an RNA template and then uses target-specific primers to amplify cDNA to detectable levels in a single-tube reaction. Using laboratory-generated West Nile virus (WNV) samples and field-collected mosquito pools, we evaluated the sensitivity and specificity of a commercially available WNV real-time RT-LAMP assay (Pro-AmpRT™ WNV; Pro-Lab Diagnostics, Inc., Round Rock, Texas) and compared the results to a validated real-time RT-PCR assay. Laboratory generated virus stock samples containing ≥ 2.3 log10 plaque-forming units (PFU)/ml and intrathoracically inoculated mosquitoes containing ≥ 2.4 log10 PFU/ml produced positive results in the Pro-AmpRT WNV assay. Of field-collected pools that were WNV positive by real-time RT-PCR, 74.5% (70 of 94) were also positive by the Pro-AmpRT WNV assay, resulting in an overall Cohen's kappa agreement of 79.4% between the 2 tests. The Pro-AmpRT WNV assay shows promise as a suitable virus screening tool for vector surveillance programs provided agencies are aware of its characteristics and limitations.

Validation of a Novel IoT and AI based Point-of-Care Testing Laboratory: Analytical Accuracy and Clinical Agreement

Point-of-care testing (POCT) offers several advantages over traditional laboratory testing. Offering less invasive testing with a faster turnaround time is not enough if not associated with an acceptable level of accuracy. Here, we show the analytical validation behind the multi-analyte POCT immunochromatography analyser, Hilab Flow (HiF). Analyses from 4,518 clinical samples were compared to College of American Pathologists accredited laboratories for ten quantitative and thirteen qualitative exams. Compatibility between methods was evaluated in terms of association/correlation and clinical agreement. Strong correlation/ concordance was observed between quantitative (CHOL, HDL-c, TG, HbA1c, Glycemia, 25-Hydroxy Vitamin D, TSH, Uric Acid, Creatinine, Urea) and qualitative methods (COVID-19 IgG/ IgM, Beta-hCG, Syphilis, Anti-HBsAg, Zika IgG/ IgM, Influenza A/B, HIV, HCV, HBsAg, Dengue NS1, COVID-19 Ag, Dengue IgG/ IgM, PSA). Approval criteria was obtaining a kappa agreement > 0.8 or a Pearson correlation > 0.9 depending on the exam. Overall percentage agreement was greater than 95% for all exams, indicating a good clinical agreement to gold-standard laboratory-based tests. Results indicate all exams are suitable for POCT and present a reliable performance. Data support the analyser is a useful tool to aid decision-making at the clinical setting, with potential to contribute with healthcare solutions in diagnostic medicine worldwide.

Central incisors shape and proportions prevalence in Argentinian university students: by visual assessment and a new standardized method

The aim of this study was to analyze the dimensions of the clinical crown of upper central incisors and the prevalence of tooth shapes by two different protocols. Assessment was performed on each of the 111 dental stone type V maxillae models of students of dentistry from Buenos Aires University [93 females and 18 males, mean age 23.70 (± 2.26) years] The mesial and distal-vestibular angles were defined on each right upper incisor, and the following segments were defined: AB (zenith - incisal edge), CD, EF, GH (apical, middle, and incisal thirds - vestibule - mesial and vestibule-distal angles) and their lengths were determined with a precision caliper. Then, the CD/AB, EF/AB and mean CD-EF/AB ratios were calculated. Shapes were assessed by four independent observers, three of whom evaluated digital images of the models, while the fourth had no access to the images, and determined the shapes using an algorithm developed from the dimensions of the studied segments. Rates and confidence intervals were determined, and Fleiss’ Kappa was calculated to assess the agreement among the evaluators who worked with the images and among all of them. Average incisor length was 10 mm, and widths at CD and EF were 7.35 mm (0.65) and 8.27 mm (0.58), respectively. Regarding shapes, 51.58% (47.90-55.20) of the incisors were identified as square, 18.02% (14.50-21.90) as ovoid and 30.41% (30.00-30.90) as triangular. Fleiss’ Kappa agreement was 0.71 (0.62-0.80). The application of the proposed algorithm provided a considerable level of agreement among the observers. Regarding tooth size, both the average segment length and the proportions were similar to those reported by various authors.

Low-count whole-body PET with deep learning in a multicenter and externally validated study

More widespread use of positron emission tomography (PET) imaging is limited by its high cost and radiation dose. Reductions in PET scan time or radiotracer dosage typically degrade diagnostic image quality (DIQ). Deep-learning-based reconstruction may improve DIQ, but such methods have not been clinically evaluated in a realistic multicenter, multivendor environment. In this study, we evaluated the performance and generalizability of a deep-learning-based image-quality enhancement algorithm applied to fourfold reduced-count whole-body PET in a realistic clinical oncologic imaging environment with multiple blinded readers, institutions, and scanner types. We demonstrate that the low-count-enhanced scans were noninferior to the standard scans in DIQ (p < 0.05) and overall diagnostic confidence (p < 0.001) independent of the underlying PET scanner used. Lesion detection for the low-count-enhanced scans had a high patient-level sensitivity of 0.94 (0.83–0.99) and specificity of 0.98 (0.95–0.99). Interscan kappa agreement of 0.85 was comparable to intrareader (0.88) and pairwise inter-reader agreements (maximum of 0.72). SUV quantification was comparable in the reference regions and lesions (lowest p-value=0.59) and had high correlation (lowest CCC = 0.94). Thus, we demonstrated that deep learning can be used to restore diagnostic image quality and maintain SUV accuracy for fourfold reduced-count PET scans, with interscan variations in lesion depiction, lower than intra- and interreader variations. This method generalized to an external validation set of clinical patients from multiple institutions and scanner types. Overall, this method may enable either dose or exam-duration reduction, increasing safety and lowering the cost of PET imaging.

WHO/ISUP grading of clear cell renal cell carcinoma and papillary renal cell carcinoma; validation of grading on the digital pathology platform and perspectives on reproducibility of grade

Background There are recognised potential pitfalls in digital diagnosis in urological pathology, including the grading of dysplasia. The World Health Organisation/International Society of Urological Pathology (WHO/ISUP) grading system for renal cell carcinoma (RCC) is prognostically important in clear cell RCC (CCRCC) and papillary RCC (PRCC), and is included in risk stratification scores for CCRCC, thus impacting on patient management. To date there are no systematic studies examining the concordance of WHO/ISUP grading between digital pathology (DP) and glass slide (GS) images. We present a validation study examining intraobserver agreement in WHO/ISUP grade of CCRCC and PRCC. Methods Fifty CCRCCs and 10 PRCCs were graded (WHO/ISUP system) by three specialist uropathologists on three separate occasions (DP once then two GS assessments; GS1 and GS2) separated by wash-out periods of at least two-weeks. The grade was recorded for each assessment, and compared using Cohen’s and Fleiss’s kappa. Results There was 65 to 78% concordance of WHO/ISUP grading on DP and GS1. Furthermore, for the individual pathologists, the comparative kappa scores for DP versus GS1, and GS1 versus GS2, were 0.70 and 0.70, 0.57 and 0.73, and 0.71 and 0.74, and with no apparent tendency to upgrade or downgrade on DP versus GS. The interobserver kappa agreement was less, at 0.58 on DP and 0.45 on GS. Conclusion Our results demonstrate that the assessment of WHO/ISUP grade on DP is noninferior to that on GS. There is an apparent slight improvement in agreement between pathologists on RCC grade when assessed on DP, which may warrant further study.

Serological Detection of SARS-CoV-2 Antibodies in Naturally-Infected Mink and Other Experimentally-Infected Animals

The recent emergence of SARS-CoV-2 in humans from a yet unidentified animal reservoir and the capacity of the virus to naturally infect pets, farmed animals and potentially wild animals has highlighted the need for serological surveillance tools. In this study, the luciferase immunoprecipitation systems (LIPS), employing the spike (S) and nucleocapsid proteins (N) of SARS-CoV-2, was used to examine the suitability of the assay for antibody detection in different animal species. Sera from SARS-CoV-2 naturally-infected mink (n = 77), SARS-CoV-2 experimentally-infected ferrets, fruit bats and hamsters and a rabbit vaccinated with a purified spike protein were examined for antibodies using the SARS-CoV-2 N and/or S proteins. From comparison with the known neutralization status of the serum samples, statistical analyses including calculation of the Spearman rank-order-correlation coefficient and Cohen’s kappa agreement were used to interpret the antibody results and diagnostic performance. The LIPS immunoassay robustly detected the presence of viral antibodies in naturally infected SARS-CoV-2 mink, experimentally infected ferrets, fruit bats and hamsters as well as in an immunized rabbit. For the SARS-CoV-2-LIPS-S assay, there was a good level of discrimination between the positive and negative samples for each of the five species tested with 100% agreement with the virus neutralization results. In contrast, the SARS-CoV-2-LIPS-N assay did not consistently differentiate between SARS-CoV-2 positive and negative sera. This study demonstrates the suitability of the SARS-CoV-2-LIPS-S assay for the sero-surveillance of SARS-CoV-2 infection in a range of animal species.

IS THE “U-SIGN” RADIOLOGIC FEATURE OF A POSTERIOR CRUCIATE LIGAMENT TIBIAL AVULSION FRACTURE?

ABSTRACT Objective: By analyzing our cases of posterior cruciate ligament (PCL) tibial avulsion fracture, we noted that a U-shaped image was present in the anteroposterior plain radiographs view of the affected knee, even in cases where the profile view of the knee had been inconclusive as to tibial PCL avulsion fracture, a “hidden” fracture. Therefore, we aimed to investigate whether there was an anatomical correlation between this radiological U sign and the tibial insertion of the PCL and to ascertain the intra- and inter-rater reliability of this sign in clinical practice. Methods: The data of the widths and heights area of the PCL tibial insertion area, and the U sign area were measured and compared to the largest width of the tibia. Two moreover, the reliability and reproducibility of this imaging were analyzed. Results: The areas height of the U-sign area and the anatomical insertion area of the posterior cruciate ligament showed no difference, and both were topographically located in the two central quarters of the proximal end of the tibia. The radiographic assessment showed excellent Kappa agreement rates between interobserver and intraobserver, with high reliability and reproducibility. Conclusion: The U sign is a radiographic feature of PCL tibial avulsion fracture seen on the radiograph AP view, there is a high association between the ratios of the U-sign area height in the X-ray and the anatomical height of the PCL tibial insertion site MRI with the largest width of the proximal tibia. The radiographic U sign showed excellent rates of interobserver and intraobserver agreement with Kappa values higher than 0.8. Level of Evidence IV; Dignostic Studies - Investigating a Diagnostic Test.

Prevalence of male circumcision in four culturally non-circumcising counties in western Kenya after 10 years of program implementation from 2008 to 2019

Introduction Kenya started implementing voluntary medical male circumcision (VMMC) for HIV prevention in 2008 and adopted the use of decision makers program planning tool version 2 (DMPPT2) in 2016, to model the impact of circumcisions performed annually on the population prevalence of male circumcision (MC) in the subsequent years. Results of initial DMPPT2 modeling included implausible MC prevalence estimates, of up to 100%, for age bands whose sustained high uptake of VMMC pointed to unmet needs. Therefore, we conducted a cross-sectional survey among adolescents and men aged 10–29 years to determine the population level MC prevalence, guide target setting for achieving the goal of 80% MC prevalence and for validating DMPPT2 modelled estimates. Methods Beginning July to September 2019, a total of 3,569 adolescents and men aged 10–29 years from households in Siaya, Kisumu, Homa Bay and Migori Counties were interviewed and examined to establish the proportion already circumcised medically or non-medically. We measured agreement between self-reported and physically verified circumcision status and computed circumcision prevalence by age band and County. All statistical were test done at 5% level of significance. Results The observed MC prevalence for 15-29-year-old men was above 75% in all four counties; Homa Bay 75.6% (95% CI [69.0–81.2]), Kisumu 77.9% (95% CI [73.1–82.1]), Siaya 80.3% (95% CI [73.7–85.5]), and Migori 85.3% (95% CI [75.3–91.7]) but were 0.9–12.4% lower than DMPPT2-modelled estimates. For young adolescents 10–14 years, the observed prevalence ranged from 55.3% (95% CI [40.2–69.5]) in Migori to 74.9% (95% CI [68.8–80.2]) in Siaya and were 25.1–32.9% lower than DMMPT 2 estimates. Nearly all respondents (95.5%) consented to physical verification of their circumcision status with an agreement rate of 99.2% between self-reported and physically verified MC status (kappa agreement p-value<0.0001). Conclusion This survey revealed overestimation of MC prevalence from DMPPT2-model compared to the observed population MC prevalence and provided new reference data for setting realistic program targets and re-calibrating inputs into DMPPT2. Periodic population-based MC prevalence surveys, especially for established programs, can help reconcile inconsistencies between VMMC program uptake data and modeled MC prevalence estimates which are based on the number of procedures reported in the program annually.

Construct validity of The Routine Assessment of Patient Index Data 3 (RAPID3) in the evaluation of axial spondyloarthritis

Objective Although there are different tools to evaluate axial spondyloarthritis (axSpA), they are hardly used in clinical routine due to time constraints. The Routine Assessment of Patient Index Data 3 (RAPID3) is a composite measure feasible to be used as a sole metric in busy clinics. We aimed to test its measurement properties in patients with axial SpA in a real clinical setting. Methods Cross-sectional study that included 131 consecutive patients with axial SpA. The convergent (Spearman's rho) and discriminant (ROC curve analysis) validity of RAPID3 were tested against several axSpA-specific measures (BASDAI, ASDAS, BASFI, mSASSS). A multivariate model was built to detect disease factors associated with RAPID3 remission (values ≤3). Results The study included 82 men and 49 women, median age of 55 years (IQR: 46-61), and median disease duration of 11 years (IQR: 6-24). Mean RAPID3 was 9.45 ± 6.7. The BASDAI showed moderate correlation with ASDAS (rho: 0.66, p < 0.0001), but higher with BASFI (rho: 0.78, p < 0.0001) and RAPID3 (rho: 0.75, p < 0.0001). The ASDAS had moderate correlations with BASFI, BASDAI, and RAPID3 (ranges from 0.66 to 0.68, p < 0.0001). Higher correlations were found between BASFI and BASDAI (rho: 0.78, p < 0.0001) and BASFI-RAPID3 (rho: 0.73, p < 0.0001). The m-SASSS did not show any correlation with any of the afore-mentioned composite measures. Kappa agreement between RAPID3 remission and other SpA remission criteria was moderate (k: 0.46-0.56). The RAPID3 thresholds to define remission ranged from values ≤2 to ≤ 6 with areas under the ROC curves between 0.86 and 0.91. Female sex (OR 0.34, 95%CI: 0.12- 0.90, p= 0.031) and NSAIDs intake (OR 0.26, 95%CI: 0.10-0.66, p= 0.005) were independently associated with lower odds of achieving RAPID3 remission. Conclusion RAPID3 demonstrated construct validity in this cross-sectional study. This index can be useful for a more comprehensive assessment of axSpA in busy clinical settings.