RT-qPCR Test Research Articles

Supervised Machine Learning Methods for Seasonal Influenza Diagnosis.

Influenza has been a stationary disease in Mexico since 2009, and this causes a high cost for the national public health system, including its detection using RT-qPCR tests, treatments, and absenteeism in the workplace. Despite influenza's relevance, the main clinical features to detect the disease defined by international institutions like the World Health Organization (WHO) and the United States Centers for Disease Control and Prevention (CDC) do not follow the same pattern in all populations. The aim of this work is to find a machine learning method to facilitate decision making in the clinical differentiation between positive and negative influenza patients, based on their symptoms and demographic features. The research sample consisted of 15480 records, including clinical and demographic data of patients with a positive/negative RT-qPCR influenza tests, from 2010 to 2020 in the public healthcare institutions of Mexico City. The performance of the methods for classifying influenza cases were evaluated with indices like accuracy, specificity, sensitivity, precision, the f1-measure and the area under the curve (AUC). Results indicate that random forest and bagging classifiers were the best supervised methods; they showed promise in supporting clinical diagnosis, especially in places where performing molecular tests might be challenging or not feasible.

Evaluation of the analytical performance of the 15-minute point-of-care DASH™ SARS-CoV-2 RT-qPCR test

Accurate and timely diagnosis for COVID-19 diagnosis allows highly effective antiviral medications to be prescribed. The DASH™ Rapid PCR System is a sample-to-answer point-of-care platform combining state-of-the-art PCR kinetics with sequence specific hybridization. The platform's first assay, the DASH™ SARS-CoV-2/S test for anterior nares direct swab specimens, received FDA Emergency Use Authorization in March 2022 for point-of-care use. Here we report the analytical characteristics of the assay including limit of detection, dynamic range, and robustness of SARS-CoV-2 variant detection. The limit of detection was determined by testing swabs contrived with one hundred copies of wild type or Omicron BA.5 virus and detecting 20/20 and 19/20, respectively. The dynamic range was assessed with contrived swabs containing 102-106 copies; the log-linear relationship between Cq and copy input was plotted, and the qPCR efficiency calculated from the slope of the line was 101.4%. Detection of seven SARS-CoV-2 variants was demonstrated.

Prevalence of SARS-CoV-2 infection in asymptomatic preoperative patients

Background: Surgeries were performed on emergency and trauma patients as well as oncology patients in many centres during the pandemic period. Screening of asymptomatic preoperative patients is very important not only in protecting healthcare personnel but also in preventing nosocomial infections and in protecting patients with deficient or suppressed immune systems. The aim of the current study was to retrospectively evaluate the prevalence of SARS-CoV-2 infection in asymptomatic preoperative patients during the pandemic period. Methods: A total of 3773 asymptomatic preoperative patients among the 23385 patients were evaluated retrospectively with Rt-qPCR test. The DS CORONEX COVID-19 PCR test (Ver.2.0, DS Bio and nano technology, Turkiye) kit used for the RT-qPCR. PCR test targets the N and Orf1ab gene region specific for SARS-CoV-2. Results: SARS-CoV-2 positivity was significantly lower in the preoperative patient group than in COVID-19 positive patients whose routine test results were positive for other reasons (2.3% vs 23.1%; p<0.0005). Distribution of preoperative SARS-CoV-2 positive patients according to clinics were 7.1% thoracic surgery, 3.7% neurosurgery, 3.5% otolaryngology, 2.6% general surgery and 2.4% urology clinic, respectively. Conclusions: SARS-CoV-2 positivity was lower in the preoperative patients but this rate never excludes pre-screening of asymptomatic preoperative patients. Applying pre-screening tests to asymptomatic preoperative patients is significant in protecting healthcare personnel and patients with deficient or suppressed immune systems and also preventing nosocomial infections.

Rapid response of a public health reference laboratory to the COVID-19 pandemic.

Introduction. Brazil was one of the most affected countries by the COVID-19 pandemic. Instituto Adolfo Lutz (IAL) is the reference laboratory for COVID-19 in São Paulo, the most populous state in Brazil. In April 2020, a secondary diagnostic pole named IAL-2 was created to enhance IAL's capacity for COVID-19 diagnosis.Hypothesis/Gap Statement. Public health laboratories must be prepared to rapidly respond to emerging epidemics or pandemics.Aim. To describe the design of IAL-2 and correlate the results of RT-qPCR tests for COVID-19 with secondary data on suspected cases of SARS-CoV-2 infection in the São Paulo state.Methodology. This is a retrospective study based on the analysis of secondary data from patients suspected of infection by SARS-CoV-2 whose clinical samples were submitted to real-time PCR after reverse transcription (RT-qPCR) at IAL-2, between 1 April 2020 and 8 March 2022. RT-qPCR Ct results of the different kits used were also analysed.Results. IAL-2 was implemented in April 2020, just over a month after the detection of the first COVID-19 case in Brazil. The laboratory performed 304,250 RT-qPCR tests during the study period, of which 98 319 (32.3 %) were positive, 205827 (67.7 %) negative, and 104 (0.03 %) inconclusive for SARS-CoV-2. RT-qPCR Ct values≤30 for E/N genes of SARS-CoV-2 were presented by 79.7 % of all the samples included in the study.Conclusion. IAL was able to rapidly implement a new laboratory structure to support the processing of an enormous number of samples for diagnosis of COVID-19, outlining strategies to carry out work with quality, using different RT-qPCR protocols.

Surveillance of SARS-CoV-2 Genetic Variants in the Polish Armed Forces Using Whole Genome Sequencing Analysis.

Military operations involve the global movement of personnel and equipment, increasing the risk of spreading infectious pathogens such as SARS-CoV-2. Given the continuous engagement of the Polish Armed Forces in overseas operations, an active surveillance program targeting Variants of Concern (VOC) of SARS-CoV-2 was implemented among military personnel. Screening using RT-qPCR tests was conducted on 1699 soldiers between November 2021 and May 2022. Of these, 84 SARS-CoV-2 positive samples met the criteria for whole genome sequencing analysis and variant identification. Whole genome sequencing was performed using two advanced next-generation sequencing (NGS) technologies: sequencing by synthesis and nanopore sequencing. Our analysis revealed eleven SARS-CoV-2 lineages belonging to 21K, 21L, and 21J. The predominant lineage was BA.1.1 (57% of the samples), followed by BA.1 (23%) and BA.2 (6%). Notably, all identified lineages detected in post-deployment screening tests were classified as VOC and were already present in Poland, showing the effectiveness of the Military Sanitary Inspection measures in mitigating the COVID-19 spread. Pre-departure and post-mission screening and isolation successfully prevented SARS-CoV-2 VOC exportation and importation. Proactive measures are vital in minimizing the impact of COVID-19 in military settings, emphasizing the need for continued vigilance and response strategies.

Facile prepared microfluidic chip for multiplexed digital RT-qPCR test.

The chip-based digital polymerase chain reaction (PCR) is an indispensable technique for amplifying and quantifying nucleic acids, which has been widely employed in molecular diagnostics at both fundamental and clinical levels. However, the previous designs have yet to achieve widespread application due to limitations in complex chip fabrication, pretreatment procedures, special surface properties, and low throughput. This study presents a facile digital microfluidic chip driven by centrifugal force for digital PCR analysis. Interestingly, regardless of the hydrophilicity or hydrophobicity of the inner chip surface, an efficient digitization process can be achieved. PCR reagents introduced into the inlet can be allocated to 9600 microchambers and subsequently isolated by the immiscible phase (silicone oil). The centrifugal priming approach offers a facile means to achieve high-throughput analysis. The design was further employed for the quantification of nucleic acids using digital PCR. The calculated result exhibited a strong correlation with the measured value at the concentrations from 1 copy/μL to 1000 copies/μL (R2 =0.99). Additionally, the chip also allowed digital multiplexed analysis, thereby indicating its potential for multi-target detection applications.

Robust and high-efficient nitrogen removal from real sewage and waste activated sludge (WAS) reduction in zero-external carbon PN/A combined with in-situ fermentation-denitrification process under decreased temperatures

Despite the advantages of the combined anammox and fermentation-driven denitrification process in nitrogen removal and energy consumption, stable performance at decreased temperatures remains a challenge. In this study, a robust and high-efficient nitrogen removal efficiency (95.0–93.1 ∼ 86.8–93.4%) with desirable effluent quality (3.0–4.1 ∼ 7.9–4.9 mg/L) under long-term decreased temperatures (30 °C→25 °C→20 °C) was achieved in a zero-external carbon Partial Nitritation/Anammox combined with in-situ sludge Fermentation-Denitrification process treating sewage. Excellent sludge reduction averaged at 14.9% assuming no microbial growth. Increased hzsB mRNA (2.2-fold) and reduced Ea (80.9 kJ/mol) proved resilient anammox to lower temperature. RT-qPCR tests revealed increased NarG/NirK (5.1) and NarG/NirS (4.9) mRNA at 20 °C, suggesting higher NO3−→NO2− over NO2−→N2 pathway. Metagenomics unraveled dominant anammox bacteria (Candidatus_Brocadia, 2.27%), increased denitritation bacteria containing more NarG (Hyphomicrobium, 0.8%), fatty acid biosynthesis and CAZymes genes. Enhanced denitritation with recovered organics from sludge reserved nitrite for anammox and facilitated higher anammox contribution to N removal at 20 °C (42.4%) than 30 °C (39.5%). This study proposed an innovative low-temperature strategy for in-situ sludge fermentation, and demonstrated stability of advanced municipal wastewater treatment and sludge disposal through energy savings and carbon recovery under decreased temperatures.

Case series on clinical applications of liquid biopsy in pediatric solid tumors: towards improved diagnostics and disease monitoring.

Solid tumors account for about 30% of all pediatric cancers. The diagnosis is typically based on histological and molecular analysis of a primary tumor biopsy. Liquid biopsies carry several advantages over conventional tissue biopsy. However, their use for genomic analysis and response monitoring of pediatric solid tumors is still in experimental stages and mostly performed retrospectively without direct impact on patient management. In this case series we discuss six clinical cases of children with a solid tumor for whom a liquid biopsy assay was performed and demonstrate the potential of liquid biopsy for future clinical decision making. We performed quantitative real-time PCR (RT-qPCR), droplet digital PCR (ddPCR) or reduced representation bisulphite sequencing of cell-free DNA (cfRRBS) on liquid biopsies collected from six pediatric patients with a solid tumor treated between 2017 and 2023 at the Princess Máxima Center for Pediatric Oncology in the Netherlands. Results were used to aid in clinical decision making by contribution to establish a diagnosis, by prognostication and response to therapy monitoring. In three patients cfRRBS helped to establish the diagnosis of a rhabdomyosarcoma, an Ewing sarcoma and a neuroblastoma (case 1-3). In two patients, liquid biopsies were used for prognostication, by MYCN ddPCR in a patient with neuroblastoma and by RT-qPCR testing rhabdomyosarcoma-specific mRNA in bone marrow of a patient with a rhabdomyosarcoma (case 4 and 5). In case 6, mRNA testing demonstrated disease progression and assisted clinical decision making. This case series illustrates the value of liquid biopsy. We further demonstrate and recommend the use of liquid biopsies to be used in conjunction with conventional methods for the determination of metastatic status, prognostication and monitoring of treatment response in patients with pediatric solid tumors.

Clinical characteristics and short-term outcomes of acute pancreatitis among patients with COVID-19

ObjectiveThe existing literature on the combination of acute pancreatitis (AP) and COVID-19 is scarce. The objective of our study is to compare the clinical outcomes and occurrence of long COVID syndrome in AP patients with and without COVID-19, while investigating the potential impact of COVID-19 on the severity, mortality rate, and long COVID syndrome in these patients.MethodsThis retrospective, observational study was conducted at a single center. It included patients aged 18 years and above who were diagnosed with AP during the pandemic. Patients were categorized into two groups based on the results of RT-qPCR testing: the SARS-CoV-2-positive group and the SARS-CoV-2-negative group. The study aimed to compare the severity of AP, mortality rate, and occurrence of long COVID syndrome between these two groups.ResultA retrospective review was conducted on 122 patients diagnosed with acute pancreatitis between December 1, 2022, and January 31, 2023. Out of these patients, 100 were included in the study. The analysis revealed no significant differences in mortality rate, severity, and sequelae between AP patients with COVID-19 and those without COVID-19 (p > 0.005). However, a statistically significant difference was observed in the occurrence of long COVID syndrome, specifically in the presence of cough (p = 0.04).ConclusionThis study demonstrates that the presence of COVID-19 in patients with pancreatitis does not lead to an increase in the mortality and severity rate of pancreatitis.

An ecological study on reinfection rates using a large dataset of RT-qPCR tests for SARS-CoV-2 in Santiago of Chile.

As the SARS-CoV-2 continues to evolve, new variants pose a significant threat by potentially overriding the immunity conferred by vaccination and natural infection. This scenario can lead to an upswing in reinfections, amplified baseline epidemic activity, and localized outbreaks. In various global regions, estimates of breakthrough cases associated with the currently circulating viral variants, such as Omicron, have been reported. Nonetheless, specific data on the reinfection rate in Chile still needs to be included. Our study has focused on estimating COVID-19 reinfections per wave based on a sample of 578,670 RT-qPCR tests conducted at the University of Santiago of Chile (USACH) from April 2020 to July 2022, encompassing 345,997 individuals. The analysis reveals that the highest rate of reinfections transpired during the fourth and fifth COVID-19 waves, primarily driven by the Omicron variant. These findings hold despite 80% of the Chilean population receiving complete vaccination under the primary scheme and 60% receiving at least one booster dose. On average, the interval between initial infection and reinfection was found to be 372 days. Interestingly, reinfection incidence was higher in women aged between 30 and 55. Additionally, the viral load during the second infection episode was lower, likely attributed to Chile's high vaccination rate. This study demonstrates that the Omicron variant is behind Chile's highest number of reinfection cases, underscoring its potential for immune evasion. This vital epidemiological information contributes to developing and implementing effective public health policies.

Icariside Ⅱ attenuates bleomycin-induced pulmonary fibrosis by modulating macrophage polarization

Ethnopharmacological relevanceNumerous studies have provided evidence supporting the significant roles of icariin, in the prevention of multiple chronic diseases like diabetes, liver fibrosis, cardiac fibrosis, renal fibrosis, and pulmonary fibrosis. In particular, Icariside II (ISE II), a prominent flavonoid glycoside derived from Epimedium brevicornum Maxim, the principal metabolite of icariin, has demonstrated noteworthy anti-inflammatory and anti-oxidant properties, along with its ability to protect against lung remodeling. However, the research exploring ISE Ⅱ's application in treating pulmonary fibrosis remains limited. Aim of the studyThe aim of this study was to assess the therapeutic efficacy of ISE II in models of pulmonary fibrosis, while also investigating its potential mechanisms of action in cell signaling pathways. Materials and methodsAn in vitro model of pulmonary fibrosis was established by treating NIH-3T3 cells with transforming growth factor-β1 (TGF-β1). Western blot, RT-qPCR, and scratch test were performed to assess the effect of ISE Ⅱ. In addition, a murine model of pulmonary fibrosis was induced by intratracheal instillation of bleomycin, and the therapeutic effect of ISE Ⅱ was tested by orally administering ISE Ⅱ at a dose of 10 mg/kg. Three weeks later, lung function, micro-CT, hydroxyproline content, pathological staining, and cytokines detection of BALF or serum were used to assess the anti-fibrosis effects of ISE Ⅱ. Next, immunofluorescence staining, flow cytometry, and in vivo transcriptomics were used to investigate the underlying mechanisms of action. ResultsOur data revealed a significant inhibitory effect of ISE Ⅱ on the upregulation of α-smooth muscle actin (α-SMA) and collagen production induced by TGF-β1 in fibroblasts. Meanwhile, ISE Ⅱ exerted a therapeutic effect against bleomycin-induced pulmonary fibrosis in mice by improving lung function, decreasing collagen deposition, and reducing the expression of interleukin (IL)-1β, tumor necrosis factor α (TNF-α), TGF-β1 and platelet-derived growth factor (PDGF) in serum and bronchoalveolar lavage fluid (BALF). Additionally, ISE Ⅱ treatment effectively attenuated the infiltration of M2 macrophages, concurrently downregulating the expression level of M2 marker genes, such as CD206, arginase-1(Arg-1), and Chitinase-Like Protein 3 (YM-1). Importantly, we observed a statistically significant reduction in the M2 phenotype of interstitial macrophages (IMs). However, the impact of ISE Ⅱ on the M2 polarization of alveolar macrophages (AMs) did not reach statistical significance. Lastly, transcriptome sequencing results suggested that the anti-pulmonary fibrosis effects of ISE Ⅱ may be mediated by the suppression of the WNT/β-catenin signaling pathway, which modulated M2 polarization in macrophages and contributed to the amelioration of pulmonary fibrosis. By immunohistochemical analysis, it was verified that ISE Ⅱ treatment dramatically inhibited the activation of β-catenin in fibrosis murine. ConclusionOur findings indicated that ISE Ⅱ exerted anti-fibrotic effects by inhibiting pro-fibrotic macrophage polarization. The underlying mechanism of action might be mediated by modulating the WNT/β-catenin signaling pathway to inhibit the M2 program in IMs.

Temporal study of wastewater surveillance from September 2020 to March 2021: an estimation of COVID-19 patients in Lahore, Pakistan.

The first aim of study was to quantify the viral load in the wastewater samples by RT-qPCR testing in Lahore population to estimate the number of patients affected and predict the next resurgence of COVID-19 wave in the city. The second aim of the study was to determine the hotspot areas of Lahore which remained positive more often for virus with high viral load. In this study, n = 420 sewage samples were collected on anaverage of two weeks intervals from 30 different sewage water disposal stations (14 sampling events) from Sept 2020 to March 2021. RNA was extracted and quantified by RT-qPCR without concentrating the virus in samples. Number of positive disposal sites (7-93%), viral load from sewage samples (100.296 to 103.034), and estimated patients (660-17,030) ranged from low to high according to the surge and restrain of 2nd and 3rd COVID-19 waves in the country. The viral load and estimated patients were reported high in January 2021 and March 2021 which were similar to the peak of 2nd and 3rd waves in Pakistan. Site 18 (Niaz Baig village DS) showed the highest viral load among all sites. Findings of the present study helped to estimate the number of patients and track the resurgence in COVID-19 waves in Lahore particularly, and in Punjab generally. Furthermore, it emphasizes the role of wastewater-based epidemiology to help policymakers strengthen the quarantine measures along with immunization to overcome enteric viral diseases. Local and national stake holders should work in collaboration to improve the environmental hygiene to control the disease.

Differential expression of meiosis and homologous recombination-related genes in the life cycle of Trypanosoma cruzi.

Trypanosoma cruzi has a complex life cycle consisting of four morphological and distinct biological stages. Although some authors suggest that T. cruzi primarily follows clonal reproduction, recent genomic and transcriptomic studies indicate an unorthodox capacity for recombination. We aimed to estimate the differential gene expression of 10 meiosis/homologous recombination-related genes during the T. cruzi life cycle, including epimastigotes, under two different types of stress (oxidative stressand pH changes). We performed RT-qPCR tests using novel-designed primers to estimate the differential gene expression (∆Ct and ∆∆Ct) of nine genes (SPO11, HAP2, RAD50, MRN complex, BRCA2, DMC1, MND1, and RPA1) and RAD51, which was previously reported. Our results show basal expression of all genes during the life cycle, indicating their hypothetical role in several cellular processes but with specific signatures of differential gene expression during the life cycle (HAP2, RPA, RAD50, BRCA2, MND1, and DMC1) and oxidative stress (RPA, MRE11, NBS1, BRCA2, MND1, and RAD51). Additionally, we found that the MRN complex has an independent level of expression in T. cruzi, with profiles of MRE11 and NBS1 upregulated in some stages. Recent studies on other trypanosomatids have highlighted the influence of HAP2 and RPA in recombination and hybridization. If T. cruzi uses the same repertoire of genes, our findings could suggest that metacyclogenesis may be the putative step that the parasite uses to undergo recombination. Likewise, our study reveals the differential profiles of genes expressed in response to oxidative and pH stress. Further studies are necessary to confirm our findings and understand the recombination mechanism in T. cruzi.

The Mutational Landscape of SARS-CoV-2.

Mutation research is crucial for detecting and treating SARS-CoV-2 and developing vaccines. Using over 5,300,000 sequences from SARS-CoV-2 genomes and custom Python programs, we analyzed the mutational landscape of SARS-CoV-2. Although almost every nucleotide in the SARS-CoV-2 genome has mutated at some time, the substantial differences in the frequency and regularity of mutations warrant further examination. C>U mutations are the most common. They are found in the largest number of variants, pangolin lineages, and countries, which indicates that they are a driving force behind the evolution of SARS-CoV-2. Not all SARS-CoV-2 genes have mutated in the same way. Fewer non-synonymous single nucleotide variations are found in genes that encode proteins with a critical role in virus replication than in genes with ancillary roles. Some genes, such as spike (S) and nucleocapsid (N), show more non-synonymous mutations than others. Although the prevalence of mutations in the target regions of COVID-19 diagnostic RT-qPCR tests is generally low, in some cases, such as for some primers that bind to the N gene, it is significant. Therefore, ongoing monitoring of SARS-CoV-2 mutations is crucial. The SARS-CoV-2 Mutation Portal provides access to a database of SARS-CoV-2 mutations.

SARS-CoV-2 RT-qPCR Ct values in saliva and nasopharyngeal swab samples for disease severity prediction

ABSTRACT Background Comparison of clinical value of RT-qPCR-based SARS-CoV-2 tests performed on saliva samples (SSs) and nasopharyngeal swab samples (NPSs) for prediction of the COVID-19 disease severity. Methods Three paired SSs and NPSs collected every 3 days from 100 hospitalised COVID-19 patients during 2020 Jul-2021 Jan were tested by RT-qPCR for the original SARS-CoV-2 virus and compared to 150 healthy controls. Cases were divided into mild+moderate (Cohort I, N = 47) and severe disease (Cohort II, N = 53) cohorts and compared. Results SARS-CoV-2 was detected in 65% (91/140) vs. 53% (82/156) of NPSs and 49% (68/139) vs. 48% (75/157) of SSs collected from Cohort I and II, respectively, resulting in the total respective detection rates of 58% (173/296) vs. 48% (143/296) (P = 0.017). Ct values of SSs were lower than those of NPSs (mean Ct = 28.01 vs. 30.07, P = 0.002). Although Ct values of the first SSs were significantly lower in Cohort I than in Cohort II (P = 0.04), it became negative earlier (mean 11.7 vs. 14.8 days, P = 0.005). Multivariate Cox proportional hazards regression analysis showed that Ct value ≤30 from SSs was the independent predictor for severe COVID-19 (HR = 10.06, 95% CI: 1.84–55.14, P = 0.008). Conclusion Salivary RT-qPCR testing is suitable for SARS-CoV-2 infection control, while simple measurement of Ct values can assist in prediction of COVID-19 severity.

What to Do if the qPCR Test for SARS-CoV-2 or Other Pathogen Lacks Endogenous Internal Control? A Simple Test on Housekeeping Genes.

Some of the products for the molecular diagnosis of infections do not have an endogenous internal control, and this is necessary to ensure that the result is not a false negative. The aim of the project was to design a simple low-cost RT-qPCR test that can confirm the expression of basic metabolism proteins, thus confirming the quality of genetic material for molecular diagnostic tests. Two successful equivalent qPCR assays for the detection of the GADPH and ACTB genes were obtained. The course of standard curves is logarithmic, with a very high correlation coefficient R2 within the range of 0.9955-0.9956. The reaction yield was between 85.5 and 109.7%, and the detection limit (LOD) with 95% positive probability was estimated at 0.0057 ng/µL for GAPDH and 0.0036 ng/µL for ACTB. These tests are universal because they function on various types of samples (swabs, cytology, etc.) and can complement the diagnosis of SARS-CoV-2 and other pathogens, as well as potentially oncological diagnostics.

Cycle threshold predicted mortality in a cohort of patients with hematologic malignancies infected with SARS-CoV-2

When a SARS-CoV-2 RT-qPCR test is performed, it may determine an indirect measure of viral load called cycle threshold (Ct). Respiratory samples with Ct <25.0 cycles are considered to contain a high viral load. We aimed to determine whether SARS-CoV-2 Ct at diagnosis could predict mortality in patients with hematologic malignancies (lymphomas, leukemias, multiple myeloma) who contracted COVID-19. We included 35 adults with COVID-19 confirmed by RT-qPCR performed at diagnosis. We evaluated mortality due to COVID-19 rather than mortality due to the hematologic neoplasm or all-cause mortality. Twenty-seven (27) patients survived and 8 died. The global mean Ct was 22.8 cycles with a median of 21.7. Among the survivors, the mean Ct was 24.2, and the median Ct value was 22.9 cycles. In the deceased patients, the mean Ct was 18.0 and the median Ct value was 17.0 cycles. Using the Wilcoxon Rank Sum test, we found a significant difference (p=0.035). SARS-CoV-2 Ct measured in nasal swabs obtained at diagnosis from patients with hematologic malignancies may be used to predict mortality.

Trends in SARS-CoV-2 cycle threshold values in the Czech Republic from April 2020 to April 2022

The inability to predict the evolution of the COVID-19 epidemic hampered abilities to respond to the crisis effectively. The cycle threshold (Ct) from the standard SARS-CoV-2 quantitative reverse transcription-PCR (RT-qPCR) clinical assay is inversely proportional to the amount of SARS-CoV-2 RNA in the sample. We were interested to see if population Ct values could predict future increases in COVID-19 cases as well as subgroups that would be more likely to be affected. This information would have been extremely helpful early in the COVID-19 epidemic. We therefore conducted a retrospective analysis of demographic data and Ct values from 2,076,887 nasopharyngeal swab RT-qPCR tests that were performed at a single diagnostic laboratory in the Czech Republic from April 2020 to April 2022 and from 221,671 tests that were performed as a part of a mandatory school surveillance testing program from March 2021 to March 2022. We found that Ct values could be helpful predictive tools in the real-time management of viral epidemics. First, early measurement of Ct values would have indicated the low viral load in children, equivalent viral load in males and females, and higher viral load in older individuals. Second, rising or falling median Ct values and differences in Ct distribution indicated changes in the transmission in the population. Third, monitoring Ct values and positivity rates would have provided early evidence as to whether prevention measures are effective. Health system authorities should thus consider collecting weekly median Ct values of positively tested samples from major diagnostic laboratories for regional epidemic surveillance.

A RT-qPCR test targeting the conserved 5´-UTR of SARS-CoV-2 overcomes major shortcomings of the first WHO-recommended RT-qPCR test

For the first time in medical history, the result of a laboratory assay (RT-qPCR) was used as the sole criterion to diagnose a disease (Covid-19) without any rating of clinical symptoms and to justify the implementation of population-wide non-pharmaceutical interventions during virus season. The aim of this study is (1) to evaluate a robust RT-qPCR test that overcomes major concerns raised within the scientific community on the first WHO-recommended RT-qPCR protocol to detect SARS-CoV-2 sequences, (2) to genomically characterize individual SARS-CoV-2 strains circulating in the Czech Republic from autumn 2020 to spring 2021 applying NGS and (3) to re-initiate the currently missing scientific dialogue and return to reason and evidence-based medicine. We present a RT-qPCR test designed for the detection of all SARS-CoV-2 variants known so far. Based on the genomic mutation profile, we demonstrate that the three individual waves culminating from autumn 2020 to spring 2021 in the Czech Republic were successive, but lacked direct genomic relationship between each other. This became obvious with the omicron variant, which did not reveal direct evolutionary connection to any of the previous SARS-CoV-2 variants. In addition, we provide evidence that neglected principles of good scientific practice resulted not only in the publication of the WHO-recommended Charité RT-qPCR protocol, but also in health-related problems. Unnecessary quarantine of false positive-tested, healthy individuals, as well as lockdowns and atrocious collateral damage on societies and economies worldwide due to a high number of false-positive “PCR-cases.” Further, some chain of infection caused by false negative-tested, symptomatic individuals lead to real Covid-19 clusters. Both our results and literature data confirm that validation of any PCR-based diagnostic test by sequencing is mandatory on a regular basis. To prevent future misconduct, science needs a reality check and must re-initiate the scientific dialogue and liberate itself from political influence and dogma.

A Novel RT-LAMP for the Detection of Different Genotypes of Crimean-Congo Haemorrhagic Fever Virus in Patients from Spain.

Crimean-Congo haemorrhagic fever (CCHF) is a potentially lethal tick-borne viral disease with a wide distribution. In Spain, 12 human cases of CCHF have been confirmed, with four deaths. The diagnosis of CCHF is hampered by the nonspecific symptoms, the high genetic diversity of CCHFV, and the biosafety requirements to manage the virus. RT-qPCR and serological tests are used for diagnosis with limitations. Reverse-transcription loop-mediated isothermal amplification (RT-LAMP) could be an effective alternative in the diagnosis of the disease. However, none of the few RT-LAMP assays developed to date has detected different CCHFV genotypes. Here, we designed a RT-LAMP using a degenerate primer set to compensate for the variability of the CCHFV target sequence. RT-LAMP was performed in colorimetric and real-time tests on RT-qPCR-confirmed CCHF patient samples notified in Spain in 2020 and 2021. Urine from an inpatient was analysed by RT-LAMP for the first time and compared with RT-qPCR. The amplicons obtained by RT-qPCR were sequenced and African III and European V genotypes were identified. RT-LAMP amplified both genotypes and was more sensitive than RT-qPCR in urine samples. We have developed a novel, rapid, specific, and sensitive RT-LAMP test that allows the detection of different CCHFV genotypes in clinical samples. This pan-CCHFV RT-LAMP detected viral RNA for the first time in urine samples. It can be easily performed as a single-tube isothermal colorimetric method on a portable platform in real time and without the need for expensive equipment, thus bringing molecular diagnostics closer to rural or resource-poor areas, where CCHF usually occurs.