Real-time Polymerase Chain Reaction Test Research Articles

The effectiveness of liver function tests in the prediction of mortality in patients with COVID-19

Objective: To evaluate the impact of liver function test abnormalities on mortality in critically ill coronavirus disease-2019 patients. Method: The single-centre, retrospective study was conducted at the Department of Anaesthesiology and Reanimation, Mersin City Training and Research Hospital, Turkiye, and comprised data from March 2020 to January 2022 of patients with positive real-time reverse transcription polymerase chain reaction test on nasopharyngeal swab sample for coronavirus disease-2019. Comorbidities, demographic data, admission to and discharge from the intensive care unit, Acute Physiology and Chronic Health Evaluation II scores, complete blood count on the first and second day of hospitalisation, coagulation parameters, biochemical analysis, 7-day, 28-day and total mortality of the patients in the intensive care unit were recorded. The patients were then divided into groups, with Group 1 having patients with normal liver enzymes, Group 2 having patients with abnormal liver enzymes, and Group 3 having patients with liver damage. Data was analysed using SPSS 20. Results: Of the 940 subjects, 587(62.4) were males and 353(37.6) were females. The overall mean age of the patients was 67.11±16.65 years. There were 395(42%) patients in Group 1, 534(56.8%) in Group 2 and 11(1.2%) in Group 3. Gamma-glutamyl transferase-1s, aspartate aminotransferase-2nd and albumin-2nd were significant predictors of mortality (p<0.05). Conclusion: High gamma-glutamyl transferase, aspartate aminotransferase and low albumin levels in critically ill coronavirus disease-2019 patients could be considered indicators of high mortality rate. Key Words: Coronavirus, Critical illness, Liver function test, Pandemic.

B-236 New real-time PCR test for APOE genotyping in patients with Alzheimer’s disease before anti-amyloid therapy

Abstract Background In 2023, the US Food and Drug Administration approved LEQEMBI, a monoclonal antibody therapy targeting beta-amyloid plaques, for patients with early-stage Alzheimer’s disease (AD). However, anti-amyloid drug trials demonstrated an elevated risk for amyloid-related imaging abnormalities with cerebral edema (ARIA-E) in carriers of the AD risk allele apolipoprotein E epsilon 4 (APOE-ɛ4), especially in ɛ4/ɛ4 homozygous individuals [1-3]. Here, we report on the evaluation of a new real-time polymerase chain reaction (PCR) test for APOE genotyping. Methods The EURORealTime APOE (EUROIMMUN), which enables simultaneous testing for all six APOE genotypes (ɛ2/ɛ2, ɛ2/ɛ3, ɛ2/ɛ4, ɛ3/ɛ3, ɛ3/ɛ4, and ɛ4/ɛ4) by real-time PCR, was performed using genomic DNA (gDNA) isolated from EDTA blood samples. The PCR amplification results were evaluated using the EURORealTime Analysis Software (EUROIMMUN). Samples from healthy blood donors (HBD) were used to assess intra-assay, inter-assay and between-lot precision (n=6) as well as analytical sensitivity (n=21), with each APOE genotype represented by at least one sample. Assay accuracy was analyzed by comparing genotype assignments to the results of the CE-IVD-marked EUROArray APOE (EUROIMMUN) and bidirectional Sanger sequencing using 110 samples (100 AD, 10 HBD). APOE genotype frequencies were compared between patients (100 AD) and controls (250 HBD). Results Precision testing of the EURORealTime APOE resulted in 100% correct genotype calls. Assessment of the analytical sensitivity revealed valid and correct genotype assignment in all 21 samples, confirming 1 ng/µl as the recommended minimum gDNA concentration. The analytical accuracy amounted to 100% based on agreement with the two reference methods in 110/110 cases. Comparison of the APOE genotype distribution showed a higher proportion of ɛ4 allele carriers (i.e., ɛ2/ɛ4, ɛ3/ɛ4, and ɛ4/ɛ4) in AD patients than in HBD (50.0% vs. 25.2%). The prevalence of the ɛ4/ɛ4 genotype in these cohorts was 25.0% for AD patients compared with 2.8% for HBD. Conclusions The well-established high prevalence of the APOE-ɛ4/ɛ4 genotype in AD patients, together with the increased risk of ARIA-E in these homozygous carriers, substantiates the relevance of APOE genotyping prior to anti-amyloid treatment. The EURORealTime APOE test provides sensitive and reliable determination of APOE alleles, thus presenting a suitable tool to improve risk stratification for potential LEQEMBI recipients.

A-279 Performance Evaluation of the Respiratory Viral for BD MAX™ System in Geriatric Population

Abstract Background Respiratory Viral Infection is one of the most common infection in the geriatric population and cause major concern in the Long-Term Care Facilities. Influenza A and B, SARS-CoV-2, and respiratory syncytial virus (RSV) are responsible for most of the hospitalization and deaths among elderly patients. Rapid identification, treatment, and isolation are among the most important steps to fight the spreading of the infection. Methods The BD Respiratory Viral Panel for BD MAX™ System is an automated multiplexed real-time reverse transcriptase polymerase chain reaction (RT- PCR) test intended for the simultaneous, qualitative detection and differentiation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A, influenza B, and/or respiratory syncytial virus (RSV) nucleic acid in nasopharyngeal swab (NPS) and anterior nasal swab (ANS) specimens from individuals with signs and symptoms of respiratory tract infection. The assay is targeting RNA from the nucleocapsid phosphoprotein gene (N1 and N2 regions) of the SARS-CoV-2, a conserved region of the matrix protein M1 gene for influenza A, conserved regions of the matrix protein M1 gene and HA gene for influenza B, conserved regions of the N and M genes for RSV, and the human RNase P gene. The assay was evaluated for: precision, reproducibility, accuracy, and correlation with BioGx for SARS-CoV-2, Solana (Quidel) for influenza A, influenza B and RSV; the percentage agreement for the evaluation steps was calculated. We ran 86 samples collected from patients residing in Long-Term Care Facilities. Statistical analyses were done using Analyse-it. Results The percentage agreement for precision, reproducibility, and accuracy were 100%. The patients’ correlation was 100% when compared to the existing molecular testing in the laboratory. Female patients accounted for 62.8% (54 female and 32 male); 2 patients were positive for both influenza A and RSV, and one patient was positive for both influenza A and SAR-CoV-2. Conclusions The Respiratory Viral panel for BD MAX™ System gave the benefit of a fully automated, high precision, accuracy and acceptable sensitivity assay; in addition, it offers easier collection (one swabs versus multiple swabs), faster turnaround time for four targets in less than three hours which allow for earlier isolation to prevent the spreading of the infection and initiating treatment when needed. As with every molecular assay avoiding contamination is very important to eliminated false positive results.

Spore PCR and qPCR Methods for Rapid Detection of Five Colletotrichum Species Responsible for Pepper Anthracnose in Korea

Pepper anthracnose, caused by <i>Colletotrichum</i> spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of <i>Colletotrichum</i> spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.

One-Tube Nested PCR Coupled with CRISPR-Cas12a for Ultrasensitive Nucleic Acid Testing.

Nucleic acid testing with high sensitivity and specificity is of great importance for accurate disease diagnostics. Here, we developed an in situ one-tube nucleic acid testing assay. In this assay, the target nucleic acid is captured using magnetic silica beads, avoiding an elution step, followed directly by the polymerase chain reaction (PCR) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a detection. This assay achieved visual readout and a sensitivity of 120 copies/mL for detecting SARS-CoV-2. More importantly, the assay demonstrated over 95% sensitivity and 100% specificity compared to the gold standard real-time quantitative PCR (RT-qPCR) test by using 75 SARS-CoV-2 clinical samples. By integrating nested PCR and Cas12a, this all-in-one nucleic acid testing approach enables ultrasensitive, highly specific, and cost-effective diagnosis at community clinics and township hospitals.

Shilajit (Mumio) Elicits Apoptosis and Suppresses Cell Migration in Oral Cancer Cells Through Targeting Urokinase-type Plasminogen Activator and Its Receptor and Chemokine Signaling Pathways

Background Shilajit a natural phytomineral has a proven record of treating many human body ailments. Purpose This study aimed to explore the influence of Shilajit on cell proliferation and apoptosis induction in oral cancer cells (OCC) and to comprehend the molecular mechanism associated with OCC migration. Materials and Methods Human gingival fibroblast (hGF) and OCC (KB-1 (KERATIN-forming tumor cell line HeLa)) were exposed to Shilajit solution dilutions. Cell growth and apoptosis were measured by MTT and Annexin-V tests (control/test group). Cellular morphology using an inverted microscope, cellular apoptosis using acridine orange/ethidium bromide dual staining, reactive oxygen species production analysis using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, and gene protein expression using real-time polymerase chain reaction test were used to measure study outcomes. The data enumerated were the average of three trial experiments. Categorical and numerical data were expressed as frequency distribution and means, respectively. Differences in groups (control/test; zero/24 h/48 h) were determined using Student’s t-test and one way analysis of variance, with probability ‘ p’ value considered significant at ≤0.05. Results The viability of OCC exhibited a concentration and time-dependent response to Shilajit. Notably, Shilajit demonstrated selectivity against cancer cells. Through an examination of the Annexin-V apoptosis assay, it was observed that Shilajit induces apoptosis by upregulating the proapoptotic gene expression ( p ≤ 0.05) and downregulating antiapoptotic proteins ( p ≤ 0.05). Furthermore, the impact of Shilajit on cell migration decreased significantly when compared to control cells through modulating the urokinase-type plasminogen activator (uPA) and its receptor (uPAR) and chemokines gene expression. Conclusion Shilajit exhibited greater cytotoxicity, decreased OCC proliferation and migration, and initiated OCC apoptosis as equivalent to normal cells. These promising outcomes indicate that Shilajit holds potential as a robust, promising option for oral cancer treatment, underscoring the need for further research in this domain.

Analiza różnic w stężeniu przeciwciał przeciw SARS-CoV-2 w przebiegu COVID-19 w zależności od ciężkości choroby, płci, wieku i statusu szczepienia

Introduction and objectiveThe role of immunoglobulin G antibodies in combating SARS-CoV-2 infection, modulation of COVID-19 disease severity, and persistence of humoral response after primary infection and vaccination is unclear. This study aimed to evaluate the role of antibodies in limiting the infection, modulating disease severity, and determining the durability of the immune response depending on the clinical status of patients, their age, sex, and vaccination status.Material and methodsThe study involved 156 patients, 99 men and 57 women, aged 58.3 ± 12.5 years old, hospitalised for pneumonia in the course of COVID-19, with infection confirmed by real-time polymerase chain reaction test. The concentration of anti-SARS-CoV-2 IgG antibodies was tested at 3, 6, and 9 months from the day of hospitalisation.ResultsThe concentration of anti-SARS-CoV-2 IgG antibodies in patients with severe COVID-19 was higher compared to the group of patients with mild to moderate disease. The level of anti-SARS-CoV-2 IgG antibodies was comparable in men and women. In patients over 60 years of age, a lower concentration of antibodies was observed than in patients under 60 years of age. In the vaccinated group, the level of antibodies was higher than in the unvaccinated group.ConclusionsThe study findings showed that the concentration of IgG anti-SARS-CoV-2 antibodies was not a parameter dependent on the sex of the patients, but rather on the severity of the disease and the age of the patients and their vaccination status.

The impact of photobiomodulation on angiogenic differentiation of two different dental derived stem cells using two irradiation protocols: an in vitro investigation

The present study aimed to compare the effect of photobiomodulation with different energy densities on the angiogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and stem cells from human exfoliated deciduous teeth (SHED). Photobiomodulation therapy with a 660 nm diode laser (2.4 J/cm2 and 3.9 J/cm2) on two consecutive days post-culture was applied to two types of stem cells (hPDLSCs and SHED). The Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) test was undertaken to investigate Vascular Endothelial Growth Factor-A (VEGF-A) and Angiopoietin I (ANG-I) genes on days 1, 3, 5, 7, and 10 after the first session of laser application. The 4′,6-diamidino-2-phenylindole (DAPI) staining and Methyl Thiazolyl Tetrazolium (MTT) test were conducted on days 1, 3, and 5 after the first session of laser application, to assess the cell viability. The Two-way ANOVA with Tukey post hoc test was used to analyze the outcomes of the MTT and RT-qPCR tests. The results of the MTT and DAPI convergently illustrated that the groups receiving photobiomodulation with 2.4 J/cm2 had higher cell viability compared to 3.9 J/cm2. All experimental groups showed an upregulation of VEGF-A and ANG-I gene expression from day 1 to 5, followed by a downregulation from day 5 to 10. The groups with cultured hPDLSCs and SHED receiving photobiomodulation using 2.4 J/cm2 had the most amounts of VEGF-A and ANG-I gene expression on day 5, respectively. In conclusion, the 660 nm mediated photobiomodulation therapy of cultured SHED and hPDLSCs with 2.4 J/cm2 energy density may be associated with higher angiogenic differentiation (the expression of VEGF-A and ANG-I) as well as higher cell viability compared to the photobiomodulation therapy with 3.9 J/cm2.

Value of CDR1-AS as a predictive and prognostic biomarker for patients with breast cancer receiving neoadjuvant chemotherapy in a prospective Chinese cohort

BackgroundNeoadjuvant chemotherapy (NAC) is an effective treatment for locally advanced breast cancer (BC). However, there are no effective biomarkers for evaluating its efficacy. CDR1-AS, well known for its important role in tumorigenesis, is a famous circular RNA involved in the chemosensitivity of cancers other than BC. However, the predictive role of CDR1-AS in the efficacy and prognosis of NAC for BC has not been fully elucidated. We herein aimed to clarify this role.MethodsThe present study included patients treated with paclitaxel-cisplatin-based NAC. The expression of CDR1-AS was detected by real-time quantitative reverse transcription polymerase chain reaction testing. The predictive value of CDR1-AS expression was examined in pathological complete response (pCR) after NAC using logistic regression analysis. The relationship between CDR1-AS expression and survival was demonstrated using the Kaplan–Meier method, and tested by log-rank test and Cox proportional hazards regression model.ResultsThe present study enrolled 106 patients with BC. Multivariate logistic regression analysis revealed that CDR1-AS expression was an independent predictive factor for pCR (odds ratio [OR] = 0.244; 95% confidence interval [CI] 0.081–0.732; p = 0.012). Furthermore, pCR benefits with low CDR1-AS expression were observed across all subgroups. The Kaplan–Meier curves and log-rank test suggested that the CDR1-AS high-expression group showed significantly better disease-free survival (DFS; log-rank p = 0.022) and relapse-free survival (RFS; log-rank p = 0.012) than the CDR1-AS low-expression group. Multivariate analysis revealed that CDR1-AS expression was an independent prognostic factor for DFS (adjusted HR = 0.177; 95% CI 0.034–0.928, p = 0.041), RFS (adjusted HR = 0.061; 95% CI 0.006–0.643, p = 0.020), and distant disease-free survival (adjusted HR = 0.061; 95% CI 0.006–0.972, p = 0.047).ConclusionsCDR1-AS may be a potential novel predictive biomarker of pCR and survival benefit in patients with locally advanced BC receiving NAC. This may help identify specific chemosensitive individuals and build personalized treatment strategies.

Two Peaks of Seasonal Influenza Epidemics - China, 2023.

Influenza is a communicable respiratory disease that causes annual epidemics and occasional unpredictable pandemics. This study examines the occurrence of two unexpected influenza peaks within the year 2023. Influenza-like illness (ILI) data were reported, and specimens of ILI were collected by designated surveillance hospitals. Positive cases were confirmed using real-time reverse transcription polymerase chain reaction (rRT-PCR) testing conducted at national surveillance network laboratories. The data were then submitted to the Chinese Influenza Surveillance Information System. From December 2022 to January 2023, influenza activity was minimal until a spring epidemic began in February, peaking in late March. The outbreak spread from the north to the south, with A(H1N1)pdm09 being the predominant strain and A(H3N2) also circulating concurrently. The positivity rate in northern and southern provincial-level administrative divisions (PLADs) reached 60.0% (week 10) and 60.2% (week 13), respectively, before dropping to below 5% from May to August. Subsequently, rates surged starting in week 41 in the south and week 44 in the north, peaking at 54.4% (week 50) and 44.0% (week 49), respectively, and remained high until the end of 2023. A(H3N2) became the prevailing strain, with a notable increase in B/Victoria lineage viruses from December, resulting in two peaks during the 2023 influenza season. The pattern of the influenza epidemic shifted due to the coronavirus disease 2019 (COVID-19) outbreak and is gradually returning to its usual seasonal and intensity patterns. It is, therefore, crucial to continuously strengthen influenza surveillance, enhance the influenza surveillance network, and lay the groundwork for advancing integrated surveillance of multiple pathogens in China.

Diagnostic utility of pharyngeal follicular structures in COVID-19: A large-scale cross-sectional study

ObjectivesPharyngeal follicles similar to those seen in influenza have been observed in patients with coronavirus disease 2019 (COVID-19), suggesting their potential as early-stage diagnostic markers. In this study, we examined the diagnostic potential of pharyngeal follicles for COVID-19, particularly the Omicron variant and its subtypes, to obtain basic data for AI-based diagnostic imaging tools. MethodsA cross-sectional study was conducted from July 21, 2022, to March 31, 2023, at the Tokyo Shinagawa Hospital's fever clinic. Participants aged ≥15 years who underwent real-time polymerase chain reaction testing for COVID-19 and pharyngeal examinations were included. Demographic details, symptom onset, throat pain, and vaccination status were also recorded. Pharyngeal structures were categorized into four groups: follicles, buds, mixed, or absent. ResultsOf the 1223 participants, 829 (67.8%) tested positive for COVID-19. Among those who tested positive, 73.6% (95% CI: 70.6%-76.6%) had follicular structures, compared to 52.8% (95% CI: 47.9%-57.7%) of those who tested negative (P = 1.0 × 10−12). Overall, 818 participants exhibited follicular structures (439 with follicles, 281 with buds, and 98 with mixed structures), while 405 lacked any follicular structures. Regression analysis identified throat pain and follicular structures as significant COVID-19 predictors (95% confidence intervals: 2.49-4.85 and 1.43-2.59, respectively). Mixed follicles were identified as a potentially characteristic feature of COVID-19. ConclusionPharyngeal follicular structures demonstrated high sensitivity for early COVID-19 diagnosis.

Comparison of diagnostic accuracy of rapid antigen tests for COVID19 compared to the viral genetic test in adults: a systematic review and meta-analysis.

The objective of this review is to determine the diagnostic accuracy of the currently available and upcoming point-of-care rapid antigen tests (RATs) used in primary care settings relative to the viral genetic real-time reverse transcriptase polymerase chain reaction (RT-PCR) test as a reference for diagnosing COVID-19/SARS-CoV-2 in adults. Accurate COVID-19 point-of-care diagnostic tests are required for real-time identification of SARS-CoV-2 infection in individuals. Real-time RT-PCR is the accepted gold standard for diagnostic testing, requiring technical expertise and expensive equipment that are unavailable in most primary care locations. RATs are immunoassays that detect the presence of a specific viral protein, which implies a current infection with SARS-CoV-2. RATs are qualitative or semi-quantitative diagnostics that lack thresholds that provide a result within a short time frame, typically within the hour following sample collection. In this systematic review, we synthesized the current evidence regarding the accuracy of RATs for detecting SARS-CoV-2 compared with RT-PCR. Studies that included nonpregnant adults (18 years or older) with suspected SARS-CoV-2 infection, regardless of symptomology or disease severity, were included. The index test was any available SARS-CoV-2 point-of-care RAT. The reference test was any commercially distributed RT-PCR-based test that detects the RNA genome of SARS-CoV-2 and has been validated by an independent third party. Custom or in-house RT-PCR tests were also considered, with appropriate validation documentation. The diagnosis of interest was COVID-19 disease and SARS-CoV-2 infection. This review considered cross-sectional and cohort studies that examined the diagnostic accuracy of COVID-19/SARS-CoV-2 infection where the participants had both index and reference tests performed. The keywords and index terms contained in relevant articles were used to develop a full search strategy for PubMed and adapted for Embase, Scopus, Qinsight, and the WHO COVID-19 databases . Studies published from November 2019 to July 12, 2022, were included, as SARS-CoV-2 emerged in late 2019 and is the cause of a continuing pandemic. Studies that met the inclusion criteria were critically appraised using QUADAS-2. Using a customized tool, data were extracted from included studies and were verified prior to analysis. The pooled sensitivity, specificity, positive predictive, and negative predictive values were calculated and presented with 95% CIs. When heterogeneity was observed, outlier analysis was conducted, and the results were generated by removing outliers. Meta-analysis was performed on 91 studies of 581 full-text articles retrieved that provided true-positive, true-negative, false-positive, and false-negative values. RATs can identify individuals who have COVID-19 with high reliability (positive predictive value 97.7%; negative predictive value 95.2%) when considering overall performance. However, the lower level of sensitivity (67.1%) suggests that negative test results likely need to be retested through an additional method. Most reported RAT brands had only a few studies comparing their performance with RT-PCR. Overall, a positive RAT result is an excellent predictor of a positive diagnosis of COVID-19. We recommend that Roche's SARS-CoV-2 Rapid Antigen Test and Abbott's BinaxNOW tests be used in primary care settings, with the understanding that negative results need to be confirmed through RT-PCR. We recommend adherence to the STARD guidelines when reporting on diagnostic data. PROSPERO CRD42020224250.

Contactless Single Cell Extraction from Monolayer Cell Culture Using A MEMS Acoustic Droplet Ejector.

To achieve a contactless and damage-less extraction of a single (or a few) human retinal pigment epithelial (RPE) cell(s) from a cell monolayer with acoustic droplet ejection. An acoustic droplet ejector based on a self-focusing acoustic transducer (SFAT) is designed, microfabricated, and placed on a precision movable stage that aligns it to the targeted cell(s) in a Petri dish. The device delivers 20.1 MHz focused ultrasound (FUS) with a focal diameter of 100 μm, which ejects droplets capable of extracting and transferring only the targeted cell(s) from a monolayer. The extraction and collection of 1-10 cells are successfully demonstrated. The number of ejected cells can be controlled by the FUS pulse width. As confirmed by fluorescence-based cell viability assays and re-culture experiments, the ejected cell(s) and remaining monolayer cells are intact without damage after cell ejection. Furthermore, real-time reverse transcription polymerase chain reaction (RT-PCR) tests for both housekeeping genes (GAPDH and β -actin) and RPE-specific genes (MITF, PEDF, and PMEL17) show no significant difference between the acoustically ejected cells and those collected manually with a micropipette. The proposed technology realizes a contactless, damage-free extraction of cells with high spatial resolution and precise control of the number of cells ejected, with a simple setup. This powerful technology not only enables efficient, high-precision cell extraction for quality check applications but also opens new avenues for other advanced biotechnologies such as bioprinting.

Role of IgM/ IgG Ratio in Distinguishing Primary and Secondary Dengue Viral Infections: A Cross-Sectional Study.

Objectives In recent years, Uttarakhand, a state in North India has become one of the prime spots for tourism all over the world. Thereby, a tremendous increase in the epidemics of dengue infection has been observed recently. Secondary dengue causes more severe disease in comparison with primary, thus to differentiate the two is very crucial. We aim to find out the cut-off values of the IgM:IgG ratio for early detection of secondary dengue which could further help clinicians to prevent the complications. Methods A cross-sectional study was conducted over one year involving around 936 suspected cases of dengue. Samples were tested using the commercially available capture enzyme linked Immunosorbent assay (ELISA) method for IgM and IgG. Real-time and nested polymerase chain reaction (PCR) tests were also done to find out the prevalent serotype. IgM:IgG ratio was evaluated by using receiver operating characteristic curve analysis for the differentiation of primary and secondary dengue. Results Among the total 91 serologically confirmed dengue patients, forty-seven (51.6%) were found to be primary, and forty-four (48.4%) were secondary dengue infections with male preponderance. Using the WHO diagnostic criteria, patients with dengue fever (DF) without warning signs added up to 51.6%, with warning signs 42.9% and severe dengue 5.5% of the total cases. The cut-off ratio of IgM:IgG ratio = 1.59 found the best discrimination between primary and secondary infection. Forty out of ninety-one (44%) patients exhibited ratios of > 1.59 whereas the rest fifty-one (56%) exhibited ratios of < 1.59. Dengue virus - 2 (DENV- 2) was found to be the most prevalent serotype. Conclusion Our study recommends the cut-off values for IgM:IgG ratio as 1.59. Therefore it is hoped that this will guide the clinicians to early distinguish between primary and secondary dengue. Furthermore, it can reduce morbidity and mortality because of dengue infections in the future.

Microbial contamination of spittoons and germicidal effect of irradiation with krypton chloride excimer lamps (Far UV-C 222 nm).

In dentistry, instruments, appliances, and body fluids such as saliva or blood are possible sources of infection. Although conventional antiseptic procedures effectively prevent infection, spittoons cannot be sanitized between each treated patient and are usually washed only with running water. However, there is currently no fast and efficient disinfection method that can be implemented between treatments. An optically filtered krypton chloride excimer lamp using ultraviolet light (Far UV-C) in the 200-230 nm wavelength range (innocuous to humans) has been recently used as a virus- and bacteria-inactivating technology. This study aimed to identify the bioburden of a dental spittoon and examine the susceptibility of two oral Streptococcus and two Enterococci to 222-nm Far UV-C by irradiating the spittoon with 222 nm Far UV-C for 5 min before evaluating the disinfection effect. Bacterial analysis and real-time polymerase-chain reaction testing was used to confirm the spittoon's biological contamination. Bacterial susceptibility to a 222-nm Far UV-C was determined with a graded dose irradiation test. After each treatment, the spittoon was irradiated with 222-nm Far UV-C for 5 min, and the disinfecting effect was evaluated. Microbial analysis of the spittoon's surface was performed using the Silva database. We found that > 97% of the microbes consisted of six bacterial phyla, whereas no viruses were found. Pseudomonas aeruginosa was frequently detected. The 1-log reduction value of two oral-derived Streptococci and two Enterococci species at 222-nm Far UV-C was 4.5-7.3 mJ/cm2. Exposure of the spittoon to 222-nm Far UV-C at 3.6-13.5 mJ/cm2 significantly decreased bacterial counts (p < 0.001). Irradiation with 222-nm Far UV-C at 3.6-13.5 mJ/cm2 significantly eliminates bacteria in spittoons, even when they are only rinsed with water. Hence, 222-nm Far UV-C irradiation may inhibit the risk of bacterial transmission from droplets in sink surfaces.

Rapid Detection of Severe Fever with Thrombocytopenia Syndrome Virus in the Acute Phase of Infection by Direct Real-Time Reverse Transcription without RNA Extraction.

No specific treatment has been developed for severe fever with thrombocytopenia syndrome (SFTS). However, the prognosis can improve with early plasma exchange. Therefore, rapid and accurate detection of SFTS virus is important for diagnosis and prognosis. Direct real-time reverse transcription polymerase chain reaction (RT-PCR) testing is easier and more time-efficient than conventional real-time RT-PCR. Our study compared direct real-time RT-PCR efficiency without the RNA extraction and purification of conventional real-time RT-PCR. Samples were collected from 18 patients with SFTS and five without SFTS. A strong correlation (r = 0.774, 95% CI: 0.652-0.857, P <0.01) was found between conventional and direct real-time RT-PCR assays. Direct real-time RT-PCR showed 84.4% sensitivity and 92.0% specificity for viral detection. Direct real-time RT-PCR is an effective diagnostic tool for patients with acute phase SFTS, but further optimization is required for viral detection.

The role of uncoupling protein 2 in experimental periodontitis-associated renal injury in rats.

This study aims to explore changes in uncoupling protein 2 (UCP2) in experimental periodontitis-associated renal injury induced by ligation and investigate the effect of UCP2 on renal injury induced by periodontitis. Twelve Wistar male rats were randomly divided into two groups: control and periodontitis groups. A periodontal model was built by ligating the maxillary first molars area with 0.2 mm orthodontic ligature wire. After 8 weeks, the intraoral condition of the rats was observed and periodontal clinical indices such as gingival bleeding index (BI), periodontal probing depth (PD), and tooth mobility (TM) were detected. The maxillary bone was scanned by Micro CT to observe the alveolar bone resorption. The tissue mineral density (TMD), bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular bone separation (Tb.Sp) were recorded, and the distance from the enamel bone boundary to the alveolar crest (CEJ-ABC) of the maxillary first molar was measured. The oxidative stress indexes such as malondialdehyde, glutathione (GSH), and superoxide dismutase (SOD) were detected using frozen rat kidney tissue. The gene expression of UCP2, nuclear factor erythroid 2-related factor 2 (Nrf2), and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) was observed by quantitative real-time polymerase chain reaction (qRT-PCR) test. The gingival tissue of the rats was used for immunohistochemical staining to observe the expression of the UCP2 protein. The fixed rat kidney tissue was used for hematoxylin-eosin (HE), periodic acid-schiff (PAS), MitoSOX Red, JC-1, and immunohistochemical staining to observe the renal histopathology, the level of reactive oxygen species (ROS), the level of mitochondrial membrane potential, and the expression of UCP2, Nrf2, and PGC-1α protein. Rat serum was collected to detect renal function indices, namely, blood urea nitrogen (BUN), creatinine (Cre), and albumin (Alb). Compared with the control group, the periodontitis group showed red, swollen, and soft gingival tissue, with gingival probing bleeding, periodontal PD increased, tooth loosening, alveolar bone resorption, decreased TMD, BMD, BV/TV, and Tb.Th indices, and increased Tb.Sp index, CEJ-ABC, and gingival UCP2 protein expression. Compared with the control group, the levels of MDA and ROS in the kidney tissue of periodontitis rats and the gene and protein expression of UCP2 increased, and the levels of MMP, GSH, and SOD and the gene and protein expression of Nrf2 and PGC-1α decreased. Renal functional indices, namely, BUN, Cre, and Alb, were not significantly different between the two groups. UCP2 may play a role in renal injury induced by periodontitis through oxidative stress.

Image Findings and Radiation Dose in Lung CT Scan of Patients With COVID-19 in Hamadan

Background: Computed tomography (CT) scan plays an important role in the diagnosis of the COVID-19. Lung involvement appears in different forms on CT images. Absorbed dose to the patients may exceed diagnostic reference levels. This study aimed to investigate clinical symptoms, findings of lung CT scan, and absorbed dose to the patients. Methods: This cross-sectional descriptive study was conducted in two CT scan centers in Hamadan: Besat and Sina. CT images of 163 patients with positive real-time polymerase chain reaction (PCR) tests were interpreted by six experienced radiologists. Volume CT dose index (CTDIvol), dose length product (DLP), and effective dose were calculated in both centers and compared using the Mann-Whitney statistical test. Results: The most common findings in the CT images were ground-glass opacification (GGO), consolidation, and the combination of GGO and consolidation. CTDIvol, DLP, and effective dose to the patients were respectively 1.64±5.24 mGy, 177.12±59.03 mGy.cm, and 3.01±1.00 mSv in Sina hospital and 4.58±1.91 mGy, and 2.60±1.02 mSv in Besat hospital. The difference between quantities in the two centers was statistically significant (P&gt;0.05). Conclusion: The most common findings in lung CT images of patients with positive polymerase chain reaction (PCR) tests were GGO and consolidation. Furthermore, there were differences in radiation dose to patients between hospitals, indicating the need for optimization.

Comparison of Cytomegalovirus Polymerase Chain Reaction Test Results Obtained with Fully Automated Systems: Roche Cobas 6800 and Qiagen NeuMoDx Experience

Viral load monitoring is important in identifying patients at risk of developing cytomegalovirus (CMV) related complications after transplantation and for this purpose, quantitative real-time polymerase chain reaction (Rt-qPCR) tests are most commonly used. The main problem in CMV DNA Rt-qPCR tests that make quantitative measurements is that there are significant differences in measurements performed with different kits in different laboratories. Comparability of viral load measurements between laboratories has increased with the introduction of quantitative PCR tests calibrated with the CMV International Quantitation Standard (IQS) developed by the World Health Organization (WHO). However, quantitative agreement between measurements made with different kits has still not been fully achieved. In this study, it was aimed to investigate the quantitative compatibility between measurements made with Cobas 6800 (Roche Diagnostics, Mannheim, Germany) and NeuMoDx (Qiagen, Ann Arbor, USA) CMV DNA Rt-qPCR tests, which are fully automated new generation systems calibrated with the WHO CMV IQS. The results of 214 plasma samples, which were studied simultaneously with Cobas 6800 CMV Rt-qPCR and NeuMoDx CMV Rt-qPCR tests were analyzed. In the tests, the extraction, amplification and detection stages were carried out fully automatically. CMV DNA was detected in 144 (67.28%) samples in both tests and was not detected in 53 (24.76%) samples. Incompatible results were obtained in a total of 17 (7.94%) samples. Good agreement was found between the qualitative results of both tests (kappa= 0.80, p< 0.001). When the quantitative results (n= 129) obtained in the dynamic measurement range of both tests were examined, the median viral load values measured by Cobas 6800 CMV Rt-qPCR and NeuMoDx CMV Rt-qPCR tests were 513 IU/mL (range= 35-37000) and 741 IU/mL (range= 68-48978), respectively. According to the correlation analysis, a very strong correlation was found between the results of both tests (r= 0.94, p< 0.001). According to Bland-Altman analysis; the average difference between the results of the NeuMoDx CMV Rt-qPCR test and the Cobas 6800 CMV Rt-qPCR test was found to be -0.14 log10 [standard deviation (SD)= 0.23] IU/mL and it was determined that the Cobas 6800 CMV Rt-qPCR test had lower measurements than the NeuMoDx CMV Rt-qPCR test. In 120 of 129 samples (93%) whose results were within the dynamic measurement range of both tests, the measurement difference was within ± 0.5 log10 IU/mL and in 9 (7%), it was detected as more than ± 0.5 log10 (median 0.54 log10 IU/ml; range= 0.51-0.81). No measurement difference of more than ± 1.0 log10 was detected in any sample. In this study, quantitative agreement was found in the measurements made in plasma samples with the fully automated Cobas 6800 CMV Rt-qPCR and NeuMoDx CMV Rt-qPCR tests calibrated with the CMV IQS. To the best of our knowledge, a study comparing viral load measurements made with Cobas 6800 and NeuMoDx fully automated systems in the detection of CMV DNA has not yet been conducted, and this is the first study on this subject.

Clinical Characteristics and Cycle Thresholds Among Discordant and True Positive Test Results for Clostridiodes difficile

Background: The diagnosis of Clostridioides difficile infection (CDI) is challenging. Despite guideline-directed, multistep testing algorithms and diagnostic stewardship, the treatment of C. difficile colonization persists. The testing algorithm at our system utilizes an initial real-time PCR test (PCR) for Toxin B gene, which if positive, reflexes to an enzyme immunoassay (EIA) for detecting Toxins A and B. Discordant results (PCR +/EIA -) are suggestive of colonization, but the majority of patients with discordant results are treated for CDI. Correlation of C. difficile EIA B polymerase chain reaction (PCR) cycle thresholds (Ct) with the presence of free EIA and disease severity has been observed, but the ability to use Ct in the decision to treat patients with discordant results is unclear. Our study assesses if Ct values and other clinical characteristics favor treatment in select patients with discordant Methods: A retrospective chart review was performed of adult patients (≥ 18-year-old) with positive C. difficile PCR results that were admitted to our health system between June 01 and August 31, 2023. C. difficile PCR and Ct results were obtained by Cepheid GeneXpert and Toxin A and B EIA results were obtained by Meridian Bioscience Immunocard. Patients with discordant (PCR+/EIA) and true positive (PCR+/EIA+) results were compared. We assessed demographics, past medical history, clinical characteristics, severity of illness, PCR Ct values, treatment, and clinical outcomes including: 30-day all-cause mortality and re-admission, and 60-day CDI repeat testing and treatment. Results Of the 122 patients identified, 89 patients had discordant results and 43 had true positive Results: Severity of illness and other clinical and laboratory characteristics were similar between both groups. Mean Ct values were significantly lower for true positive results compared to discordant results, 24.28 vs 29.27, respectively (p26 (p=.08). Of the patients with discordant results, 73 completed treatment for CDI and no difference in clinical outcomes was observed compared to patients with discordant results that were not treated. Conclusion Ct values were lower among patients with true positive results compared to patients with discordant Conclusion: There were no statistically significant different rates of severe or fulminant CDI among patients with discordant results and Ct values &lt; 26, although this finding may be limited by sample size and Ct may be helpful in deciding which discordant patients to treat.