RNA-Binding Proteins and Alternative Splicing Genes Are Coregulated in Human Retinal Endothelial Cells Treated with High Glucose.

To explore the relevant RNA-binding proteins (RBPs) and alternative splicing events (ASEs) in diabetic retinopathy (DR). We devised a comprehensive work to integrate analyses of the differentially expressed genes, including differential RBPs, and variable splicing characteristics related to DR in human retinal endothelial cells induced by low glucose and high glucose in dataset GSE117238. A total of 2320 differentially expressed genes (DEGs) were identified, including 1228 upregulated genes and 1092 downregulated genes. Further analysis screened out 232 RBP genes, and 42 AS genes overlapped DEGs. We selected high expression and consistency six RBP genes (FUS, HNRNPA2B1, CANX, EIF1, CALR, and POLR2A) for coexpression analysis. Through analysis, we found eight RASGs (MDM2, GOLGA2P7, NFE2L1, KDM4A, FAM111A, CIRBP, IDH1, and MCM7) that could be regulated by RBP. The coexpression network was conducted to further elucidate the regulatory and interaction relationship between RBPs and AS. Apoptotic progress, protein phosphorylation, and NF-kappaB cascade revealed by the functional enrichment analysis of RASGs regulated by RBPs were closely related to diabetic retinopathy. Furthermore, the expression of differentially expressed RBPs was validated by qRT-PCR in mouse retinal microvascular endothelial cells and retinas from the streptozotocin mouse model. The results showed that Fus, Hnrnpa2b1, Canx, Calr, and Polr2a were remarkedly difference in high-glucose-treated retinal microvascular endothelial cells and Fus, Hnrnpa2b1, Canx, and Calr were remarkedly difference in retinas from streptozotocin-induced diabetic mice compared to control. The regulatory network between identified RBPs and RASGs suggests the presence of several signaling pathways possibly involved in the pathogenesis of DR. The verified RBPs should be further addressed by future studies investigating associations between RBPs and the downstream of AS, as they could serve as potential biomarkers and targets for DR.

Improvement of Lipotoxicity-Induced Islet ß Cellular Insulin Secretion Disorder by Osteocalcin.

Background: Osteocalcin (OCN) has been proved to be closely related with the development of type 2 diabetes mellitus (T2DM). We aimed to study if OCN could improve the disorder of islet cell caused by lipotoxicity. Methods: Alizarin red staining was used to investigate the mineralization. Western blotting and ELISA methods were used to measure protein expression. Immunofluorescence staining was used to investigate the protein nuclear transfer. Results: High glucose and high fat inhibited the differentiation of osteoblast precursors. Overexpression of insulin receptor (InsROE) significantly promoted the Runx2 and OCN expression. The increase of insulin, Gprc6a, and Glut2 by osteoblast culture medium overexpressing insulin receptor was reversed by osteocalcin neutralizing antibody. Undercarboxylated osteocalcin (ucOC) suppressed the lipotoxic islet ß-cell damage caused by palmitic acid. The FOXO1 from intranuclear to extranuclear was also significantly increased after ucOC treatment compared with the group PA. Knockdown of Gprc6a or suppression of PI3K/AKT signal pathway could reverse the upregulation of GPRC6A/PI3K/AKT/FoxO1/Pdx1 caused by ucOC. Conclusion: OCN could activate the FOXO1 signaling pathway to regulate GLUT2 expression and improve the insulin secretion disorder caused by lipotoxicity.

EEF2K silencing inhibits tumour progression through repressing SPP1 and synergises with BET inhibitors in melanoma.

BACKGROUND: Despite the remarkable breakthroughs achieved in the management of metastatic melanoma using immunotherapy and targeted therapies, long-term clinical efficacy is often compromised due to dose-limiting toxicity and innate or acquired resistance. Therefore, it is of vital importance to further explore the molecular mechanisms underlying melanoma progression and identify new targeted therapeutic approaches. METHODS: The function of eukaryotic elongation factor-2 kinase (EEF2K) in melanoma were investigated in vitro and in vivo. RNA-seq and chromatin immunoprecipitation (ChIP) assay were undertaken to explore the mechanisms. The antitumor effect of bromodomain and extra terminal domain (BET) inhibitors combined with cytarabine were assessed in melanoma both in vitro and in vivo. RESULTS: EEF2K silencing markedly attenuated the malignant phenotypes of melanoma cells, including proliferation, migration, invasion and metastasis. In contrast, EEF2K overexpression promoted melanoma cell proliferation, migration and invasion. Mechanistically, we demonstrated that EEF2K upregulates the phosphorylation of STAT3 (p-STAT3) at Tyr705, which binds to the promoter region of SPP1 and enhances its transcription, thus facilitating melanoma progression. Transfection-induced re-expression of SPP1 partly negated the inhibitory effect of EEF2K silencing on melanoma, whereas inhibition of SPP1 or STAT3 significantly abolished the efficacy of EEF2K on melanoma cells. Intriguingly, EEF2K silencing combined with BET inhibitor treatment further inhibited cell proliferation and promoted apoptosis in melanoma. We further screened the US FDA-approved antitumour drug library and identified cytarabine as a potential clinically applicable EEF2K inhibitor that could synergise with BET inhibitors in melanoma treatment. CONCLUSION: EEF2K/p-STAT3/SPP1 may be a novel oncogenic pathway in melanoma progression, which could be a target for novel combination therapy for melanoma.

Distinct histologic and genetic characteristics of round cell sarcoma with CIC-DUX4 fusion and comparison with ewing sarcoma.

CIC-DUX4 fusion gene associated sarcoma is a new emerging subgroup of round cell sarcoma with Ewing sarcoma-like morphology. Distinguishing these tumors from Ewing sarcoma family tumors (ESFT) is critical because of the clinical impact but is still challenging due to the overlapped histological and immunohistochemical phenotypes of each subtype. The present study investigated small round cell sarcoma to identify CIC-DUX4 fusion positive sarcoma, examined clinical, histopathologic and immunohistochemical characteristics of CIC-DUX4 sarcoma, and evaluated parameters to differentiate Ewing sarcoma family tumors. Seventy patients with undifferentiated round cell sarcoma or Ewing-like sarcoma were retrieved. Molecular tests including EWSR1, CIC break apart FISH, and RT-PCR for CIC-DUX4 gene fusion were performed and immunohistochemistry was performed. Six cases (8.6%) of CIC-DUX4 sarcomas were detected. Histologically, CIC-DUX4 sarcomas composed of heterogeneous round, plasmacytoid, and spindle cells and more commonly showed cytologic pleomorphism with bizarre nuclei and multinucleated cells and myxoid stoma unlike ESFT. CIC-DUX4 sarcomas didn't show overall survival differences (p = 0.325) compared to ESFT but they demonstrated short disease-free survival (p = 0.034) and poor response to treatment (p = 0.007). Therefore, molecular analysis to detect the distinctive genetic alteration is mandatory in tumors with atypical histologic, immunohistochemical and/or clinical presentation for accurate diagnosis and treatment.

Evaluating performance of multiplex real time PCR for the diagnosis of malaria at elimination targeted low transmission settings of Ethiopia.

BACKGROUND: Malaria incidence has declined in Ethiopia in the past 10 years. Current malaria diagnostic tests, including light microscopy and rapid antigen-detecting diagnostic tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. This study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. METHODS: A health facility-based, cross-sectional survey was conducted in selected malaria sentinel sites. Malaria-suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 was enrolled into this study. Sociodemographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood spot (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at Ethiopian Public Health Institute (EPHI) malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other. RESULTS: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as Plasmodium falciparum infection, 16 as Plasmodium vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as P. falciparum, 18 as P. vivax, and RDT detected 43 as P. falciparum, 17 as P. vivax, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI (93-100)) and 83.2% (95% CI (77.6-87.9)), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI 46.9-68.4) and 67% (95% CI 56.2-76.7); and 100% (95% CI 98-100) and 98.9% (95% CI 96-99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. CONCLUSIONS: Multiplex real-time PCR had an advanced performance in parasite detection and species identification on febrile patients' samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination programme, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

One-step and sequential SARSCOV-2 polymerase chain reaction tests would not work every time.

INTRODUCTION: RT-PCR is widely used as a diagnostic test for the detection of SARS-CoV-2. In this study, we aim to describe the clinical utility of serial PCR testing in the final detection of COVID-19. METHOD: We collected multiple nasopharyngeal swab samples from patients who had negative RT-PCR test on the first day after hospitalization. RT-PCR tests were performed on the second day for all patients with initial negative result. For the patients with secondary negative results on day 2, tertiary RT-PCR tests were performed on day 3 after hospitalization. RESULT: Among 68 patients with initial negative test results, at the end of follow-up, the mortality number was 20 (29.4%). About 33.8% of patients had subsequent positive PCR test results for the second time and 17.4% of the patients who performed third PCR test had positive result. CONCLUSION: Based on this study, serial RT-PCR testing is unlikely to yield additional information.

CIC rearranged sarcomas: A single institution experience of the potential pitfalls in interpreting CIC FISH results.

AIM: The aim of this study was to establish how reliable FISH CIC analysis using an IVD (in vitro diagnostic) commercial probe is. METHODS AND RESULTS: A series of 19 CIC-DUX4 sarcomas were evaluated. The samples presenting CIC-DUX4 fusion transcript detected by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Sanger sequencing and/or Next Generation Sequencing were selected for Fluorescent in Situ Hybridization (FISH) CIC analysis with CIC break-apart IVD probe and compared to molecular analysis. CIC FISH analysis showed 26% of false negatives. CONCLUSION: Our results indicate that, in the setting of CIC-DUX4 fusion positive small round cell sarcomas, CIC FISH using IVD commercial probe may lead to false-negative results. This novel study evaluates the diagnostic use of a commercial IVD CIC probe for FISH.

Multi-omic landscaping of human midbrains identifies disease-relevant molecular targets and pathways in advanced-stage Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) is the second most common neurodegenerative disorder whose prevalence is rapidly increasing worldwide. The molecular mechanisms underpinning the pathophysiology of sporadic PD remain incompletely understood. Therefore, causative therapies are still elusive. To obtain a more integrative view of disease-mediated alterations, we investigated the molecular landscape of PD in human post-mortem midbrains, a region that is highly affected during the disease process. METHODS: Tissue from 19 PD patients and 12 controls were obtained from the Parkinson's UK Brain Bank and subjected to multi-omic analyses: small and total RNA sequencing was performed on an Illumina's HiSeq4000, while proteomics experiments were performed in a hybrid triple quadrupole-time of flight mass spectrometer (TripleTOF5600+) following quantitative sequential window acquisition of all theoretical mass spectra. Differential expression analyses were performed with customized frameworks based on DESeq2 (for RNA sequencing) and with Perseus v.1.5.6.0 (for proteomics). Custom pipelines in R were used for integrative studies. RESULTS: Our analyses revealed multiple deregulated molecular targets linked to known disease mechanisms in PD as well as to novel processes. We have identified and experimentally validated (quantitative real-time polymerase chain reaction/western blotting) several PD-deregulated molecular candidates, including miR-539-3p, miR-376a-5p, miR-218-5p and miR-369-3p, the valid miRNA-mRNA interacting pairs miR-218-5p/RAB6C and miR-369-3p/GTF2H3, as well as multiple proteins, such as CHI3L1, HSPA1B, FNIP2 and TH. Vertical integration of multi-omic analyses allowed validating disease-mediated alterations across different molecular layers. Next to the identification of individual molecular targets in all explored omics layers, functional annotation of differentially expressed molecules showed an enrichment of pathways related to neuroinflammation, mitochondrial dysfunction and defects in synaptic function. CONCLUSIONS: This comprehensive assessment of PD-affected and control human midbrains revealed multiple molecular targets and networks that are relevant to the disease mechanism of advanced PD. The integrative analyses of multiple omics layers underscore the importance of neuroinflammation, immune response activation, mitochondrial and synaptic dysfunction as putative therapeutic targets for advanced PD.

Biophysical analysis of the Mycobacteria tuberculosis peptide binding protein DppA reveals a stringent peptide binding pocket.

The peptide binding protein DppA is an ABC transporter found in prokaryotes that has the potential to be used as drug delivery tool for hybrid antibiotic compounds. Understanding the motifs and structures that bind to DppA is critical to the development of these bivalent compounds. This study focused on the biophysical analysis of the MtDppA from M. tuberculosis. Analysis of the crystal structure revealed a SVA tripeptide was co-crystallized with the protein. Further peptide analysis demonstrated MtDppA shows very little affinity for dipeptides but rather preferentially binds to peptides that are 3-4 amino acids in length. The structure-activity relationships (SAR) between MtDppA and tripeptides with varied amino acid substitutions were evaluated using thermal shift, SPR, and molecular dynamics simulations. Efforts to identify novel ligands for use as alternative scaffolds through the thermal shift screening of 35,000 compounds against MtDppA were unsuccessful, indicating that the MtDppA binding pocket is highly specialized for uptake of peptides. Future development of compounds that seek to utilize MtDppA as a drug delivery mechanism, will likely require a tri- or tetrapeptide component with a hydrophobic -non-acidic peptide sequence.

Circ_0059354 aggravates the progression of papillary thyroid carcinoma by elevating ARFGEF1 through sponging miR-766-3p.

BACKGROUND: Circular RNAs (circRNAs) have been identified as vital players in tumors, including papillary thyroid carcinoma (PTC). The purpose of this study is to explore the functions of circ_0059354 on PTC development. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the levels of circ_0059354, microRNA-766-3p (miR-766-3p) and ADP ribosylation factor guanine nucleotide exchange factor 1 (ARFGEF1). Cell Counting Kit-8 (CCK-8) assay and colony formation assay were proceeded for cell proliferation ability. Transwell assay was conducted for cell migration and invasion. Tube formation assay was employed to examine the angiogenesis ability. Flow cytometry analysis was adopted for cell apoptosis. Western blot assay was conducted for protein levels. Dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were utilized to verify the relationships among circ_0059354, miR-766-3p and ARFGEF1. The murine xenograft model was constructed to analyze the function of circ_0059354 in vivo. RESULTS: Circ_0059354 level was abnormally increased in PTC tissues and cells. Functionally, circ_0059354 silencing suppressed cell proliferation, migration, invasion and angiogenesis and facilitated apoptosis in PTC cells. Circ_0059354 was identified to sponge miR-766-3p, which directly targeted ARFGEF1. Moreover, circ_0059354 directly targeted miR-766-3p to positively regulated ARFGEF1 expression. MiR-766-3p inhibition reversed circ_0059354 knockdown-mediated effect of PTC cell malignant behaviors. Overexpression of miR-766-3p restrained the malignant behaviors of PTC cells, whereas ARFGEF1 elevation reversed the effects. Additionally, circ_0059354 deficiency blocked tumor growth in vivo. CONCLUSION: Circ_0059354 served as an oncogene in PTC progression through regulating miR-766-3p/ARFGEF1 axis.

Hyper-prevalence of submicroscopic Plasmodium falciparum infections in a rural area of western Kenya with declining malaria cases.

BACKGROUND: The gold standard for diagnosing Plasmodium falciparum infection is microscopic examination of Giemsa-stained peripheral blood smears. The effectiveness of this procedure for infection surveillance and malaria control may be limited by a relatively high parasitaemia detection threshold. Persons with microscopically undetectable infections may go untreated, contributing to ongoing transmission to mosquito vectors. The purpose of this study was to determine the magnitude and determinants of undiagnosed submicroscopic P. falciparum infections in a rural area of western Kenya. METHODS: A health facility-based survey was conducted, and 367 patients seeking treatment for symptoms consistent with uncomplicated malaria in Homa Bay County were enrolled. The frequency of submicroscopic P. falciparum infection was measured by comparing the prevalence of infection based on light microscopic inspection of thick blood smears versus real-time polymerase chain reaction (RT-PCR) targeting P. falciparum 18S rRNA gene. Long-lasting insecticidal net (LLIN) use, participation in nocturnal outdoor activities, and gender were considered as potential determinants of submicroscopic infections. RESULTS: Microscopic inspection of blood smears was positive for asexual P. falciparum parasites in 14.7% (54/367) of cases. All of these samples were confirmed by RT-PCR. 35.8% (112/313) of blood smear negative cases were positive by RT-PCR, i.e., submicroscopic infection, resulting in an overall prevalence by RT-PCR alone of 45.2% compared to 14.7% for blood smear alone. Females had a higher prevalence of submicroscopic infections (35.6% or 72 out of 202 individuals, 95% CI 28.9-42.3) compared to males (24.2%, 40 of 165 individuals, 95% CI 17.6-30.8). The risk of submicroscopic infections in LLIN users was about half that of non-LLIN users (OR = 0.59). There was no difference in the prevalence of submicroscopic infections of study participants who were active in nocturnal outdoor activities versus those who were not active (OR = 0.91). Patients who participated in nocturnal outdoor activities and use LLINs while indoors had a slightly higher risk of submicroscopic infection than those who did not use LLINs (OR = 1.48). CONCLUSION: Microscopic inspection of blood smears from persons with malaria symptoms for asexual stage P. falciparum should be supplemented by more sensitive diagnostic tests in order to reduce ongoing transmission of P. falciparum parasites to local mosquito vectors.

Rapid Full-Cycle Technique to Control Adulteration of Meat Products: Integration of Accelerated Sample Preparation, Recombinase Polymerase Amplification, and Test-Strip Detection.

Verifying the authenticity of food products is essential due to the recent increase in counterfeit meat-containing food products. The existing methods of detection have a number of disadvantages. Therefore, simple, cheap, and sensitive methods for detecting various types of meat are required. In this study, we propose a rapid full-cycle technique to control the chicken or pig adulteration of meat products, including 3 min of crude DNA extraction, 20 min of recombinase polymerase amplification (RPA) at 39 °C, and 10 min of lateral flow assay (LFA) detection. The cytochrome B gene was used in the developed RPA-based test for chicken and pig identification. The selected primers provided specific RPA without DNA nuclease and an additional oligonucleotide probe. As a result, RPA-LFA, based on designed fluorescein- and biotin-labeled primers, detected up to 0.2 pg total DNA per µL, which provided up to 0.001% w/w identification of the target meat component in the composite meat. The RPA-LFA of the chicken and pig meat identification was successfully applied to processed meat products and to meat after heating. The results were confirmed by real-time PCR. Ultimately, the developed analysis is specific and enables the detection of pork and chicken impurities with high accuracy in raw and processed meat mixtures. The proposed rapid full-cycle technique could be adopted for the authentication of other meat products.

Baicalein resensitizes tamoxifen-resistant breast cancer cells by reducing aerobic glycolysis and reversing mitochondrial dysfunction via inhibition of hypoxia-inducible factor-1α.

Drug resistance is a major hurdle for the effectiveness of tamoxifen (TAM) to provide clinical benefit. Therefore, it is essential to identify a sensitizer that could be used to improve TAM efficacy in treating TAM-resistant breast cancer. Here, we investigated the ability of baicalein to reverse TAM resistance. We found that baicalein increased the efficacy of TAM in inhibiting proliferation and inducing apoptosis of TAM-resistant cells. It also enhanced the TAM-induced growth reduction of resistant cells from NOD/SCID mouse mammary fat pads, without causing obvious systemic toxicity. Analyses using the CellMiner tool and the Kaplan-Meier plotter database showed that HIF-1α expression was inversely correlated with TAM therapeutic response in NCI-60 cancer cells and breast cancer patients. HIF-1α expression was increased in TAM-resistant cells due to an increase in mRNA levels and reduced ubiquitin-mediated degradation. Baicalein reduced HIF-1α expression by promoting its interaction with PHD2 and pVHL, thus facilitating ubiquitin ligase-mediated proteasomal degradation and thereby suppressing the nuclear translocation, binding to the hypoxia-response element, and transcriptional activity of HIF-1α. As a result, baicalein downregulated aerobic glycolysis by restricting glucose uptake, lactate production, ATP generation, lactate/pyruvate ratio and expression of HIF-1α-targeted glycolytic genes, thereby enhancing the antiproliferative efficacy of TAM. Furthermore, baicalein interfered with HIF-1α inhibition of mitochondrial biosynthesis, which increased mitochondrial DNA content and mitochondrial numbers, restored the generation of reactive oxygen species in mitochondria, and thus enhanced the TAM-induced mitochondrial apoptotic pathway. The HIF-1α stabilizer dimethyloxallyl glycine prevented the baicalein-induced downregulation of glycolysis and mitochondrial biosynthesis and reduced the effects of baicalein on reversing TAM resistance. Our results indicate that baicalein is a promising candidate to help overcome TAM resistance by sensitizing resistant cells to TAM-induced growth inhibition and apoptosis. The mechanism underlying the effects of baicalein consists of inhibition of HIF-1α-mediated aerobic glycolysis and mitochondrial dysfunction.

A retrospective analysis of 12,400 SARS-CoV-2 RNA tests in patients with COVID-19 in Wuhan.

ABSTRACT: The outbreak and widely spread of coronavirus disease 2019 (COVID-19) has become a global public health concern. COVID-19 has caused an unprecedented and profound impact on the whole world, and the prevention and control of COVID-19 is a global public health challenge remains to be solved. The retrospective analysis of the large scale tests of SARS-CoV-2 RNA may indicate some important information of this pandemic. We selected 12400 SARS-CoV-2 tests detected in Wuhan in the first semester of 2020 and made a systematic analysis of them, in order to find some beneficial clue for the consistent prevention and control of COVID-19.SARS-CoV-2 RNA was detected in suspected COVID-19 patients with real-time fluorescence quantitative PCR (RT-qPCR). The patients' features including gender, age, type of specimen, source of patients, and the dynamic changes of the clinical symptoms were recorded and statistically analyzed. Quantitative and qualitive statistical analysis were carried out after laboratory detection.The positive rate of SARS-CoV-2 was 33.02% in 12,400 suspected patients' specimens in Wuhan at the first months of COVID-19 epidemics. SARS-CoV-2 RT-qPCR test of nasopharyngeal swabs might produce 4.79% (594/12400) presumptive results. The positive rate of SARS-CoV-2 RNA was significantly different between gender, age, type of specimen, source of patients, respectively (P < .05). The median window period from the occurrence of clinical symptom or close contact with COVID-19 patient to the first detection of positive PCR was 2 days (interquartile range, 1-4 days). The median interval time from the first SARS-CoV-2 positive to the turning negative was 14 days (interquartile range, 8-19.25 days).This study reveals the comprehensive characteristics of the SARS-CoV-2 RNA detection from multiple perspectives, and it provides important clues and may also supply useful suggestions for future work of the prevention and treatment of COVID-19.

Severe Acute Respiratory Syndrome Coronavirus 2 Total and Subgenomic RNA Viral Load in Hospitalized Patients.

BACKGROUND: Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS: We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS: We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS: Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Consideration of the Cycle Threshold Values from Real-Time RT-PCR SARS-CoV-2 Interpretation for the Clinicians: Analysis of 339 Positive Cases from a Referral Laboratory in Jakarta, Indonesia.

BACKGROUND: real-time RT-PCR was recommended by WHO for COVID-19 diagnosis. The cycle threshold (Ct) values were expected to have an association with clinical manifestation. However, the diagnostic modalities such as quantitative molecular detection and virus isolation were not yet available for the routine test. This study has been conducted to analyze the relationship between the Ct values of qualitative rRT-PCR and the clinical manifestation and to describe the factors determining the result. METHODS: from March to April 2020, specimens were sent to our laboratory from different healthcare centers in Jakarta. The patient's characteristic and clinical manifestation were extracted from the specimen's epidemiology forms. The specimens extracted and tested using rRT-PCR, and the Ct value were collected. The data were analyzed using the appropriate statistic test. RESULTS: from 339 positive results, the mild to moderate case was 176 (52%) and the severe cases was 163 (48%). Female was dominant in the mild to moderate cases (58%), while the male was prevalent in the severe cases (60%). The median age for mild to moderate case was 35 years old and severe cases was 49 years old. Statistical analysis found relationship between both group with gender (p = 0.001) and age (p < 0.001), but not with the Ct value. CONCLUSION: many variables in specimen sampling and processing could affect the Ct value result. In addition, the disease's severity was depended with the host immune response, regardless the number of virus. There was suggested no significant difference between the Ct values of mild-moderate and severe COVID-19, and thus should not be loosely interpreted.

Diagnostic Accuracy Estimates for COVID-19 Real-Time Polymerase Chain Reaction and Lateral Flow Immunoassay Tests With Bayesian Latent-Class Models.

Our objective was to estimate the diagnostic accuracy of real-time polymerase chain reaction (RT-PCR) and lateral flow immunoassay (LFIA) tests for coronavirus disease 2019 (COVID-19), depending on the time after symptom onset. Based on the cross-classified results of RT-PCR and LFIA, we used Bayesian latent-class models, which do not require a gold standard for the evaluation of diagnostics. Data were extracted from studies that evaluated LFIA (immunoglobulin G (IgG) and/or immunoglobulin M (IgM)) assays using RT-PCR as the reference method. The sensitivity of RT-PCR was 0.68 (95% probability interval (PrI): 0.63, 0.73). IgG/M sensitivity was 0.32 (95% PrI :0.23; 0.41) for the first week and increased steadily. It was 0.75 (95% PrI: 0.67; 0.83) and 0.93 (95% PrI: 0.88; 0.97) for the second and third weeks after symptom onset, respectively. Both tests had a high to absolute specificity, with higher point median estimates for RT-PCR specificity and narrower probability intervals. The specificity of RT-PCR was 0.99 (95% PrI: 0.98; 1.00). and the specificity of IgG/IgM was 0.97 (95% PrI: 0.92, 1.00), 0.98 (95% PrI: 0.95, 1.00) and 0.98 (95% PrI: 0.94, 1.00) for the first, second, and third weeks after symptom onset. The diagnostic accuracy of LFIA varies with time after symptom onset. Bayesian latent-class models provide a valid and efficient alternative for evaluating the rapidly evolving diagnostics for COVID-19, under various clinical settings and different risk profiles.

Performance of a sensitive haemozoin-based malaria diagnostic test validated for vivax malaria diagnosis in Brazilian Amazon.

BACKGROUND: Vivax malaria diagnosis remains a challenge in malaria elimination, with current point of care rapid diagnostic tests (RDT) missing many clinically significant infections because of usually lower peripheral parasitaemia. Haemozoin-detecting assays have been suggested as an alternative to immunoassay platforms but to date have not reached successful field deployment. Haemozoin is a paramagnetic crystal by-product of haemoglobin digestion by malaria parasites and is present in the food vacuole of malaria parasite-infected erythrocytes. This study aimed to compare the diagnostic capability of a new haemozoin-detecting platform, the Gazelle™ device with optical microscopy, RDT and PCR in a vivax malaria-endemic region. METHODS: A comparative, double-blind study evaluating symptomatic malaria patients seeking medical care was conducted at an infectious diseases reference hospital in the western Brazilian Amazon. Optical microscopy, PCR, RDT, and Gazelle™ were used to analyse blood samples. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and Kappa values were calculated. RESULTS: Out of 300 patients, 24 test results were excluded from the final analysis due to protocol violation (6) and inconclusive and/or irretrievable results (18). Gazelle™ sensitivity was 96.1 % (91.3-98.3) and 72.1 % (65.0-78.3) when compared to optical microscopy and PCR, respectively whereas it was 83.9 % and 62.8 % for RDTs. The platform presented specificity of 100 % (97.4-100), and 99.0 % (94.8-99.9) when compared to optical microscopy, and PCR, respectively, which  was the same for RDTs. Its correct classification rate was 98.2 % when compared to optical microscopy and 82.3 % for PCR; the test's accuracy when compared to optical microscopy was 98.1 % (96.4-99.7), when compared to RDT was 95.2 % (93.0-97.5), and when compared to PCR was 85.6 % (82.1-89.1). Kappa (95 % CI) values for Gazelle™ were 96.4 (93.2-99.5), 88.2 (82.6-93.8) and 65.3 (57.0-73.6) for optical microscopy, RDT and PCR, respectively. CONCLUSIONS: The Gazelle™ device was shown to have faster, easier, good sensitivity, specificity, and accuracy when compared to microscopy and was superior to RDT, demonstrating to be an alternative for vivax malaria screening particularly in areas where malaria is concomitant with other febrile infections (including dengue fever, zika, chikungunya, Chagas, yellow fever, babesiosis).

Measurement of Telomere Length for Longitudinal Analysis: Implications of Assay Precision.

Researchers increasingly wish to test hypotheses concerning the impact of environmental or disease exposures on telomere length (TL), and they use longitudinal study designs to do so. In population studies, TL is usually measured with a quantitative polymerase chain reaction (qPCR)-based method. This method has been validated by calculating its correlation with a gold standard method such as Southern blotting (SB) in cross-sectional data sets. However, in a cross-section, the range of true variation in TL is large, and measurement error is introduced only once. In a longitudinal study, the target variation of interest is small, and measurement error is introduced at both baseline and follow-up. In this paper, we present results from a small data set (n = 20) in which leukocyte TL was measured twice 6.6 years apart by means of both qPCR and SB. The cross-sectional correlations between qPCR and SB were high at both baseline (r = 0.90) and follow-up (r = 0.85), yet their correlation for TL change was poor (r = 0.48). Moreover, the qPCR data but not the SB data showed strong signatures of measurement error. Through simulation, we show that the statistical power gain from performing a longitudinal analysis is much greater for SB than for qPCR. We discuss implications for optimal study design and analysis.

A simple lung ultrasound protocol for the screening of COVID-19 pneumonia in the emergency department.

The most relevant manifestation of coronavirus disease 2019 (COVID-19) is interstitial pneumonia. Several lung ultrasound (US) protocols for pneumonia diagnosis are used in clinical practice, but none has been proposed for COVID-19 patients' screening in the emergency department. We adopted a simplified 6-scan lung US protocol for COVID-19 pneumonia diagnosis (LUSCOP) and compared its sensitivity with high resolution computed tomography (HRCT) in patients suspected for COVID-19, presenting to one Emergency Department from February 21st to March 15th, 2020, during the outbreak burst in northern Italy. Patients were retrospectively enrolled if both LUSCOP protocol and HRCT were performed in the Emergency Department. The sensitivity of LUSCOP protocol and HRCT were compared. COVID-19 pneumonia's final diagnosis was based on real-time reverse-transcription polymerase chain reaction from nasal-pharyngeal swab and on clinical data. Out of 150 suspected COVID-19 patients, 131 were included in the study, and 130 had a final diagnosis of COVID-19 pneumonia. The most frequent lung ultrasonographic features were: bilateral B-pattern in 101 patients (77%), B-pattern with subpleural consolidations in 26 (19.8%) and lung consolidations in 2 (1.5%). LUSCOP Protocol was consistent with HRCT in correctly screening 130 out of the 131 COVID-19 pneumonia cases (99.2%). In one case COVID-19 pneumonia was excluded by both HRCT and lung US. LUSCOP protocol showed optimal sensitivity and can be proposed as a simple screening tool for COVID-19 pneumonia diagnosis in the context of outbreak burst areas where prompt isolation of suspected patients is crucial for patients' and operators' safety.