Search Results (32)

Background/Objectives: Aseptic loosening (AL) is among the most common causes of late failure following total hip arthroplasty (THA), often necessitating complex revision surgery. Current diagnostic tools, mainly based on clinical and radiological findings, are primarily able to identify advanced changes of periprosthetic osteolysis (PPOL). Therefore, early detection of AL remains a challenge. Circulating microRNAs (miRNAs) have emerged as promising, minimally invasive biomarkers in musculoskeletal disorders. This study investigates the expression of inflammation-related miRNAs let-7i-5p, let-7e-5p, miR-15a-5p, miR-30a-3p and miR-130a-3p in patients with confirmed AL after THA to evaluate their potential role in AL. Methods: AL patients undergoing revision were compared with asymptomatic post-THA individuals and controls with degenerative osteoarthritis. Preoperative, peripheral blood samples were collected; total RNA was extracted; and quantitative real-time PCR (qRT-PCR) was performed to quantify miRNA expression. The relative expression of miRNAs was calculated using the 2–ΔΔCt method after proper normalization of Ct values. Statistical analysis assessed differences between groups. Results: The under investigation miRNAs exhibited distinct expression patterns. Several targets demonstrated significant downregulation in AL patients, suggesting a potential link to inflammatory and osteolytic pathways like Toll-like receptor 4 (TLR4)–Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, NLRP3 inflammasome activation and macrophage polarization. Conclusions: The observed alterations in circulating miRNAs support their capability as biomarkers for early detection of AL following THA. Larger cohorts could facilitate translation into routine clinical diagnostics. Full article

Background/Objectives: Acute respiratory infections remain a major global public health concern affecting individuals across all ages. Accurate and rapid diagnosis of respiratory pathogens is crucial for effective patient management and infection control. Multiplex real-time polymerase chain reaction (PCR) assays have gained prominence over conventional methods for routine viral detection in clinical laboratories owing to their enhanced sensitivity and specificity; however, comparative performance data for PowerChek™ RVP remain limited. This study aimed to evaluate the diagnostic performance of the PowerChek™ Respiratory Virus Panel 1/2/3/4, which detects 16 respiratory viruses, including SARS-CoV-2, in nasopharyngeal swab (NPS) specimens. Methods: Overall, 336 NPS specimens were analyzed using the PowerChek™ RVP, BioFire^® RP 2.1plus, and Allplex™ RP assays, with nucleic acid extraction performed using the Advansure™ E3 system. The performance metrics were calculated using two-by-two contingency tables. Results: Among 336 NPS specimens (232 positive, 104 negative), PowerChek™ RVP detected 226 positives with minimal discrepancies, showing high concordance with BioFire^® RP 2.1plus (accuracy 94.6%, kappa 0.843–1.000). Fifteen discordant cases were identified in this study. Eleven could not be sequenced because of amplification failure and most had high Ct values (>30). Sequencing of four samples confirmed concordance with BioFire^® RP 2.1plus and PowerChek™ RVP, whereas Allplex™ RP showed false-negative results. Conclusions: The PowerChek™ RVP assay demonstrated a high level of relative sensitivity, specificity, accuracy, diagnostic predictive values and strong concordance with comparable reference assays in identifying its targets. This assay is a reliable and efficient diagnostic tool for clinical laboratories to facilitate the accurate identification of respiratory pathogens. Full article

Background/Objectives: Monitoring community health and tracking SARS-CoV-2 evolution were critical priorities throughout the COVID-19 pandemic. However, widespread shortages of personal protective equipment, the necessity for social distancing, and the redeployment of healthcare personnel to clinical duties presented significant barriers to traditional sample collection. Methods: In this study, we evaluated the feasibility of using self-collected saliva specimens for the qualitative detection of SARS-CoV-2 infection. Following confirmation of reliable viral detection in saliva, we established a large-scale surveillance program in Arizona, USA, to enable clinical diagnosis and genomic sequencing from self-collected samples. Between April 2020 and December 2023, we tested approximately 1.4 million saliva samples using RT-PCR, identifying 94,330 SARS-CoV-2 infections. Whole genome sequencing was performed on 69,595 samples, yielding 54,040 high-quality consensus genomes. Results: This surveillance approach enabled real-time monitoring of general infection trends that matched regional case counts. We monitored multiple wave-like introductions of viral lineages over the course of the pandemic. We identified three periods of S gene target failure on a commercial assay and assessed its ability to make fast, genotyping assignment during the pandemic (PPV = 0.98, 95% CI = 0.97–0.99; NPV = 0.94, 95% CI = 0.94–0.96). The co-location of clinical testing and sequencing capabilities within the same facility resulted in low turnaround time from the sample collection to the generation of sequencing data (median = 12 days, IQR: 9.0–19.75). Conclusions: Our findings support the use of self-collected saliva as a scalable, cost-effective, and practical strategy for infectious disease surveillance in future pandemics. Full article

Antibiotic resistance in Helicobacter pylori is increasing rapidly and emerging as a major factor in treatment failure. We aimed to identify genetic mutations associated with resistance to clarithromycin (23S rRNA peptidyl transferase), fluoroquinolones (gyrA), and metronidazole (rdxA), and to explore their mechanisms of action through molecular modeling. H. pylori detection and the molecular characterization of genes were conducted directly on gastric biopsies by real-time PCR followed by nucleotide sequencing. A 3D model was used to evaluate molecular interactions between the antibiotics and respective target proteins. H. pylori was identified in 66.7% of 33 patients. An analysis of 23SrRNA revealed novel mutations that, by in silico analysis, do not appear to contribute to clarithromycin resistance. In gyrA, mutations in amino acid residues 87 and 91 had an incidence of 27%, and the in silico analysis revealed that these positions are relevant in the binding and resistance to fluoroquinolones. It is also reported for other mutations, some of which are never described. All rdxA mutations were missense, with R16H, M56V, H97T, G98S, A118T, V123T, and R131K predicted by in silico analysis to impact metronidazole resistance. Monitoring H. pylori gene mutations is crucial for tailoring effective antibiotic therapies. Our study advances personalized medicine by introducing novel methods to detect resistance-related mutations and uncovering the molecular mechanisms driving this resistance. Full article

MicroRNAs (miR) are a group of small, non-coding RNAs of 17–25 nucleotides that regulate gene expression at the post-transcriptional level. Dysregulation of miRNA expression or function may contribute to abnormal gene expression and signaling pathways, leading to disease pathology. Lagovirus europaeus (L. europaeus) causes severe disease in rabbits called rabbit hemorrhagic disease (RHD). The symptoms of liver, lung, kidney, and spleen degeneration observed during RHD are similar to those of acute liver failure (ALF) and multi-organ failure (MOF) in humans. In this study, we assessed the expression of miRs and their target genes involved in the innate immune and inflammatory response. Also, we assessed their potential impact on pathways in L. europaeus infection—two genotypes (GI.1 and GI.2)—in the liver, lungs, kidneys, and spleen. The expression of miRs and target genes was determined using quantitative real-time PCR (qPCR). We assessed the expression of miR-155 (MyD88, TAB2, p65, NLRP3), miR-146a (IRAK1, TRAF6), miR-223 (TLR4, IKKα, NLRP3), and miR-125b (MyD88). We also examined biomarkers of inflammation: IL-1β, IL-6, TNF-α, and IL-18 in four tissues at the mRNA level. Our study shows that the main regulators of the innate immune and inflammatory response in L. europaeus/GI.1 and GI.2 infection, as well as RHD, are miR-155, miR-223, and miR-146a. During infection with L. europaeus/RHD, miR-155 has both pro- and anti-inflammatory effects in the liver and anti-inflammatory effects in the kidneys and spleen; miR-146a has anti-inflammatory effects in the liver, lungs and kidneys; miR-223 has anti-inflammatory effects in all tissues; however, miR-125b has anti-inflammatory effects only in the liver. In each case, such an effect may be a determinant of the pathogenesis of RHD. Our research shows that miRs may regulate three innate immune and inflammatory response pathways in L. europaeus infection. However, the result of this regulation may be influenced by the tissue microenvironment. Our research shows that infection of rabbits with L. europaeus/GI.1 and GI.2 genotypes causes an overexpression of two critical acute phase cytokines: IL-6 in all examined tissues and TNF-α (in the liver, lungs, and spleen). IL-1β was highly expressed only in the lungs after L. europaeus infection. These facts indicate a strong and rapid involvement of the local innate immune and inflammatory response in L. europaeus infection—two genotypes (GI.1 and GI.2)—and in the pathogenesis of RHD. Profile of biomarkers of inflammation in rabbits infected with L. europaeus/GI.1 and GI.2 genotypes are similar regarding the nature of changes but are different for individual tissues. Therefore, we propose three inflammation profiles for L. europaeus infection for both GI.1 and GI.2 genotypes (pulmonary, renal, liver, and spleen). Full article

Lung cancer is the leading cause of cancer mortality worldwide. Fortunately, the advent of precision medicine, which includes targeted therapy and immunotherapy, offers hope. However, identifying specific mutations is imperative before initiating precise medications. Traditional methods, such as real-time PCR examination of individual mutations, are time-consuming. Contemporary techniques, such as tissue- and plasma-based next-generation sequencing (NGS), allow comprehensive genome analysis concurrently. Notably, plasma-based NGS has a shorter turnaround time (TAT) and thus a shorter time-to-treatment (TTT). In this case report, we demonstrate the benefits of plasma-based NGS before pathological diagnosis in a patient with image-suspected non-small cell lung cancer (NSCLC). An 82-year-old Taiwanese woman presented with lower back pain persisting for one month and left-sided weakness for two weeks. Whole-body computed tomography (CT) revealed lesions suspicious for brain and bone metastases, along with a mass consistent with a primary tumor in the left upper lobe, indicative of advanced NSCLC with T4N3M1c staging. The patient underwent a bronchoscopic biopsy on Day 0, and the preliminary report that came out on Day 1 was suggestive of metastatic NSCLC. Blood was also collected for plasma-based NGS on Day 0. The patient was Coronavirus disease 2019-positive and was treated with molnupiravir on Day 6. On Day 7, pathology confirmed pulmonary adenocarcinoma, and the results of plasma-based NGS included EGFR L858R mutation. The patient was started on targeted therapy (afatinib) on Day 9. Unfortunately, the patient died of hypoxic respiratory failure on Day 26, a complication of underlying viral infection. Plasma-based NGS offers a rapid and efficient means of mutation detection in NSCLC, streamlining treatment initiation and potentially improving the negative emotions of patients. Its utility, particularly in regions with a high prevalence of specific mutations, such as EGFR alterations in East Asian populations, highlights its relevance in guiding personalized therapy decisions. Full article

Arrhythmogenic right ventricular cardiomyopathy (ARVC) can lead to sudden cardiac death and life-threatening heart failure. Due to its high fatality rate and limited therapies, the pathogenesis and diagnosis biomarker of ARVC needs to be explored urgently. This study aimed to explore the lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network in ARVC. The mRNA and lncRNA expression datasets obtained from the Gene Expression Omnibus (GEO) database were used to analyze differentially expressed mRNA (DEM) and lncRNA (DElnc) between ARVC and non-failing controls. Differentially expressed miRNAs (DEmiRs) were obtained from the previous profiling work. Using starBase to predict targets of DEmiRs and intersecting with DEM and DElnc, a ceRNA network of lncRNA-miRNA-mRNA was constructed. The DEM and DElnc were validated by real-time quantitative PCR in human heart tissue. Protein–protein interaction network and weighted gene co-expression network analyses were used to identify hub genes. A logistic regression model for ARVC diagnostic prediction was established with the hub genes and their ceRNA pairs in the network. A total of 448 DEMs (282 upregulated and 166 downregulated) were identified, mainly enriched in extracellular matrix and fibrosis-related GO terms and KEGG pathways, such as extracellular matrix organization and collagen fibril organization. Four mRNAs and two lncRNAs, including COL1A1, COL5A1, FBN1, BGN, XIST, and LINC00173 identified through the ceRNA network, were validated by real-time quantitative PCR in human heart tissue and used to construct a logistic regression model. Good ARVC diagnostic prediction performance for the model was shown in both the training set and the validation set. The potential lncRNA-miRNA-mRNA regulatory network and logistic regression model established in our study may provide promising diagnostic methods for ARVC. Full article

The primary goal of this investigation was to identify mRNA targets affected by dysregulated miRNAs in RIF. This was accomplished by comprehensively analyzing mRNA and miRNA expression profiles in two groups: female subjects with normal reproductive function (control, n = 5) and female subjects experiencing recurrent implantation failure (RIF, n = 5). We conducted transcriptome sequencing and small RNA sequencing on endometrial tissue samples from these cohorts. Subsequently, we validated a selection of intriguing findings using real-time PCR with samples from the same cohort. In total, our analysis revealed that 929 mRNAs exhibited differential expression patterns between the control and RIF patient groups. Notably, our investigation confirmed the significant involvement of dysregulated genes in the context of RIF. Furthermore, we uncovered promising correlation patterns within these mRNA/miRNA pairs. Functional categorization of these miRNA/mRNA pairs highlighted that the differentially expressed genes were predominantly associated with processes such as angiogenesis and cell adhesion. We identified new target genes that are regulated by miR-665, including Blood Vessel Epicardial Substance (BVES) and Adenosylhomocysteinase like 2 (AHCYL2). Our findings suggest that abnormal regulation of genes involved in angiogenesis and cell adhesion, including BVES and AHCYL2, contributes to the endometrial dysfunction observed in women with recurrent implantation failure (RIF) compared to healthy women. Full article

During the SARS-CoV-2 pandemic, the Dr. Risch medical group employed the multiplex TaqPath^TM COVID-19 CE-IVD RT-PCR Kit for large-scale routine diagnostic testing in Switzerland and the principality of Liechtenstein. The TaqPath Kit is a widely used multiplex assay targeting three genes (i.e., ORF1AB, N, S). With emergence of the B.1.1.7 (Alpha) variant, a diagnostic flaw became apparent as the amplification of the S-gene target was absent in these samples due to a deletion (ΔH69/V70) in the Alpha variant genome. This S-gene target failure (SGTF) was the earliest indication of a new variant emerging and was also observed in subsequent variants such as Omicron BA.1 and BA4/BA.5. The Delta variant and Omicron BA.2 did not present with SGTF. From September 2020 to November 2022, we investigated the applicability of the SGTF as a surrogate marker for emerging variants such as B.1.1.7, B.1.617.2 (Delta), and Omicron BA.1, BA.2, and BA.4/BA.5 in samples with cycle threshold (Ct) values < 30. Next to true SGTF-positive and SGTF-negative samples, there were also samples presenting with delayed-type S-gene amplification (higher Ct value for S-gene than ORF1ab gene). Among these, a difference of 3.8 Ct values between the S- and ORF1ab genes was found to best distinguish between “true” SGTF and the cycle threshold variability of the assay. Samples above the cutoff were subsequently termed partial SGTF (pSGTF). Variant confirmation was performed by whole-genome sequencing (Oxford Nanopore Technology, Oxford, UK) or mutation-specific PCR (TIB MOLBIOL). In total, 17,724 (7.4%) samples among 240,896 positives were variant-confirmed, resulting in an overall sensitivity and specificity of 93.2% [92.7%, 93.7%] and 99.3% [99.2%, 99.5%], respectively. Sensitivity was increased to 98.2% [97.9% to 98.4%] and specificity lowered to 98.9% [98.6% to 99.1%] when samples with pSGTF were included. Furthermore, weekly logistic growth rates (α) and sigmoid’s midpoint (t₀) were calculated based on SGTF data and did not significantly differ from calculations based on comprehensive data from GISAID. The SGTF therefore allowed for a valid real-time estimate for the introduction of all dominant variants in Switzerland and Liechtenstein. Full article

G-protein-coupled receptors (GPCRs) are critical regulators of cardiac physiology and a key therapeutic target for the treatment of heart disease. Ectopic olfactory receptors (ORs) are GPCRs expressed in extra-nasal tissues which have recently emerged as new mediators in the metabolic control of cardiac function. The goals of this study were to profile OR gene expression in the human heart, to identify ORs dysregulated by heart failure caused by ischemic cardiomyopathy, and to provide evidence suggestive of a role for those altered ORs in the pathogenesis of heart failure. Left ventricular tissue from heart failure patients (n = 18) and non-failing heart samples (n = 4) were subjected to a two-step transcriptome analysis consisting of the quantification of 372 distinct OR transcripts on real-time PCR arrays and simultaneous determination of global cardiac gene expression by RNA sequencing. This strategy led to the identification of >160 ORs expressed in the human heart, including 38 receptors differentially regulated with heart failure. Co-expression analyses predicted the involvement of dysregulated ORs in the alteration of mitochondrial function, extracellular matrix remodeling, and inflammation. We provide this dataset as a resource for investigating roles of ORs in the human heart, with the hope that it will assist in the identification of new therapeutic targets for the treatment of heart failure. Full article

The high demand for SARS-CoV-2 tests but limited supply to South African laboratories early in the COVID-19 pandemic resulted in a heterogenous diagnostic footprint of open and closed molecular testing platforms being implemented. Ongoing monitoring of the performance of these multiple and varied systems required novel approaches, especially during the circulation of variants. The National Health Laboratory Service centrally collected cycle threshold (Ct) values from 1,497,669 test results reported from 6 commonly used PCR assays in 36 months, and visually monitored changes in their median Ct within a 28-day centered moving average for each assays’ gene targets. This continuous quality monitoring rapidly identified delayed hybridization of RdRp in the Allplex™ SARS-CoV-2 assay due to the Delta (B.1.617.2) variant; S-gene target failure in the TaqPath™ COVID-19 assay due to B.1.1.7 (Alpha) and the B.1.1.529 (Omicron); and recently E-gene delayed hybridization in the Xpert^® Xpress SARS-CoV-2 due to XBB.1.5. This near “real-time” monitoring helped inform the need for sequencing and the importance of multiplex molecular nucleic acid amplification technology designs used in diagnostics for patient care. This continuous quality monitoring approach at the granularity of Ct values should be included in ongoing surveillance and with application to other disease use cases that rely on molecular diagnostics. Full article

The aim of this study was to investigate whether the presence of Staphylococcus aureus (SA) producing the Panton–Valentine leukocidin (PVL) affects the outcome of Prosthetic Joint Infection (PJI). Patients with acute and chronic PJI sustained by SA were prospectively enrolled at the orthopedic unit of “Casa di Cura Santa Maria Maddalena”, from January 2019 to October 2021. PJI diagnosis was reached according to the diagnostic criteria of the International Consensus Meeting on PJI of Philadelphia. Synovial fluid obtained via joint aspirations was collected in order to isolate SA. The detection of PVL was performed via real-time quantitative PCR (RT-qPCR). The outcome assessment was performed using the criteria of the Delphi-based International Multidisciplinary Consensus. Twelve cases of PJI caused by SA were included. Nine (75%) cases were acute PJI treated using debridement, antibiotic and implant retention (DAIR); the remaining three (25%) were chronic PJI treated using two-stage (n = 2) and one-stage revision (n = 1), respectively. The SA strains that tested positive for PVL genes were 5/12 (41.6%,). Treatment failure was documented in three cases of acute PJI treated using DAIR, all supported by SA–PVL strains (p < 0.045). The remaining two cases were chronic PJI treated with a revision arthroplasty (one and two stage, respectively), with a 100% eradication rate in a medium follow-up of 24 months. Although a small case series, our study showed a 100% failure rate in acute PJI, probably caused by SA PVL-producing strains treated conservatively (p < 0.04). In this setting, toxin research should guide radical surgical treatment and targeted antibiotic therapy. Full article

In the absence of validated biomarkers to control the cure of Chagas disease, PCR-based diagnosis is being used as the main tool for an early indication of therapeutic failure. However, since it is considered a technique of complex reproducibility, mainly due to difficulties in establishing accurate controls to guarantee the quality of the reaction, the use of PCR for Chagas disease diagnosis is restricted to specialized centers. In an effort to disseminate the molecular diagnosis of Chagas disease and its applications, new diagnostic kits based on qPCR have been made available in the market in recent years. Here, we show the results of the validation of the NAT Chagas kit (Nucleic Acid Test for Chagas Disease) for the detection and quantification of T. cruzi in blood samples of patients suspected of Chagas disease infection. The kit, composed of a TaqMan duplex reaction targeting the T. cruzi satellite nuclear DNA and an exogenous internal amplification control, presented a reportable range from 10⁴ to 0.5 parasite equivalents/mL and a limit of detection (LOD) of 0.16 parasite equivalents/mL of blood. In addition, the NAT Chagas kit detected T. cruzi belonging to all six discrete typing units (DTUs—TcI to TcVI), similarly to the in-house real-time PCR performed with commercial reagents, which has been selected as the best performance assay in the international consensus for the validation of qPCR for Chagas disease. In the clinical validation presented here, the kit showed 100% sensitivity and 100% specificity when compared to the consensus in-house real-time PCR assay. Thus, the NAT Chagas kit, which is produced entirely in Brazil under the international standards of good manufacturing practices (GMP), appears as an excellent alternative to enable the molecular diagnosis of Chagas disease in public and private diagnostic centers, as well as to facilitate the monitoring of patients under etiological treatment participating in clinical trials. Full article

Periprosthetic joint infection (PJI) is a devastating complication. This study aimed to unravel the veil of the N⁶-methyladenine (m⁶A) modification in PJI. Synovium, synovial fluid, sonication fluid and bone samples were collected intraoperatively from Staphylococcus aureus PJI and aseptic failure (AF) patients. The overall m⁶A level was detected by the m⁶A RNA methylation quantification kit, and the expression of m⁶A-related genes was quantified by real-time PCR and Western blot. Finally, an epitranscriptomic microarray and bioinformatics analysis were performed. We showed that there was a significant difference in overall m⁶A level between the PJI group and the AF group (PJI group had a higher overall m⁶A level). The expression level of METTL3 was higher in the PJI group than that in the AF group. There were 2802 differential m⁶A-modified mRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that differential m⁶A-modified mRNAs were significantly enriched in the NOD-like receptor signaling pathway, Th17 cell differentiation and the IL-17 signaling pathway, which indicates that the m⁶A modification might be involved in the processes of infection and immune response, bone metabolism and programmed cell death in PJI. In summary, the present work demonstrated that m⁶A modification plays a role in PJI and might be a therapeutic target for developing effective treatment strategies. Full article

Shortly after its emergence, Omicron and its sub-variants have quickly replaced the Delta variant during the current COVID-19 outbreaks in Vietnam and around the world. To enable the rapid and timely detection of existing and future variants for epidemiological surveillance and diagnostic applications, a robust, economical real-time PCR method that can specifically and sensitively detect and identify multiple different circulating variants is needed. The principle of target- failure (TF) real-time PCR is simple. If a target contains a deletion mutation, then there is a mismatch with the primer or probe, and the real-time PCR will fail to amplify the target. In this study, we designed and evaluated a novel multiplex RT real-time PCR (MPL RT-rPCR) based on the principle of target failure to detect and identify different variants of SARS-CoV-2 directly from the nasopharyngeal swabs collected from COVID-19 suspected cases. The primers and probes were designed based on the specific deletion mutations of current circulating variants. To evaluate the results from the MPL RT-rPCR, this study also designed nine pairs of primers for amplifying and sequencing of nine fragments from the S gene containing mutations of known variants. We demonstrated that (i) our MPL RT-rPCR was able to accurately detect multiple variants that existed in a single sample; (ii) the limit of detection of the MPL RT-rPCR in the detection of the variants ranged from 1 to 10 copies for Omicron BA.2 and BA.5, and from 10 to 100 copies for Delta, Omicron BA.1, recombination of BA.1 and BA.2, and BA.4; (iii) between January and September 2022, Omicron BA.1 emerged and co-existed with the Delta variant during the early period, both of which were rapidly replaced by Omicron BA.2, and this was followed by Omicron BA.5 as the dominant variant toward the later period. Our results showed that SARS-CoV-2 variants rapidly evolved within a short period of time, proving the importance of a robust, economical, and easy-to-access method not just for epidemiological surveillance but also for diagnoses around the world where SARS-CoV-2 variants remain the WHO’s highest health concern. Our highly sensitive and specific MPL RT-rPCR is considered suitable for further implementation in many laboratories, especially in developing countries. Full article