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UNLABELLED: Given the challenges that SARS-CoV-2 variants have caused in terms of rapid spread and reduced vaccine efficacy, a rapid and cost-effective assay that can detect new and emerging variants is greatly needed worldwide. We have successfully applied the xenonucleic acid-based molecular-clamping technology to develop a multiplex reverse-transcription quantitative real-time PCR assay for SARS-CoV-2 multivariant detection. The assay was used to test 649 nasopharyngeal swab samples that were collected for clinical diagnosis or surveillance. The assay was able to correctly identify all 36 Delta variant samples as it accurately detected the D614G, T478K, and L452R mutations. In addition, the assay was able to correctly identify all 34 Omicron samples by detecting the K417N, T478K, N501Y, and D614G mutations. This technique reliably detects a variety of variants and has an analytical sensitivity of 100 copies/mL. In conclusion, this novel assay can serve as a rapid and cost-effective tool to facilitate large-scale detection of SARS-CoV-2 variants. IMPORTANCE: We have developed a multiplex reverse-transcription quantitative real-time PCR (RT-qPCR) testing platform for the rapid detection of SARS-CoV-2 variants using the xenonucleic acid (XNA)-based molecular-clamping technology. The XNA-based RT-qPCR assay can achieve high sensitivity with a limit of detection of about 100 copies/mL for variant detection which is much better than the next-generation sequencing (NGS) assay. Its turnaround time is about 4 hours with lower cost and a lot of Clinical Laboratory Improvement Amendments (CLIA) labs own the instrument and meet skillset requirements. This assay provides a rapid, reliable, and cost-effective testing platform for rapid detection and monitoring of known and emerging SARS-CoV-2 variants. This testing platform can be adopted by laboratories that perform routine SARS-CoV-2 PCR testing, providing a rapid and cost-effective method in lieu of NGS-based assays, for detecting, differentiating, and monitoring SARS-CoV-2 variants. This assay is easily scalable to any new variant(s) should it emerge.

The grapevine fleck virus (GFkV) is a ubiquitous grapevine-infecting virus found worldwide, is associated with the grapevine fleck complex, and is often found in mixed infections with viruses of the grapevine leafroll complex and/or vitiviruses. Although GFkV has been studied for a long time, limited sequence information is available in the public databases. In this study, the GFkV sequence data available in GenBank and data generated at the Foundation Plant Services, University of California, Davis, were used to perform nucleotide sequence comparisons, construct a phylogenetic tree, and develop a new RT-qPCR assay. Sequence comparisons showed high genetic diversity among the GFkV isolates, and the phylogenetic analyses revealed a new group comprised of GFkV isolates identified in the present study. A new assay, referred to as GFkV-CP, was designed and validated using an existing GFkV positive control together with 11 samples known to be infected with combinations of different marafiviruses and maculaviruses but not GFkV. In addition, the newly designed assay was used in a field survey to screen grapevines from diverse geographical locations that are maintained at the United States Department of Agriculture (USDA) National Clonal Germplasm Repository (NCGR) in Winters, CA.

BackgroundSputum-based testing is a barrier to increasing access to molecular diagnostics for tuberculosis (TB). Many people with TB are unable to produce sputum, and sputum processing increases assay complexity and cost. Tongue swabs are emerging as an alternative to sputum, but performance limits are uncertain.MethodsFrom June 2022 to July 2023, we enrolled 397 consecutive adults with cough >2 weeks at 2 health centers in Kampala, Uganda. We collected demographic and clinical information, sputum for TB testing (Xpert MTB/RIF Ultra and 2 liquid cultures), and tongue swabs for same-day quantitative polymerase chain reaction (qPCR) testing. We evaluated tongue swab qPCR diagnostic accuracy versus sputum TB test results, quantified TB targets per swab, assessed the impact of serial swabbing, and compared 2 swab types (Copan FLOQSWAB and Steripack spun polyester).ResultsAmong 397 participants, 43.1% were female, median age was 33 years, 23.5% were diagnosed with human immunodeficiency virus, and 32.0% had confirmed TB. Sputum Xpert Ultra and tongue swab qPCR results were concordant for 98.2% (95% confidence interval [CI]: 96.2-99.1) of participants. Tongue swab qPCR sensitivity was 92.6% (95% CI: 86.5 to 96.0) and specificity was 99.1% (95% CI: 96.9 to 99.8) versus microbiological reference standard. A single tongue swab recovered a 7-log range of TB copies, with a decreasing recovery trend among 4 serial swabs. Swab types performed equivalently.ConclusionsTongue swabs are a promising alternative to sputum for molecular diagnosis of TB, with sensitivity approaching sputum-based molecular tests. Our results provide valuable insights for developing successful tongue swab-based TB diagnostics.

The obligate intracellular bacterium Chlamydia has a unique developmental cycle that alternates between two contrasting cell types. With a hardy envelope and highly condensed genome, the small elementary body (EB) maintains limited metabolic activities yet survives in extracellular environments and is infectious. After entering host cells, EBs differentiate into larger and proliferating reticulate bodies (RBs). Progeny EBs are derived from RBs in late developmental stages and eventually exit host cells. How expression of the chlamydial genome consisting of nearly 1,000 genes governs the chlamydial developmental cycle is unclear. A previous microarray study identified only 29 Chlamydia trachomatis immediate early genes, defined as genes with increased expression during the first hour postinoculation in cultured cells. In this study, we performed more sensitive RNA sequencing (RNA-Seq) analysis for C. trachomatis cultures with high multiplicities of infection. Remarkably, we observed well over 700 C. trachomatis genes that underwent 2- to 900-fold activation within 1 hour postinoculation. Quantitative reverse transcription real-time PCR analysis was further used to validate the activated expression of a large subset of the genes identified by RNA-Seq. Importantly, our results demonstrate that the immediate early transcriptome is over 20 times more extensive than previously realized. Gene ontology analysis indicates that the activated expression spans all functional categories. We conclude that over 70% of C. trachomatis genes are activated in EBs almost immediately upon entry into host cells, thus implicating their importance in initiating rapid differentiation into RBs and establishing an intracellular niche conducive with chlamydial development and growth.

Propolis, a resinous substance collected by honeybees from various botanical sources, is known for its diverse biological activities. This study aimed to investigate the health-promoting properties of Turkish propolis by assessing its antioxidant capacity and antimicrobial activity against various pathogens. Additionally, its antiviral effects were evaluated against human herpes simplex virus type 1 (HSV-1) using MTT and quantitative real-time polymerase chain reaction (qRT-PCR) assays. The antiproliferative potential of propolis was also tested against cancer cell lines (MDA-MB-231, AR42J, A549) and a normal epithelial cell line (Vero) through the MTT method. The ethanolic extract exhibited notable bioactive properties, as indicated by its high total phenolic content (TPC) of 235.86 ± 1.35 mg GAE/g, total flavonoid content (TFC) of 43.93 ± 1.14 mg QE/g, ferric reducing antioxidant power (FRAP) of 332.04 ± 16.78 µmol FeSO4.7H2O/g, and a 2,2-diphenyl-1-picrylhydrazyl (DPPH)• radical scavenging activity (SC50) of 0.01 ± 0.00 mg/mL. High-performance liquid chromatography (HPLC–PDA) revealed that pinocembrin, chrysin, and caffeic acid phenethyl ester (CAPE) were the predominant compounds. The propolis extract showed antimicrobial activity against 7 of 14 tested microorganisms using the agar well diffusion method. While the extract inhibited quorum sensing activity, it did not exhibit anti-swarming or anti-biofilm effects. Cytotoxicity was observed in Vero and A549 cells at a concentration of 200 µg/mL, and in MDA-MB-231 and AR42J cells at 50 µg/mL. Moreover, the propolis extract displayed antiviral activity against HSV-1 at a concentration of 100 µg/mL. These findings underscore the broad-spectrum biological activity of Turkish propolis, reinforcing its potential as a nutraceutical agent with antioxidant, antimicrobial, antiviral, and anticancer properties. [ABSTRACT FROM AUTHOR]

ABSTRACT: Objectives: Guo Min decoction (GMD) is a Chinese traditional medicine that can regulate allergy-related symptoms. Although GMD treatment was reported to treat allergy-associated symptoms by regulating the immune response, the rationale between GMD treatment and angiogenesis has not been reported yet. Our objective is to investigate the angiogenesis-modulating activity of GMD. Materials and Methods: In this study, we used fluorescence recording, alkaline phosphatase (AP) activity staining, and real-time polymerase chain reaction (PCR) experiments to examine the effects of GMD on angiogenesis in a zebrafish model. Results: GMD-treated zebrafish embryos exhibited more intercapillary spaces in the caudal vein plexus (Mock: 11.1 ± 1.8 [ n = 20; n : numbers of embryos]; GMD-treated: 16.2 ± 1.9 [ n = 20]). AP activity staining showed that treatment with GMD and liquorice (Gan Cao, a component of GMD) induced subintestinal vein outgrowth. However, glycyrrhizin (a component of Gan Cao) had no obvious pro-angiogenic effects on zebrafish. Furthermore, real-time PCR experiments indicated that GMD exposure might be through regulating angiogenesis-related genes (cdh5 , nrp1a , and flt1) expressions. Conclusion: Based on these observations, we proposed that GMD had pro-angiogenic activity in a zebrafish model, and it might partially be contributed by one of the components, liquorice. [ABSTRACT FROM AUTHOR]

Although diagnostic and therapeutic advances have been made in the treatment of breast cancer, the challenge of effectively controlling tumor progression persists. The objective of this study was to determine the relationship between IDO1 and TDO2 expression in breast cancer by immunohistochemistry and real-time polymerase chain reaction, hormone receptor status, Ki67 proliferation index, molecular classification, metastasis, and to investigate whether IDO1 and TDO2 expression can be used in combination with targeted therapy or as a marker to increase treatment efficacy in selected cases. The study included 74 cases of breast cancer and 14 cases of normal breast tissue as controls. All cases were analyzed for IDO1 and TDO2 by both immunohistochemistry and real-time polymerase chain reaction. The immunohistochemical analysis revealed that IDO1 immunoreactivity was significantly higher in tumor tissue compared to normal breast tissue. A statistically significant correlation was observed between IDO1 immunoreactivity and histologic subtypes. Furthermore, IDO1 gene expression was correlated with IDO1 immunoreactivity. TDO2 immune reactivity did not differ between tumor and non-tumor tissues and no correlation was found between histological subtypes. There was no correlation between TDO2 immunoreactivity and gene expression. The significant increase in IDO1 levels in tumor tissue and high positivity in age, HER2 positive and triple negative cases compared to other cases suggest that IDO1 inhibitors may be suitable for the target patient group in treatment selection. [ABSTRACT FROM AUTHOR]

Objectives: Guo Min decoction (GMD) is a Chinese traditional medicine that can regulate allergy-related symptoms. Although GMD treatment was reported to treat allergy-associated symptoms by regulating the immune response, the rationale between GMD treatment and angiogenesis has not been reported yet. Our objective is to investigate the angiogenesis-modulating activity of GMD. Materials and Methods: In this study, we used fluorescence recording, alkaline phosphatase (AP) activity staining, and real-time polymerase chain reaction (PCR) experiments to examine the effects of GMD on angiogenesis in a zebrafish model. Results: GMD-treated zebrafish embryos exhibited more intercapillary spaces in the caudal vein plexus (Mock: 11.1 ± 1.8 [n = 20; n: numbers of embryos]; GMD-treated: 16.2 ± 1.9 [n = 20]). AP activity staining showed that treatment with GMD and liquorice (Gan Cao, a component of GMD) induced subintestinal vein outgrowth. However, glycyrrhizin (a component of Gan Cao) had no obvious pro-angiogenic effects on zebrafish. Furthermore, real-time PCR experiments indicated that GMD exposure might be through regulating angiogenesis-related genes (cdh5, nrp1a, and flt1) expressions. Conclusion: Based on these observations, we proposed that GMD had pro-angiogenic activity in a zebrafish model, and it might partially be contributed by one of the components, liquorice.

Aims: The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Methods: Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. Results: LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. Conclusion: LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism. Cite this article: Bone Joint Res 2024;13(12):779–789.

BACKGROUND: C-FLICE-like inhibitory protein (c-FLIP) is a protein that does not merely block apoptosis signaling but also adjusts further pathways of cell death. Acute myeloid leukemia (AML), a nonhomogeneous hematologic malignancy, is the highly common form of AML among adults and is described through the clonal enlargement of myeloid blasts in the bone marrow (BM), peripheral blood, and/or else tissues. OBJECTIVES: The aims of this study were to investigate the role of c-FLIP and microRNA (miRNA) 708-5p as a prospective prognostic biomarker as well as the therapeutic goal in AML. PATIENTS MATERIALS AND METHODS: This study includes two groups of patients (40) individuals newly diagnosed AML patients, (20) AML taking chemotherapy, and (50) apparently healthy volunteers. The study was conducted at the National Center of Hematology/Mustansiriyah University. The methods employed in the analysis include total RNA extraction, complementary cDNA synthesis, and quantitative real-time polymerase chain reaction (PCR) were used to evaluate the gene expression of cFLIP and miRNA-708-5p. Complete blood count to estimate some hematological parameters. RESULTS: The expression of c-FLIP was notably higher in both newly diagnosed and patients under chemotherapy compared to controls with fold expression (3.291 and 2.92), respectively, with a highly significant (P = 0.0001). The increase in miRNA 708-5p expression in newly diagnosed patients with fold expression (5.345), whereas downregulation in patients under chemotherapy with fold expression (0.789) indicates that treatment may restore its levels, contributing to the suppression of c-FLIP and promoting apoptosis. CONCLUSION: The c-FLIP and miRNA708-5p gene might be used as a biological marker for the AML initial diagnosis. The researches emphasize the role of miRNA 708-5p as a tumor suppressor, which negatively regulates the antiapoptotic protein c-FLIP, indicating its potential as a therapeutic target in AML treatment. By modulating the levels of miRNA708-5p, it may be possible to regulate the expression of c-FLIP, thus enhancing the effectiveness of apoptosis-inducing therapies. This suggests the promising development of miRNA-based therapies as part of AML treatment strategies. Furthermore, miRNA 708-5p can act as a prognostic indicator in AML, with its expression levels offering valuable insights into disease progression and patient response to treatment. Further research into miRNA 708-5p can lead to a better understanding of its role in AML pathogenesis and its potential applications in clinical practice.

Detection sensitivity of aquatic species using environmental DNA (eDNA) generally decreases in turbid water but is poorly characterized. In this study, eDNA detection targeted delta smelt (Hypomesus transpacificus), a critically endangered estuarine fish associated with turbid water. eDNA sampling in the field was first paired with a trawl survey. Species-specific detection using a Taqman qPCR assay showed concordance between the methods, but a weak eDNA signal. Informed by the results of field sampling, an experiment was designed to assess how turbidity and filtration methods influence detection of a rare target. Water from non-turbid (5 NTU) and turbid (50 NTU) estuarine sites was spiked with small volumes (0.5 and 1 mL) of water from a delta smelt tank to generate low eDNA concentrations. Samples were filtered using four filter types: cartridge filters (pore size 0.45 μm) and 47 mm filters (glass fiber, pore size 1.6 μm and polycarbonate, pore sizes 5 and 10 μm). Prefiltration was also tested as an addition to the filtration protocol for turbid water samples. eDNA copy numbers were analyzed using a censored data method for qPCR data. The assay limits and lack of PCR inhibition indicated an optimized assay. Glass fiber filters yielded the highest detection rates and eDNA copies in non-turbid and turbid water. Prefiltration improved detection in turbid water only when used with cartridge and polycarbonate filters. Statistical analysis identified turbidity as a significant effect on detection probability and eDNA copies detected; filter type and an interaction between filter type and prefilter were significant effects on eDNA copies detected, suggesting that particulate-filter interactions can affect detection sensitivity. Pilot experiments and transparent criteria for positive detection could improve eDNA surveys of rare species in turbid environments.

Newcastle disease (ND) is among the most important poultry diseases worldwide. It is the major threat to poultry production in Africa and causes major economic losses for both local and commercial chickens. To date, half of ND class II genotypes have been reported in Africa (I, IV, V, VI, VII, XI, XIII, XIV, XVII, XVIII, and XXI). The information on the circulating NDV genotypes is still scarce despite the endemic nature of ND in most countries on the African continent.A total of 659 oro-cloacal swabs were collected from local chickens in Mawenzi live bird market located in Morogoro, Tanzania, between June 2020 and May 2021. Newcastle disease virus was detected by using reverse transcription real-time polymerase chain reaction (RT-qPCR) and conventional PCR followed by sequencing of PCR products. The prevalence of NDV in the surveilled live bird markets was 23.5%. Sequencing and phylogenetic analysis revealed the presence of sub-genotype VII.2. The detected sub-genotype VII.2 has phylogenetic links to Zambian NDV strains implying a Southeast dissemination of the virus, considering that it was first detected in Mozambique. This study underscores the need of active NDV surveillance to determine the distribution of this NDV genotype in the country and monitor its spread and contribution to the emergence of new ND viruses.

Background/Aims: Bismuth-based quadruple therapy (BQT) is a treatment option for clarithromycin-resistant Helicobacter pylori (HP) infection. The aim of this study was to compare the efficacy of 7-day BQT with that of 14-day BQT as first-line treatment for clarithromycin-resistant HP infection. Methods: A total of 162 treatment-naïve patients with peptic ulcer disease and clarithromycin-resistant HP infection confirmed by real-time polymerase chain reaction (RT-PCR) were enrolled. The enrolled patients were prospectively randomized to receive BQT for either 7 or 14 days of treatment. Eradication of HP infection was assessed using a 13C-urea breath test. Eradication and adverse event rates of the two groups were assessed. Results: The overall eradication rates in the intention-to-treat (ITT) and per-protocol (PP) analyses were 83.0% (95% confidence interval [CI], 77.2% to 88.9%; 132/159) and 89.8% (95% CI, 84.9% to 94.7%; 132/147), respectively. The eradication rates in the ITT analysis were 79.0% (64/81) in the 7-day group and 87.2% (68/78) in the 14-day group (p=0.170). The eradication rates in the PP analysis were 86.5% (64/74) in the 7-day group and 93.2% (68/73) in the 14-day group (p=0.182). Clinically significant adverse events occurred in 18.2% of patients. There was no statistically significant difference in the rates of individual or all adverse events between the two groups. Conclusions: Both 7-day and 14-day BQT were effective and safe as first-line therapy for HP infections identified as resistant to clarithromycin by RT-PCR. For clarithromycin-resistant HP infections, 7-day BQT may be sufficient as first-line therapy.

Summary Background Treatment failure (TF) in leprosy following multidrug therapy (MDT) presents a significant challenge. The current World Health Organization (WHO) fixed-duration MDT regimen, based on lesion count, might not be adequate. Leprosy lacks clear-cut objective cure criteria, and the predictive value of post-MDT histopathological findings remains uncertain. This study aims to identify predictive factors for TF among leprosy patients who have completed the WHO-recommended MDT. Methods An analysis was conducted on 80 individuals from a national leprosy reference center, comprising 40 TF cases (with a mean relapse at 13.0 months) and 40 controls (with a mean of 113.1 months without disease signs). Various epidemiological and clinical-laboratory parameters were assessed post-MDT. Results In skin samples, the presence of foamy granuloma (OR = 7.36; 95%CI2.20-24.60; p = 0.0012) and histological bacillary index (hBI) ≥ 1+ (OR = 1.55; 95%CI1. 22-1.99; p = 0.0004) were significantly associated with TF, with odds ratios of 7.36 and 1.55, respectively. Individuals who experienced TF had a mean hBI of 3.02+ (SD ± 2.02), while the control group exhibited a mean hBI of 1.8+ (SD ± 1.88). An hBI ≥ 3 + showed a sensitivity of 73% and a specificity of 78% for TF detection (AUC: 0.75; p = 0.0001). Other histopathological features like epithelioid granulomas, and skin changes did not show significant associations (p > 0.05). Additionally, higher anti-phenolic glycolipid-I (anti-PGL-I) ELISA index (EI) levels were linked to a 1.4-fold increased likelihood for TF (OR = 1.4; 95%CI1.13-1.74; p = 0.0019). A mean EI of 4.48 (SD ± 2.80) was observed, with an EI ≥ 3.95 showing a sensitivity of 79% and a specificity of 59% for TF detection (AUC: 0.74; p = 0.0001). Moreover, the presence of Mycobacterium leprae (M. leprae) DNA in real-time polymerase chain reaction (qPCR) was associated with a 3.43-fold higher likelihood of TF. Multivariate regression analysis indicated that concurrent presentation of neural/perineural lymphocytic infiltrate, foamy granuloma, hBI ≥ 1+, and EI ≥ 1 markedly increased the likelihood of TF by up to 95.41%. Conclusion Persistence of nerve-selective lymphocytic infiltrate, foamy granulomas, and bacilli in skin biopsies, and elevated EI post-MDT, may serve as predictive factors for identifying individuals at higher probability of TF.

Introduction: Clinically significant High-risk Human Papillomavirus (HR-HPV) infection in Oral Cavity Squamous Cell Carcinoma (OCSCC) is not clearly defined. There is uncertainty regarding the true impact of HPV-related variability (ranging from 0% to 100%) when using the GP5+/GP6+/MY09/11 (L1) primer. The identification of HPV DNA in tumours is insufficient to establish a causal viral association; therefore, a gold standard test is required to determine HPV’s role in malignant cells by identifying the transcripts of the E6/E7 mRNA viral oncogene and the p16ink4a biomarker. Aim: To assess the diagnostic performance of p16ink4a in the presence of E6/E7 mRNA in Formalin-fixed, Paraffin-embedded (FFPE) specimens. Materials and Methods: This cross-sectional study was conducted in the Department of Pathology at JIPMER in Puducherry, India, from February 2019 to April 2022. Combined molecular markers p16ink4a and E6/E7 mRNA (considered the gold standard) from FFPE specimens of newly diagnosed Squamous Cell Carcinoma (SCC) were analysed to identify biologically and clinically relevant Infections using Immunohistochemistry (IHC) and real-time Polymerase Chain Reaction (PCR). Results: This study indicates that tissue samples with p16 positive expression (score of 2 or higher) showed the presence of HPV E6/E7 mRNA, with a sensitivity of 100% and a specificity of 91.67%. The relationship between p16ink4a and E6/E7 mRNA expression in all OCSCC cases was found to be significant (p-value=0.003). Conclusion: The study found a significant relationship between mRNA and p16ink4a overexpression in tongue SCC. This finding suggests that alterations in mRNA expression levels are associated with changes in p16ink4a expression in tongue cancer. Such understanding could potentially aid in the development of diagnostic or therapeutic strategies targeting these molecular pathways in tongue SCC.

Ongoing outbreaks and often rapid spread of infections caused by coronaviruses, influenza, Nipah, Dengue, Marburg, monkeypox, and other viruses are a concern for health authorities in most countries. Therefore, the search for and study of new antiviral compounds are in great demand today. Since almost all viruses with pandemic potential have immunotoxic properties of various origins, particular attention is paid to the search and development of immunomodulatory drugs. We have synthesised a new compound related to indole-3-carboxylic acid derivatives (hereinafter referred to as the XXV) that has antiviral and interferon-inducing activity. The purpose of this work is to study the effect of the XXV on the stimulation of the expression of toll-like receptor genes, interferons, and immunoregulatory cytokines in a macrophage-like cell model. In this study, real-time PCR methods were used to obtain data on the transcriptional activity of genes in macrophage-like cells. Stimulation of the genes of toll-like receptors TLR2, TLR3, TLR4, TLR7, TLR8, and TLR9 was detected. A high-fold increase in stimulation (from 6.5 to 16,000) of the expression of the TLR3 and TLR4 genes was detected after 4 h of exposure to the XXV. Increased activity of interferon (IFNA1, IFNA2, IFNB1, IFNK, and IFNλ1) genes with simultaneous stimulation of the expression of interferon receptor (IFNAR1 and IFNAR2) genes and signalling molecule (JAK1 and ISG15) genes was detected. Increased fold stimulation of the expression of the cytokine genes IL6, TNFA, IL12A, and IL12B was also observed. Thus, it is shown that the XXV is an activator of TLR genes of innate immunity, which trigger signalling mechanisms of pathogen "recognition" and lead to stimulation of the expression of genes of proinflammatory cytokines and interferons. [ABSTRACT FROM AUTHOR]

Aim: Cyclooxygenase-2 (COX-2) is a crucial enzyme in the prostaglandin cascade. Overexpression of the COX-2 enzyme has been associated with the pathogenesis and progression of certain cancers, including oral cancer. This study aimed to investigate the quantitative expression and immunohistochemistry (IHC) expression of COX-2 in tissue samples from patients with oral squamous cell carcinoma (OSCC) and oral lichen planus (OLP) to gain insight into its potential role in the pathogenesis of these oral diseases. Methods: A total of 76 samples were analyzed, consisting of 25 cases of OSCC, 27 cases of OLP, and 24 cases of normal oral mucosa. COX-2 expression was analyzed using IHC staining and quantitative polymerase chain reaction (qPCR). Statistical analysis was performed using the Chi-squared, Spearman's, and Fisher's exact tests, with significance declared at a P value of <.05. Results: The mRNA expression of COX-2 in OSCC tissues (3.17 ± 1.02) was found to be significantly higher than in OLP (1.65 ± 0.72) and normal (1.08 ± 0.79; P <.01). In addition, protein expression of COX-2 was significantly higher in cancer tissues than in normal and OLP (P <.05). The oral cavity was the most common tumor site in the studied cases, representing 56% of OSCC cases, as determined by qPCR analysis (P <.05). Conclusion: Understanding the involvement of COX-2 in these oral diseases has implications for the development of targeted therapies and personalized treatment approaches. Also, the identification of COX-2 as a potential biomarker may contribute to improved diagnostic accuracy and prognosis in patients with OSCC and OLP. Moreover, the qPCR method could be as effective a technique as IHC to evaluate COX-2 amplification. [ABSTRACT FROM AUTHOR]

The aim of the study was to assess glucocorticoid sensitivity in survivors of childhood acute lymphoblastic leukemia using in vivo and in vitro tests. Thirty leukemia survivors of both sexes aged ≥18 years participated in the study and at least two years after therapy withdrawal. In vivo tests comprised: a) a very low dose intravenous dexamethasone suppression test for measurement of serum cortisol before, after, and % suppression, compared with 32 age-matched controls; and b) 0.25 mg overnight oral dexamethasone suppression test for assessment of salivary cortisol before, after, and % suppression. In vitro methods comprised: c) glucocorticoid receptor polymorphisms: BcI1-NR3C1 and A3669G; and d) splicing variant of glucocorticoid receptor GR-α mRNA by real-time quantitative polymerase chain reaction, compared with 32 controls. There was a reduction in salivary cortisol, and 73.3% of leukemia survivors showed high sensitivity according to % suppression after oral dexamethasone (p<0.05). Serum cortisol at baseline, after the test, % suppression after intravenous dexamethasone, and the percentage of high sensitivity were reduced in the leukemia group (%F=36.7; p<0.05). The BcI1-NR3C1 and A3669G polymorphisms were present in 11/30 (36.7%) and 5/30 (16.7%) patients, respectively. GR-α mRNA levels were lower in the leukemia group than in the controls (p<0.05). Survivors of acute lymphoblastic leukemia presented with reduced glucocorticoid sensitivity. Glucocorticoid sensitivity allows individualized treatment to avoid adverse effects and may be involved in cardiovascular disease risk among this particular group of cancer survivors. [ABSTRACT FROM AUTHOR]

Background and Objectives: Graft-versus-host disease (GvHD) frequently complicates hematopoietic stem cell transplantation (HSCT). Emerging evidence suggests a correlation between gut microbiota and GvHD risk. This study aims to elucidate the microbiota profiles in HSCT patients before and after transplantation and their association with GvHD. Materials and Methods: This study, conducted from December 2022 to December 2023, involved the collection of 15 stool samples from HSCT patients. Bacterial content was quantified using real-time PCR, while interleukin-6 levels were assessed via ELISA. Results: Among the 15 participants (8 male, 7 female), 9 underwent allogeneic HSCT (allo-HSCT) and 6 received autologous HSCT. In the aGvHD group, there was a significant reduction in the abundance of Bacteroides and Bifidobacterium compared to those without aGvHD. Additionally, declines were observed in Clostridium and Firmicutes populations. The genus Blautia also showed reduced prevalence in the aGvHD group, whereas no significant differences were noted in the uncomplicated group. ELISA analysis revealed that interleukin-6 levels remained within the normal range (30-960 pg/ml) with no significant elevation in the aGvHD group. Conclusion: The study highlights a notable association between alterations in gut microbiota, specifically reductions in certain bacterial populations and the development of aGvHD following allo-HSCT. [ABSTRACT FROM AUTHOR]

Aims: The study aimed to assess the antimicrobial effectiveness of green tea and neem extract compared to sodium hypochlorite (NaOCl) against various root canal microorganisms. Materials and Methods: Thirty patients with pulpal necrosis were selected, providing 60 samples before and after irrigation. Groups were assigned as follows: Group A: 3% NaOCl (control), Group B: green tea, and Group C: neem extract. Samples were collected before and after irrigation in two phases. Samples were collected maintaining a strict sterile condition and stored in buffer solution at −80°C for bacterial-load measurement through real-time Polymerase chain reaction (PCR). Statistical analysis included within-group comparisons using Wilcoxon's test and the paired t-test and inter-group comparisons using the Kruskal–Wallis test with post hoc Dunn's test and one-way analysis of variance with post hoc Tukey's honestly significant difference test (P ≤ 0.05). Results: While no irrigant achieved complete bacterial eradication, all solutions exhibited significant antimicrobial activity postirrigation. NaOCl yielded the most effective results, with green tea nearly comparable, and neem extract demonstrating the lowest efficacy. Conclusions: Herbal irrigants, particularly green tea, can serve as effective alternatives to chemical solutions. However, neem extract proved less effective than both green tea and NaOCl, indicating its inferiority in root canal disinfection. [ABSTRACT FROM AUTHOR]

Summary: Background: Treatment failure (TF) in leprosy following multidrug therapy (MDT) presents a significant challenge. The current World Health Organization (WHO) fixed-duration MDT regimen, based on lesion count, might not be adequate. Leprosy lacks clear-cut objective cure criteria, and the predictive value of post-MDT histopathological findings remains uncertain. This study aims to identify predictive factors for TF among leprosy patients who have completed the WHO-recommended MDT. Methods: An analysis was conducted on 80 individuals from a national leprosy reference center, comprising 40 TF cases (with a mean relapse at 13.0 months) and 40 controls (with a mean of 113.1 months without disease signs). Various epidemiological and clinical-laboratory parameters were assessed post-MDT. Results: In skin samples, the presence of foamy granuloma (OR = 7.36; 95%CI2.20-24.60; p = 0.0012) and histological bacillary index (hBI) ≥ 1+ (OR = 1.55; 95%CI1. 22-1.99; p = 0.0004) were significantly associated with TF, with odds ratios of 7.36 and 1.55, respectively. Individuals who experienced TF had a mean hBI of 3.02+ (SD ± 2.02), while the control group exhibited a mean hBI of 1.8+ (SD ± 1.88). An hBI ≥ 3 + showed a sensitivity of 73% and a specificity of 78% for TF detection (AUC: 0.75; p = 0.0001). Other histopathological features like epithelioid granulomas, and skin changes did not show significant associations (p > 0.05). Additionally, higher anti-phenolic glycolipid-I (anti-PGL-I) ELISA index (EI) levels were linked to a 1.4-fold increased likelihood for TF (OR = 1.4; 95%CI1.13-1.74; p = 0.0019). A mean EI of 4.48 (SD ± 2.80) was observed, with an EI ≥ 3.95 showing a sensitivity of 79% and a specificity of 59% for TF detection (AUC: 0.74; p = 0.0001). Moreover, the presence of Mycobacterium leprae (M. leprae) DNA in real-time polymerase chain reaction (qPCR) was associated with a 3.43-fold higher likelihood of TF. Multivariate regression analysis indicated that concurrent presentation of neural/perineural lymphocytic infiltrate, foamy granuloma, hBI ≥ 1+, and EI ≥ 1 markedly increased the likelihood of TF by up to 95.41%. Conclusion: Persistence of nerve-selective lymphocytic infiltrate, foamy granulomas, and bacilli in skin biopsies, and elevated EI post-MDT, may serve as predictive factors for identifying individuals at higher probability of TF. [ABSTRACT FROM AUTHOR]

Introduction: Clinically significant High-risk Human Papillomavirus (HR-HPV) infection in Oral Cavity Squamous Cell Carcinoma (OCSCC) is not clearly defined. There is uncertainty regarding the true impact of HPV-related variability (ranging from 0% to 100%) when using the GP5+/GP6+/MY09/11 (L1) primer. The identification of HPV DNA in tumours is insufficient to establish a causal viral association; therefore, a gold standard test is required to determine HPV's role in malignant cells by identifying the transcripts of the E6/E7 mRNA viral oncogene and the p16ink4a biomarker. Aim: To assess the diagnostic performance of p16ink4a in the presence of E6/E7 mRNA in Formalin-fixed, Paraffin-embedded (FFPE) specimens. Materials and Methods: This cross-sectional study was conducted in the Department of Pathology at JIPMER in Puducherry, India, from February 2019 to April 2022. Combined molecular markers p16ink4a and E6/E7 mRNA (considered the gold standard) from FFPE specimens of newly diagnosed Squamous Cell Carcinoma (SCC) were analysed to identify biologically and clinically relevant Infections using Immunohistochemistry (IHC) and real-time Polymerase Chain Reaction (PCR). Results: This study indicates that tissue samples with p16 positive expression (score of 2 or higher) showed the presence of HPV E6/E7 mRNA, with a sensitivity of 100% and a specificity of 91.67%. The relationship between p16ink4a and E6/E7 mRNA expression in all OCSCC cases was found to be significant (p-value=0.003). Conclusion: The study found a significant relationship between mRNA and p16ink4a overexpression in tongue SCC. This finding suggests that alterations in mRNA expression levels are associated with changes in p16ink4a expression in tongue cancer. Such understanding could potentially aid in the development of diagnostic or therapeutic strategies targeting these molecular pathways in tongue SCC. [ABSTRACT FROM AUTHOR]

Curcumin has been employed in traditional medicine for over a millennium to treat various ailments, and its global use is now widespread. Chinese medicine relies heavily on curcumin as a primary element and uses it to cure infectious diseases, skin disorders, depression, and stress. It has cardioprotective, neuroprotective, and anti-diabetic properties, as well as pharmacological effects on disorders like type II diabetes, atherosclerosis, and human immunodeficiency virus replication. The anti-cancer activity of curcumin has been studied extensively with notable improvements in gastrointestinal, melanoma, urogenital, breast, and lung malignancies. We investigated the anti-inflammatory effects of curcumin on expression of tumor necrosis factor (TNF)-α, c-Fos, and interleukin (IL)-6 genes in brain and liver tissue owing to the effects of ketamine anesthesia on postnatal rats. The thalamic and hepatic tissues were collected without anesthesia, immediately after anesthesia, and 4 and 12 hr after anesthesia in control and curcumin treated postnatal rats. The results showed that glucose, triglyceride, high- and low-density lipoprotein levels were lowered with curcumin treatment. We also found that ketamine increased c-Fos and inflammatory cytokines like TNF-α and IL-6, all of which contribute to inflammation. Brain and liver immunohistochemistry studies confirmed the real-time polymerase chain reaction findings. Curcumin injections alone may be effective in decreasing ketamine-induced inflammation in both brain and liver tissues. [ABSTRACT FROM AUTHOR]

The accurate diagnosis and identification of Leishmania species are crucial for the therapeutic selection and effective treatment of leishmaniasis. This study aims to develop and evaluate the use of high-resolution melting curve analysis (HRM)-PCR for Leishmania species identification causing visceral leishmaniasis (VL), post-kala-azar dermal leishmaniasis (PKDL) and cutaneous leishmaniasis (CL) in the Indian subcontinent. Two multi-copy targets (ITS-1 and 7SL-RNA genes) were selected, and an HRM-PCR assay was established using L. donovani, L. major, and L. tropica standard strain DNA. The assay was applied on 93 clinical samples with confirmed Leishmania infection, including VL (n = 30), PKDL (n = 50), and CL (n = 13) cases. The ITS-1 HRM-PCR assay detected as little as 0.01 pg of template DNA for L. major and up to 0.1 pg for L. donovani and L. tropica. The detection limit for the 7SL-RNA HRM-PCR was 1 pg for L. major and 10 pg for L. donovani and L. tropica. The ITS-1 HRM-PCR identified 68 out of 93 (73.11%) leishmaniasis cases, whereas 7SL-RNA HRM-PCR could only detect 18 out of 93 (19.35%) cases. A significant correlation was observed between the kDNA-based low Ct values and ITS-1 HRM-PCR positivity in the VL (p = 0.007), PKDL (p = 0.0002), and CL (p = 0.03) samples. The ITS-1 HRM-PCR assay could identify Leishmania spp. causing different clinical forms of leishmaniasis in the Indian subcontinent, providing rapid and accurate results that can guide clinical management and treatment decisions. [ABSTRACT FROM AUTHOR]

Bacterial commensals of the human genitourinary tract, Mycoplasma hominis and Ureaplasma species (parvum and urealyticum) can be sexually transmitted, and may cause nongonococcal urethritis, pelvic inflammatory disease, and infertility. Mycoplasma hominis and Ureaplasma species may also cause severe invasive infections in immunocompromised patients. Current culture-based methods for Mycoplasma/Ureaplasma identification are costly and laborious, with a turnaround time between 1 and 2 weeks. We developed a high-throughput, real-time multiplex PCR assay for the rapid detection of M. hominis and Ureaplasma species in urine, genital swab, body fluid, and tissue. In total, 282 specimens were tested by PCR and compared with historic culture results; a molecular reference method was used to moderate discrepancies. Overall result agreement was 99% for M. hominis (97% positive percentage agreement and 100% negative percentage agreement) and 96% for Ureaplasma species (96% positive percentage agreement and 97% negative percentage agreement). Specimen stability was validated for up to 7 days at room temperature. This multiplex molecular assay was designed for implementation in a high-complexity clinical microbiology laboratory. With this method, >90 samples can be tested in one run, with a turnaround time of 4 to 5 hours from specimen extraction to reporting of results. This PCR test is also more labor effective and cheaper than the conventional culture-based test, thus improving laboratory efficiency and alleviating labor shortages.

The aim of this study was to use environmental sampling to determine the frequency of detection of selected equine respiratory viruses and bacteria in horses attending a multi-week equestrian show during the winter months. At four time points during showing, environmental sponge samples were collected from all stalls on the property and tested for the presence of equine herpesvirus-1 (EHV-1), EHV-2, EHV-4, equine influenza virus (EIV), equine rhinitis B virus (ERBV), Streptococcus equi ss. equi (S. equi), and S. equi ss. zooepidemicus (S. zooepidemicus) using real-time PCR (PCR). Environmental sponges were collected from all 53 barns by using one sponge for up to 10 stalls. Further, 2/53 barns were randomly selected for individual stall sampling in order to compare the results between individual and pooled stall samples. A total of 333/948 (35.13%, 95% CI 32.09-38.26%) pooled environmental stall sponges tested PCR-positive for at least one of the selected respiratory pathogens. Streptococcus zooepidemicus was the most commonly detected pathogen in pooled samples (28.69%, 95% CI 25.83-31.69%), followed by EHV-2 (14.45%, 95% CI 12.27-16.85%), EHV-4 (1.37%, 95% CI 0.73-2.33%), and a very small percentage of pooled stall sponges tested PCR-positive for EHV-1, ERBV, EIV, and S. equi. In individual samples, 171/464 (36.85%, 95% CI 32.45-41.42%) environmental stall sponges tested PCR-positive for at least one of the selected pathogens, following a similar frequency of pathogen detection as pooled samples. The detection frequency of true respiratory pathogens from environmental samples was higher during the winter months compared to previous studies performed during spring and summer, and this testing highlights that such pathogens circulate with greater frequency during the colder months of the year. The strategy of monitoring environmental stall samples for respiratory pathogens circumvents the often labor-intensive collection of respiratory secretions from healthy horses and allows for a more efficient assessment of pathogen buildup over time. However, environmental stall testing for respiratory pathogens should not replace proper biosecurity protocols, but it should instead be considered as an additional tool to monitor the silent circulation of respiratory pathogens in at-risk horses.

Canine parvovirus infections pose a significant global threat to canine health, necessitating effective vaccination strategies. This study evaluates the viral content in 20 batches of live attenuated canine parvovirus vaccines using real-time polymerase chain reaction and compares the results with the immunofluorescence test. The study involved canine parvovirus strain 39, adapted to Madin-Darby canine kidney cells, and vaccine batches sourced from various local and international suppliers. The immunofluorescence test results showed 18 batches met the permissible titer level of 3 log10 fluorescent antibody infective dose 50%/mL, while two batches (3 and 18) did not. Similarly, real-time polymerase chain reaction analysis confirmed the same 18 batches met the permissible titer level, with no significant difference between the methods as indicated by the 95% confidence interval for the difference in results (lower: 4.3949, upper: 5.3600). The findings support integrating advanced diagnostic technologies like real-time polymerase chain reaction into routine vaccine evaluation protocols, ensuring higher standards of veterinary biologics assessment; this transition aims to enhance the accuracy, efficiency, and overall quality of canine parvovirus vaccine evaluation, ultimately improving canine health protection.

Background: Microbial keratitis is a major preventable cause of visual impairment. In developed countries viral infections are the leading cause of corneal ulcer whereas bacteria and fungi attributes to maximum number of cases in the developing countries. There is paucity of recent data, risk factors and demographic distribution of patients for all three leading etiological agents of keratitis. Material and Methods: This study included all clinically diagnosed cases of infective keratitis from 01 Jan 2021 to 01 Jan 2022. In brief, four sets of corneal scrapings were taken from patients of infectious keratitis and processed for bacterial and fungal culture, microscopy and RTPCR of viral agents. Results: It was observed that rural elderly males were the most predisposed to occurrence of infectious keratitis and trauma being the leading cause. Out of total 151 samples processed, 49(32%) samples were found to have definitive evidence of bacterial, fungal and viral pathogens. Fungi were found to be the most common 37 (24%) etiological agent leading to keratitis followed by bacterial 7(5%) and viral agents 5(3%). Conclusion: Clinical signs alone are not sufficient to diagnose infectious keratitis, the importance of microbiology in the identification of pathogen and aiming at definitive therapy of infectious keratitis helps in rescuing the eye from this reversible cause of blindness.

Abstract Background The AmoyDx Pan lung cancer PCR panel (AmoyDx PLC panel) has been approved as a companion diagnostic tool for multiple anticancer agents in patients with non‐small cell lung cancer (NSCLC). However, the suitability of cytology specimens as samples for the AmoyDx PLC panel remains unclear. We evaluated the performance of frozen cell pellets from cytology specimens (FCPs) in the Amoy 9‐in‐1 assay, a preapproval assay of the AmoyDx PLC panel. Methods We retrospectively collected data of NSCLC patients enrolled in LC‐SCRUM‐Asia from the Shizuoka Cancer Center between September 2019 and May 2021. Results A total of 49 cases submitted FCPs for evaluation of oncogenic driver alterations and were assessed using Amoy 9‐in‐1 and next‐generation sequencing (NGS) assays. The success rates of DNA and RNA analyses using the Amoy 9‐in‐1 were both 100%, compared with 86% and 45%, respectively, using NGS assays. Oncogenic driver alterations were detected in 27 (55%) and 23 (47%) patients using Amoy 9‐in‐1 and NGS, respectively. No inconsistent results were observed among 19 cases in which both assays showed successful detection. In the remaining 30 cases, 10 had inconsistent results: nine oncogenic driver alterations (3 MET, 2 ALK, 2 ROS1, and 2 KRAS) were detectable only in Amoy 9‐in‐1, and one epidermal growth factor receptor (EGFR) mutation was detectable only in NGS. Conclusion FCPs can be successfully used in the AmoyDx PLC panel, with higher success rate compared with the NGS assay. The AmoyDx PLC panel may be an option in cases when insufficient tissue sample is available for the NGS assay.

Activation of Toll-like receptors (TLRs) is one of the earliest indicators of functional activation of the innate immune system. Therefore, the development of drugs that stimulate the transcription of TLR/RLR genes and at the same time are “multi-target” drugs is an important task of modern immunopharmacology. In this regard, antiviral drugs that combine the properties of interferonogens and immunomodulators, which also include Cycloferon® and its analogues, are of great interest. The purpose of this study was to assess the expression of genes that determine the TLR/RLR signalling reactions of the innate immune system under the influence of immunomodulatory antiviral drugs based on acridoneacetic acid (Cycloferon® and Cycloferon L). The study was conducted using a model of immunocompetent cells: THP-1, differentiated by phorbol ester into macrophage-like cells. Gene expression analysis was performed using real-time polymerase chain reaction. The expression level of genes encoding TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and RIG-I was studied under the influence of the drugs Cycloferon® and Cycloferon L in three concentrations (156 μg/mL, 312 μg/mL and 625 μg/mL) on 1 hour, 4 hours and 24 hours. It was shown that the drug Cycloferon® at concentrations of 156, 312 and 625 μg/mL at 24 hours of exposure dose-dependently stimulated the expression of TLR2, TLR3, TLR4, TLR7, TLR8 receptor genes. A stable stimulation of the expression of RIG1 receptor genes was found upon exposure 4 hours to the drug in all studied concentrations. For the first time, it was revealed that the drug Cycloferon L stimulated a stable increase in the expression of TLR2, TLR3, TLR4, TLR7, TLR8 genes at an exposure period of 24 hours. Hereby, it was shown that the drugs Cycloferon® and Cycloferon L stimulated the expression of the TLR2, TLR3, TLR4, TLR7, TLR8 genes (and RIG1 for the drug Cycloferon), which are responsible for the synthesis of innate immune receptors.

Background: Reverse transcription polymerase chain reaction (RT-PCR) is the main technique used to identify COVID-19 from respiratory samples. It has been suggested in several articles that chest CTs could offer a possible alternate diagnostic tool for COVID-19; however, no professional medical body recommends using chest CTs as an early COVID-19 detection modality. This literature review examines the use of CT scans as a diagnostic tool for COVID-19. Method: A comprehensive search of research works published in peer-reviewed journals was carried out utilizing precisely stated criteria. The search was limited to English-language publications, and studies of COVID-19-positive patients diagnosed using both chest CT scans and RT-PCR tests were sought. For this review, four databases were consulted: these were the Cochrane and ScienceDirect catalogs, and the CINAHL and Medline databases made available by EBSCOhost. Findings: In total, 285 possibly pertinent studies were found during an initial search. After applying inclusion and exclusion criteria, six studies remained for analysis. According to the included studies, chest CT scans were shown to have a 44 to 98% sensitivity and 25 to 96% specificity in terms of COVID-19 diagnosis. However, methodological limitations were identified in all studies included in this review. Conclusion: RT-PCR is still the suggested first-line diagnostic technique for COVID-19; while chest CT is adequate for use in symptomatic patients, it is not a sufficiently robust diagnostic tool for the primary screening of COVID-19.

Background: Periodontitis and peri-implantitis are diseases caused by pathogenic microorganisms that cause tissue damage and alveolar bone destruction resulting in the loss of teeth and implants. Due to the biological differences in the tissues surrounding the implants, peri-implantitis progresses more rapidly and intensely than periodontitis, underscoring the importance of understanding the characteristics and interactions of pathogenic bacteria. This study aimed to quantitatively analyze the pathogenic microorganisms associated with periodontitis and peri-implantitis in Korean patients and evaluate the correlation between these bacteria. Methods: A total of 98 (52 males and 46 females) were randomly selected and classified into three groups (healthy group [HG]=25; periodontitis group [PG]=31; and peri-implantitis group [PIG]=42). The relative expression levels of 11 pathogenic microorganisms collected from the gingival sulcus fluid were determined using multiplex real-time polymerase chain reaction. Results: Eikenella corrodens, Fusobacterium nucleatum, and Prevotella nigrescens were highly prevalent in the HG, PG, and PIG patients. The results of the relative quantitative analysis of microorganisms showed that all bacteria belonging to the green, orange, and red complexes were significantly more abundant in the PG and PIG than in the HG (p＜0.05). Porphyromonas gingivalis in the red complex showed a positive correlation with all microorganisms in the orange complex (p＜0.05). Campylobacter rectus in the orange complex showed a significant positive correlation with all microorganisms in the red complex, and with F. nucleatum, P. nigrescens, Prevotella intermedia, and Eubacterium nodatum (p＜0.05). Conclusion: P. gingivalis, C. rectus, and F. nucleatum exhibit strong interactions. Removing these bacteria can block complex formation and enhance the prevention and treatment of periodontitis and peri-implantitis.

Background: Industrial radiography uses gamma or X-ray radionuclide sources to investigate the safety of industrial materials. Industrial radiation workers receive the highest occupational radiation doses. Objective: The present study investigates the relationship between Bax and Bcl-2 gene expression variables in industrial radiation workers. Material and Methods: In this case-control study, data was collected using blood sampling from 40 workers, including two groups of non-radiation and radiation workers employed at the location. Expression levels of Bax and Bcl-2 genes were assessed in the laboratory. The environmental and absorbed doses of workers were measured using environmental and pen dosimeters. Results: Statistical analysis showed that the radiation group’s Bcl-2 gene expression level was significantly higher. Findings also demonstrated a correlation between Bcl-2 gene expression and the number of workdays. Also, the Bax gene expression did not show a significant change, and the expression ratio of Bax/Bcl-2 was insignificant in the two groups. Conclusion: Exposure to low doses of radiation could promote an adaptive response in cells by increasing Bcl-2 gene expression.

The aim of this review is to provide practical guidance for the molecular diagnosis of parasitic diseases in Korea in 2024. Specifically, the prevalence of parasitic diseases, commercially available molecular diagnostic kits, and reference laboratories for molecular diagnosis are presented. It is based on the literature and the medical diagnosis device database of the Korea Disease Control and Prevention Agency. In Korea, molecular diagnostic kits are available for intestinal protozoa (Giardia lamblia, Entamoeba histolytica, Cryptosporidium hominis, and Cryptosporidium parvum), Trichomonas vaginalis, and malarial parasites. Molecular diagnosis of other parasites is also possible; however, there is no commercially available kit. Therefore, parasite samples or derivatives for molecular diagnosis should be sent to specific laboratories, the parasitology departments of medical schools, or the Division of Vectors and Parasitic Diseases, Bureau of Infectious Disease Diagnosis Control at the Korea Disease Control and Prevention Agency. In commercial diagnostic kits, multiplex real-time polymerase chain reaction (PCR) is used to rapidly and easily detect the amplified parasitic DNA. The loop-mediated isothermal amplification (LAMP) was developed to diagnose T. vaginalis and Acanthamoeba infections. Its merits are that it does not require a PCR machine and has a short test time of approximately 60 min. Although LAMP is not commercially available, it may be widely used to screen for parasitic diseases. Commercial molecular diagnostic kits for parasitic diseases are limited to the clinical setting in Korea. Available kits are used to diagnose certain intestinal protozoa, T. vaginalis, and to differentiate Plasmodium species using multiplex PCR.

Objective: To establish a molecular method for the authentication of spices that can be used under different conditions for qualitative or semi-quantitative analyses. Methods: The real-time polymerase chain reaction (PCR) assay was developed based on SYBR Green I. Specific primers were successfully designed for the detection of star anise, fennel, pepper and prickly ash. Results: This method was able to specifically detect star anise, fennel, pepper and prickly ash with good linearity being found between Ct values and standard volume fraction in the range of 0.001%–10%. The limit of detection (LOD) was 1% for star anise standard, and 0.001% for fennel, pepper and prickly ash standards. The developed method was used to detect five actual samples. Star anise, fennel, pepper and prickly ash were not detected in sample 1; Star anise, fennel and pepper were detected in samples 2, 3 and 5; Star anise, fennel, pepper and prickly ash were detected in sample 4. Conclusion: This method has high sensitivity and good repeatability, and can be applied to the detection of real samples.

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Background: The AmoyDx Pan lung cancer PCR panel (AmoyDx PLC panel) has been approved as a companion diagnostic tool for multiple anticancer agents in patients with non‐small cell lung cancer (NSCLC). However, the suitability of cytology specimens as samples for the AmoyDx PLC panel remains unclear. We evaluated the performance of frozen cell pellets from cytology specimens (FCPs) in the Amoy 9‐in‐1 assay, a preapproval assay of the AmoyDx PLC panel. Methods: We retrospectively collected data of NSCLC patients enrolled in LC‐SCRUM‐Asia from the Shizuoka Cancer Center between September 2019 and May 2021. Results: A total of 49 cases submitted FCPs for evaluation of oncogenic driver alterations and were assessed using Amoy 9‐in‐1 and next‐generation sequencing (NGS) assays. The success rates of DNA and RNA analyses using the Amoy 9‐in‐1 were both 100%, compared with 86% and 45%, respectively, using NGS assays. Oncogenic driver alterations were detected in 27 (55%) and 23 (47%) patients using Amoy 9‐in‐1 and NGS, respectively. No inconsistent results were observed among 19 cases in which both assays showed successful detection. In the remaining 30 cases, 10 had inconsistent results: nine oncogenic driver alterations (3 MET, 2 ALK, 2 ROS1, and 2 KRAS) were detectable only in Amoy 9‐in‐1, and one epidermal growth factor receptor (EGFR) mutation was detectable only in NGS. Conclusion: FCPs can be successfully used in the AmoyDx PLC panel, with higher success rate compared with the NGS assay. The AmoyDx PLC panel may be an option in cases when insufficient tissue sample is available for the NGS assay. [ABSTRACT FROM AUTHOR]

Objective: This study aims to determine the frequency of pathogen detection by real-time polymerase chain reaction (PCR), the frequency of pathogen isolation by culture; and compare the value of real-time PCR and culture of nasopharyngeal aspiration samples in patients with severe community-acquired pneumonia (sCAP). Materials and Methods: It was a prospective and descriptive study. All pediatric patients diagnosed with sCAP were performed real-time PCR and culture of nasopharyngeal aspiration samples. Results: A total of 336 patient samples were obtained from children with sCAP. Real-time PCR detected pathogens in 312 patients (92.9%), while culture isolated bacteria in 228 patients (67.9%). Coinfections were reported in 279 cases (83.0%) through real-time PCR. The frequency of agreement between culture and real-time PCR was quite high (P < .001). Conclusion: Real-time PCR demonstrated more ability for detecting microorganisms than culture. This finding highlighted the value of real-time PCR for targeting pathogens in children with sCAP, particularly in cases involving complex pathogens or those requiring timely identification. [ABSTRACT FROM AUTHOR]

Background: Ameloblastoma is a benign but aggressive epithelial odontogenic neoplasm of unknown etiology. The role of human papilloma virus (HPV) in the etiology of oral squamous cell carcinoma has prompted the investigation of HPV as an etiologic factor in ameloblastoma. This study aimed to determine the frequency of high-risk (HR) HPV in conventional ameloblastoma and the clinical parameters associated with infection. Materials and Methods: The study was approved by the ethical review boards of the institution. DNA was extracted from fresh tissue collected 750 μL of DNA/RNA Shield (Zymo Research, United States) using Invitrogen PureLink Viral RNA/DNA Mini Kit (Invitrogen, USA). The extracted DNA was assayed for the detection of 14 HR HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68) using Anyplex™ II HPV HR Detection kit (Cat. No. HP7E00X) (Seegene Inc., Republic of Korea) on CFX-96TM Real-Time Polymerase Chain Reaction (PCR) System (Bio-Rad). Data on gender, age of patient, site of lesion, clinicohistological types of ameloblastoma and history of smoking, alcohol consumption, and practice of oral sex were collected. Data analysis was performed using analysis program SPSS version 25 and statistical significance was set at P < 0.05. Results: Two cases of conventional ameloblastoma were positive with HPV and none of the ameloblastic carcinoma cases were positive. The HPV 16 serotype was observed in both cases. While 5 of the cases had a history of alcohol consumption, none of these cases were positive for HPV serotype. Conclusions: HPV 16 positivity was detected in two cases of conventional ameloblastomas and none in ameloblastic carcinoma using real-time PCR. There was no effect of exposure to smoking, alcohol consumption, and practice of oral sex and HPV in the etiology of ameloblastoma. Data available are suggestive of a limited role of HPV in the etiology of ameloblastoma. [ABSTRACT FROM AUTHOR]

Cytomegalovirus (CMV) infection and disease is a condition usually described in immunocompromised patients, but among them, those with connective tissue diseases are poorly represented. Here we present the clinical, laboratory characteristics, management and outcomes of systemic lupus erythematosus (SLE) patients who presented with a CMV infection/disease to a high complexity hospital in southwestern Colombia between 2011 and 2020. 16 SLE patients were found to have a CMV infection. SLE was predominantly characterized by renal involvement (10 patients; 62.50%), and 14 patients (87.5%) were receiving steroids previous to the CMV infection. The entire sample required hospital admission, mainly related to acute kidney injury, and nine patients were admitted to the intensive care unit (ICU). Gastrointestinal organ damage was the most common CMV disease manifestation. All patients received ganciclovir, five of them (31.25%) suffered from septic shock, and seven (43.75%) died. Age ≥38 years and the presence of septic shock at admission were correlated to the mortality outcome. To our knowledge, this is the first publication evaluating SLE patients with CMV infection/disease in a Colombian population. [ABSTRACT FROM AUTHOR]

OBJECTIVE: The non-invasive detection of Helicobacter pylori (H. pylori) and its resistance to clarithromycin and levofloxacin significantly improves the management of infected patients by enabling tailored eradication treatments without the need for endoscopic procedures. This study aimed to assess the effectiveness of real-time PCR (RT-PCR) assays in identifying H. pylori infection and antibiotic resistance in stool and gastric biopsy specimens. PATIENTS AND METHODS: Stool and gastric biopsy samples were collected from patients within three days of post-hospitalization. A total of 115 samples were analyzed for H. pylori infection, and an additional 115 samples were evaluated for resistance to clarithromycin and levofloxacin using an RT-PCR-based molecular test. Statistical analyses were performed using (SPSS 26.0 IBM Corp., Armonk, NY, USA). RESULTS: Among 115 patients (53 males, average age 50.8±13.2 years), H. pylori was detected in 93.1% of stool samples and 93.9% of gastric biopsies. The RT-PCR assay demonstrated a sensitivity of 99.1% and a specificity of 100%, with an overall diagnostic accuracy of 99.1%. Clarithromycin resistance was found in 37.3% of stool and 46.9% of gastric biopsy specimens, with the assay showing 79.6% sensitivity and 98.4% specificity. Levofloxacin resistance was identified in 32.1% of stool samples and 31.3% of gastric biopsies, with 86.3% sensitivity and 91.1% specificity of the molecular test. CONCLUSIONS: The RT-PCR-based detection of H. pylori and its resistance to clarithromycin and levofloxacin in stool samples represents a promising approach to enhance eradication therapy outcomes, potentially improving treatment efficacy. [ABSTRACT FROM AUTHOR]

Introduction: Clinical significance of coagulase-negative staphylococci (CoNS) has been gradually acknowledged in both healthcare and clinical research, but approaches for their precise discrimination at the species level remain scarce. The current study aimed to evaluate the association of CoNS with orthopedic infections, where accurate and prompt identification of etiology is crucial for appropriate diagnosis and treatment decision-making. Methods: A 16S rRNA-based quantitative PCR (qPCR) assay was developed for the detection of Staphylococcus genus and two panels of 3-plex qPCR assays for further differentiation of six CoNS species with remarkable clinical significance, including S. epidermidis, S. haemolyticus, S. simulans, S. hominis, S. capitis, and S. caprae. All the assays exhibited excellent analytical performance. ΔCq (quantification cycle) between 16S rRNA and CoNS species-specific targets was established to determine the primary CoNS. These methods were applied to detect CoNS in wound samples from orthopedic patients with and without infection. Results and discussion: Overall, CoNS were detected in 17.8% (21/118) of patients with clinically suspected infection and in 9.8% (12/123) of patients without any infection symptom (p < 0.05). Moreover, the association with infection was found to be bacterial quantity dependent. S. epidermidis was identified as the predominant species, followed by S. simulans, S. haemolyticus, and S. hominis. Male sex, open injury, trauma, and lower extremity were determined as risk factors for CoNS infections. CoNS-positive patients had significantly longer hospitalization duration (20  days (15, 33) versus 13  days (7, 22) for Staphylococcus-negative patients, p = 0.003), which could be a considerable burden for healthcare and individual patients. Considering the complex characteristics and devastating consequences of orthopedic infections, further expanding the detection scope for CoNS may be pursued to better understand the etiology of orthopedic infections and to improve therapeutic strategies. [ABSTRACT FROM AUTHOR]

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Objectives: To explore the application of microRNA (miRNA) expression profile in prenatal diagnosis of fetal congenital heart disease (CHD). Methods: 30 pregnant women diagnosed with CHD by ultrasound (case group) and 10 pregnant women who required amniocentesis (control group) were collected from January 2021 to December 2022 at Tianjin Central Hospital of Gynecology Obstetrics. The amniotic fluid supernatant of the two groups of pregnant women was sequenced by Illumina sequencing platform. All miRNAs of the two groups of pregnant women were normalized and the differentially expressed miRNAs were analyzed. The miRNA with P＜0.05 and |log2 FC|＞3 (Fold Change, FC) were selected from the differentially expressed miRNAs for further validation by real time fluorescence quantitative polymerase chain reaction (RT-qPCR) in amniotic fluid and peripheral blood. The difference between miRNA sequencing and RT-qPCR in amniotic fluid was compared, and the miRNAs with consistent expression regulation direction in peripheral blood and amniotic fluid were selected. Results: A total of 138 differentially expressed miRNAs were found, of which 85 were up -regulated and 53 were down - regulated. Further, 15 differentially expressed miRNAs were selected, and the results of miRNA sequencing in amniotic fluid were consistent with those of RT -qPCR. There were two miRNAs with the same expression and regulation direction in peripheral blood and amniotic fluid, which were miR-222-3p and miR-189-5p, respectively. The expression of these two miRNAs in maternal blood of the case group was significantly higher than that of the control group, and the difference was statistically significant (all P＜0.05). Conclusions: As a new serological marker, miRNA in maternal blood can be preliminarily applied to the screening of fetal CHD. [ABSTRACT FROM AUTHOR]

Background: Reverse transcription polymerase chain reaction (RT-PCR) is the main technique used to identify COVID-19 from respiratory samples. It has been suggested in several articles that chest CTs could offer a possible alternate diagnostic tool for COVID-19; however, no professional medical body recommends using chest CTs as an early COVID-19 detection modality. This literature review examines the use of CT scans as a diagnostic tool for COVID-19. Method: A comprehensive search of research works published in peer-reviewed journals was carried out utilizing precisely stated criteria. The search was limited to English-language publications, and studies of COVID-19-positive patients diagnosed using both chest CT scans and RT-PCR tests were sought. For this review, four databases were consulted: these were the Cochrane and ScienceDirect catalogs, and the CINAHL and Medline databases made available by EBSCOhost. Findings: In total, 285 possibly pertinent studies were found during an initial search. After applying inclusion and exclusion criteria, six studies remained for analysis. According to the included studies, chest CT scans were shown to have a 44 to 98% sensitivity and 25 to 96% specificity in terms of COVID-19 diagnosis. However, methodological limitations were identified in all studies included in this review. Conclusion: RT-PCR is still the suggested first-line diagnostic technique for COVID-19; while chest CT is adequate for use in symptomatic patients, it is not a sufficiently robust diagnostic tool for the primary screening of COVID-19. [ABSTRACT FROM AUTHOR]

The zebrafish (Danio rerio) is one of the promising objects for modeling pathological conditions. A large number of such works are carried out at the transcriptomic level. Transcriptomic studies require the use of stable housekeeping genes (HKGs), and so we selected candidate HKGs and analyzed their stability in D. rerio under normal conditions. The work was carried out using D. rerio of the AB line whose embryos were sampled at three time points: the hatching stage (2 days postfertilization, dpf), and early larval stages (5 and 9 dpf). Analysis of HKG expression levels was performed using real-time polymerase chain reaction. Further analysis of the obtained Ct results was carried out in the RefFinder program. Different HKGs are suitable for assessing changes in gene expression at different stages of D. rerio development. When assessing changes in gene expression at the hatching stage (2 dpf), the optimal pair of HKGs is aars1 and polr2f; for the early larval stage (5 dpf), it is aars1 and psmd6; and for 9 dpf, it is bcat2 and actb1. During transcriptomic studies conducted at several developmental stages simultaneously, it is necessary to use a single selected pair of HKGs for all the studied stages of zebrafish development: aars1 and lsm12b. According to the data obtained, aars1 and lsm12b are the most stable HKGs for all the early developmental stages of D. rerio studied. At the same time, we first showed that aars1 is the most stable HKG for the early stages of the development. The second most stable HKG is lsm12b. Our data on the stability of the lsm12b gene are consistent with previously published data. [ABSTRACT FROM AUTHOR]

Background and Objectives: Graft-versus-host disease (GvHD) frequently complicates hematopoietic stem cell transplantation (HSCT). Emerging evidence suggests a correlation between gut microbiota and GvHD risk. This study aims to elucidate the microbiota profiles in HSCT patients before and after transplantation and their association with GvHD. Materials and Methods: This study, conducted from December 2022 to December 2023, involved the collection of 15 stool samples from HSCT patients. Bacterial content was quantified using real-time PCR, while interleukin-6 levels were assessed via ELISA. Results: Among the 15 participants (8 male, 7 female), 9 underwent allogeneic HSCT (allo-HSCT) and 6 received autologous HSCT. In the aGvHD group, there was a significant reduction in the abundance of Bacteroides and Bifidobacterium compared to those without aGvHD. Additionally, declines were observed in Clostridium and Firmicutes populations. The genus Blautia also showed reduced prevalence in the aGvHD group, whereas no significant differences were noted in the uncomplicated group. ELISA analysis revealed that interleukin-6 levels remained within the normal range (30-960 pg/ml) with no significant elevation in the aGvHD group. Conclusion: The study highlights a notable association between alterations in gut microbiota, specifically reductions in certain bacterial populations and the development of aGvHD following allo-HSCT.