The dynamicity of light-up aptamers in one-pot in vitro diagnostic assays

The direct modulation of a light-up aptamer that engenders an analyte-specific aptamer-light-up aptamer chimera is readily applicable in any diagnostic assay for a targeted detection.

Fast, Reliable, and Simple Point-of-Care-like Adaptation of RT-qPCR for the Detection of SARS-CoV-2 for Use in Hospital Emergency Departments

During COVID-19 pandemics, the availability of testing has often been a limiting factor during patient admissions into the hospital. To circumvent this problem, we adapted an existing diagnostic assay, Seegene Allplex SARS-CoV-2, into a point-of-care-style direct qPCR (POC dqPCR) assay and implemented it in the Emergency Department of Clinical Hospital Center Rijeka, Croatia. In a 4-month analysis, we tested over 10,000 patients and demonstrated that POC-dqPCR is robust and reliable and can be successfully implemented in emergency departments and similar near-patient settings and can be performed by medical personnel with little prior experience in qPCR.

Androgen Receptor Pathway Activity Assay for Sepsis Diagnosis and Prediction of Favorable Prognosis

Introduction: Sepsis is a life-threatening complication of a bacterial infection. It is hard to predict which patients with a bacterial infection will develop sepsis, and accurate and timely diagnosis as well as assessment of prognosis is difficult. Aside from antibiotics-based treatment of the causative infection and supportive measures, treatment options have remained limited. Better understanding of the immuno-pathophysiology of sepsis is expected to lead to improved diagnostic and therapeutic solutions.Functional activity of the innate (inflammatory) and adaptive immune response is controlled by a dedicated set of cellular signal transduction pathways, that are active in the various immune cell types. To develop an immune response-based diagnostic assay for sepsis and provide novel therapeutic targets, signal transduction pathway activities have been analyzed in whole blood samples from patients with sepsis.Methods: A validated and previously published set of signal transduction pathway (STP) assays, enabling determination of immune cell function, was used to analyze public Affymetrix expression microarray data from clinical studies containing data from pediatric and adult patients with sepsis. STP assays enable quantitative measurement of STP activity on individual patient sample data, and were used to calculate activity of androgen receptor (AR), estrogen receptor (ER), JAK-STAT1/2, JAK-STAT3, Notch, Hedgehog, TGFβ, FOXO-PI3K, MAPK-AP1, and NFκB signal transduction pathways.Results: Activity of AR and TGFβ pathways was increased in children and adults with sepsis. Using the mean plus two standard deviations of normal pathway activity (in healthy individuals) as threshold for abnormal STP activity, diagnostic assay parameters were determined. For diagnosis of pediatric sepsis, the AR pathway assay showed high sensitivity (77%) and specificity (97%), with a positive prediction value (PPV) of 99% and negative prediction value (NPV) of 50%. For prediction of favorable prognosis (survival), PPV was 95%, NPV was 21%. The TGFβ pathway activity assay performed slightly less for diagnosing sepsis, with a sensitivity of 64% and specificity of 98% (PPV 99%, NPV 39%).Conclusion: The AR and TGFβ pathways have an immunosuppressive role, suggesting a causal relation between increased pathway activity and sepsis immunopathology. STP assays have been converted to qPCR assays for further evaluation of clinical utility for sepsis diagnosis and prediction of prognosis, as well as for prediction of risk at developing sepsis in patients with a bacterial infection. STPs may present novel therapeutic targets in sepsis.

Single-Point Mutations in the N Gene of SARS-CoV-2 Adversely Impact Detection by a Commercial Dual Target Diagnostic Assay

This paper reports the identification of single-point mutations in the N gene of SARS-CoV-2 associated with a gene target failure by the Cepheid Xpert commercial system. In order to determine the mutation(s) responsible for the N gene detection failures, the genomic products from the Cepheid Xpert system were sequenced and compared to whole genomes of SARS-CoV-2 from clinical cases.

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.

Long COVID following mild SARS-CoV-2 infection: characteristic T cell alterations and response to antihistamines

Long COVID is characterized by the emergence of multiple debilitating symptoms following SARS-CoV-2 infection. Its etiology is unclear and it often follows a mild acute illness. Anecdotal reports of gradual clinical responses to histamine receptor antagonists (HRAs) suggest a histamine-dependent mechanism that is distinct from anaphylaxis, possibly mediated by T cells, which are also regulated by histamine. T cell perturbations have been previously reported in post-viral syndromes, but the T cell landscape in patients who have recovered from mild COVID-19 and its relationship to both long COVID symptoms and any symptomatic response to HRA remain underexplored. We addressed these questions in an observational study of 65 individuals who had recovered from mild COVID-19. Participants were surveyed between 87 and 408 days after the onset of acute symptoms; none had required hospitalization, 16 had recovered uneventfully, and 49 had developed long COVID. Symptoms were quantified using a structured questionnaire and T cell subsets enumerated in a standard diagnostic assay. Patients with long-COVID had reduced CD4+ and CD8+ effector memory (EM) cell numbers and increased PD-1 (programmed cell death protein 1) expression on central memory (CM) cells, whereas the asymptomatic participants had reduced CD8+ EM cells only and increased CD28 expression on CM cells. 72% of patients with long COVID who received HRA reported clinical improvement, although T cell profiling did not clearly distinguish those who responded to HRA. This study demonstrates that T cell perturbations persist for several months after mild COVID-19 and are associated with long COVID symptoms.

Reduced amplification efficiency of the RNA-dependent-RNA-polymerase (RdRp) target enables tracking of the Delta SARS-CoV-2 variant using routine diagnostic tests

Routine SARS-CoV-2 surveillance in the Western Cape region of South Africa (January-August 2021) found a reduced PCR amplification efficiency of the RdRp gene target of the Seegene, Allplex 2019-nCoV diagnostic assay when detecting the Delta variant. We propose that this can be used as a surrogate for variant detection.

Evaluation of a novel Nano diagnostic assay for Assessing the Prevalence of Schistosoma haematobium Infection in Areas at Risk in Upper Egypt

Background Urogenital schistosomiasis caused by Schistosoma heamatobium is one of the major public health problems worldwide. It is thought that despite extensive efforts and integrated control programs implicated over the last few decades, the global disease burden of schistosomiasis remains unacceptably high. This persistence of the disease may be due to in part the lack of accurate diagnostic tools for case detection and community screening in endemic areas. Aim of the work The present work aims to develop a novel nano-diagnostic assay using gold nanoparticles (nanomagnetic beads based- ELISA) which can utilize larger surface area, achieving a higher sensitivity for detection of urinary schistosomal egg antigen (SEA) in urine of human schistosomiasis haematobium and comparing it with the traditional sandwich ELISA and direct microscopic examination of urine sediments together with indirect screening by chemical reagent strips for microhaematiria and proteinuria for assessing prevalence of urinary schistosomiasis in some villages in Beni-Suef governorate. Subjects and methods A cross sectional study was conducted on 290 students (192 male and 98 female) selected randomly from Primary and Preparatory schools in four villages in Beni-Suef governorate; The participating children were aged 8–15 years old. A simple questionnaire was designed based on the key indicators of urinary schistosomiasis then, terminal urine samples were collected between 10 am and 2 pm in clean container from each participant to be screened by chemical reagent strips (Combi 10) and examined by urine microscopy and sandwich ELISA techniques (traditional and IMB) for S. haematobium detection. Soluble egg antigen (SEA) was used to produce specific polyclonal antibodies (pAbs) which were then used as a primary capture in the sandwich ELISA techniques. The anti-SEA pAbs were labeled with horse-radish peroxidase (HRP) and used as a secondary capture. Results Out of the 290 participants, 39 children (13.4%) were positive by UM, 53 were positive by traditional sandwich ELISA, with diagnostic sensitivity (87.2%) and specificity (92.4%) and 50 were positive by IMB-sandwich ELISA with diagnostic sensitivity (94.9%) and specificity (95.2%)based on UM results. Micro-haematuria and proteinuria were assessed by chemical reagent strips which gave sensitivity of 29.5%, specificity of 90.8% for micro-haematuria alone, sensitivity of 18.4%, specificity of 92.4% for proteinuria alone, while sensitivity of 35.9%, specificity of 94.9% for combined micro-haematuria and proteinuria which indicated a highly significant association with S. haematobium infection (p value<0.001). Conclusion Combination of both clinical and epidemiological data in addition to sensitive diagnostic tools is essential for diagnosis. The present study as with other studies revealed that, IMB-ELISA based on gold nanoparticles provides more rapid and sensitive detection for SEA in urine samples of patient with active schistosomiasis. Simplicity and fast detection (10 min) are its main advantages. Moreover, its high sensitivity and specificity ensure its application with greater precision and rapid detection. Also, in addition, the prevalence of urinary schistosomiasis in these regions is considered relatively high requiring rapid implementation of control programs to decrease the prevalence and improve the community's health status.