Real-time Polymerase Chain Reaction System Research Articles

Evaluation of the Seegene AllplexTM RV Master Assay for One-Step Simultaneous Detection of Eight Respiratory Viruses in Nasopharyngeal Specimens

The Seegene AllplexTM RV Master (RVM) assay is a one-step multiplex real-time reverse transcription polymerase chain reaction (RT-PCR) system for detecting eight viral respiratory pathogens from nasopharyngeal swab, aspirate, and bronchoalveolar lavage specimens. The eight RVM targets are: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Influenza A (Flu A), Influenza B (Flu B), Human respiratory syncytial virus (RSV), adenovirus (AdV), rhinovirus (HRV), parainfluenza virus (PIV), and metapneumovirus (MPV). The assay is based on Seegene's multiple detection temperature (MuDT) technology and provides cycle threshold (Ct) values for each of its viral targets upon PCR completion. We aimed to evaluate the diagnostic performance of the RVM assay by calculating sensitivity, specificity, accuracy, Positive Predictive Value (PPV), Negative Predictive Value (NPV), Positive Percent Agreement (PPA), Negative Percent Agreement (NPA), and Overall Percent Agreement (OPA) compared to definite diagnosis and analogous reference assays. Diagnostic sensitivity, specificity, accuracy, PPV, and NPV were calculated by comparing the results of the RVM assay to that of definite diagnosis assays; while PPA, NPA, and OPA were calculated by comparing results of the RVM assay to that of analogous reference products. Definite diagnosis and reference methods comprised whole genome sequencing and PCR genotyping, the AllplexTM SARS-CoV-2/FluA/FluB/RSV and Respiratory Panels 1, 2, and 3 assays (Seegene), and the Xpert® Xpress SARS-CoV-2/FluA/FluB/RSV Plus assay (Cepheid). Reproducibility of the RVM assay using fully-automated and semi-automated nucleic acid (NA) extraction workflows and as performed by independent operators was also assessed. In total, 249 positive respiratory specimens and at least 50 negative specimens for each target tested were used for this evaluation study. Sensitivity, specificity, accuracy, PPV, NPV, PPA, NPA, and OPA ranged from 95.7% to 100% for detecting all eight targets tested on the RVM assay. Reproducibility PPA, NPA, and OPA between automated and semi-automated NA extraction workflows were all >97.9%, while the reproducibility PPA, NPA and OPA between independent operators were all 100%. These results demon6strate a high level of sensitivity, specificity, accuracy and diagnostic predictive value of the RVM assay and high agreement with comparable reference assays in identifying all eight of its targets. Taken together, our study underscores the diagnostic utility of the RVM assay in detecting eight viral respiratory pathogens.

SMAD3 rs17228212 Polymorphism Is Associated with Advanced Carotid Atherosclerosis in a Slovenian Population.

Smad proteins influence the TGFβ signaling pathway, which plays an important role in the progression of atherosclerosis. The aim of our study was to investigate the association between the rs17228212 polymorphism of the SMAD3 gene and advanced carotid atherosclerosis in Slovenian subjects and to investigate the effect of the rs17228212 SMAD3 polymorphism on the expression of SMAD3 in endarterectomy sequesters. In this cross-sectional case-control study, 881 unrelated Caucasians were divided into two groups. The first group included 308 patients with advanced carotid atherosclerosis of the common or internal carotid artery with stenosis greater than 75% that underwent a revascularization procedure (cases). The control group consisted of 573 subjects without hemodynamically significant carotid atherosclerosis. We analyzed the rs17228212 polymorphism of the SMAD3 gene using the StepOne real-time polymerase chain reaction system and TaqMan SNP genotyping assay. The results in the two genetic models showed a statistically significant association, codominant (OR 4.05; CI 1.10-17.75; p = 0.037) and dominant (OR 3.60; CI 1.15-15.45; p = 0.045). An immunohistochemical analysis of SMAD3 expression was conducted for 26 endarterectomy specimens. The T allele of the rs17228212 SMAD3 gene was shown to be associated with an increased numerical area density of SMAD3-positive cells in carotid plaques.

Double-Tube Multiplex TaqMan Real-Time PCR for the Detection of Eight Animal-Derived Dairy Ingredients.

Milk and dairy products represent important sources of nutrition in our daily lives. The identification of species within dairy products holds importance for monitoring food adulteration and ensuring traceability. This study presented a method that integrated double-tube and duplex real-time polymerase chain reaction (PCR) with multiplex TaqMan probes to enable the high-throughput detection of animal-derived ingredients in milk and dairy products. The detection system utilized one pair of universal primers, two pairs of specific primers, and eight animal-derived specific probes for cow, buffalo, goat, sheep, camel, yak, horse, and donkey. These components were optimized within a double-tube and four-probe PCR multiplex system. The developed double-tube detection system could simultaneously identify the above eight targets with a detection limit of 10-0.1 pg/μL. Validation using simulated adulterated milk samples demonstrated a detection limit of 0.1%. The primary advantage of this method lies in the simplification of the multiplex quantitative real-time PCR (qPCR) system through the use of universal primers. This method provides an efficient approach for detecting ingredients in dairy products, providing powerful technical support for market supervision.

Low Detection of High-risk Human Papilloma Virus in Individuals with Ameloblastoma in a Tertiary Hospital in Lagos, Nigeria.

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. 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. 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. 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.

Ov-RPA-CRISPR/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces.

Opisthorchis viverrini infection is traditionally diagnosed using the Kato-Katz method and formalin ethyl-acetate concentration technique. However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect O.viverrini infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) (RPA-CRISPR/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and Mycobacterium tuberculosis. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA-CRISPR/Cas assay to detect O.viverrini infection in field-collected human feces. To detect O.viverrini infection in fecal samples, we developed a CRISPR/Cas12a (RNA-guided endonuclease) system combined with RPA (Ov-RPA-CRISPR/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR/Cas detection conditions, detection limits, and specificity of the RPA-CRISPR/Cas12a assay for detecting O.viverrini infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the Ov-RPA-CRISPR/Cas12a assay was evaluated on the basis of comparison with standard methods. The Ov-RPA-CRISPR/Cas12a assay exhibited high specificity for detecting O.viverrini DNA. On the basis of the detection limit, the assay could detect O.viverrini DNA at concentrations as low as 10-1ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1ng. To validate the Ov-RPA-CRISPR/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for O.viverrini-like eggs. Of these, 18 were confirmed as true positives on the basis of the Ov-RPA-CRISPR/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections. The Ov-RPA-CRISPR/Cas12a assay developed in this study can successfully detect O.viverrini infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm O.viverrini infection, marking an initial step in the development of point-of-care diagnosis.

Immunochromatographic Detection of SARS CoV-2 Antigen In Comparison With the Polymerase Chain Reaction for Laboratory Diagnosis of Covid-19 in Karachi, Pakistan

Introduction: The Covid-19 pandemic raised the question of which laboratory test was relevant for a rapid and accurate diagnosis of the disease. The current two methods for the detection of SARS CoV-2 are the ICT (Immunochromatographic Technique) and the PCR (Polymerase Chain Reaction) tests; therefore comparative analysis of test performance has to be done. Objective: To perform a comparative analysis of two different techniques (Immunochromatographic technique for Antigen and Polymerase Chain Reaction for RNA) which are implicated for the detection of SARS CoV-2. Materials &amp; Methods: The Rapid Antigen Test (RAT) and Polymerase Chain Reaction (PCR) were performed for the detection of Covid-19 on individuals having the signs and symptoms of SARS Covid-19. Immunochromatographic technique (ICT) was performed by SD Biosensor kits, while for Polymerase Chain Reaction RNA extraction was performed by Liferiver® auto extraction analyzer, and amplification was performed by SLAN®-48P Real-Time PCR System. Results: Among of 100 samples tested, 62 (23.56%) were positive on ICT while PCR had 66 (22.44%) and 38 were negative on ICT while PCR had 34. Thus the sensitivity of ICT was 90.77% (95% CI, 80.98% - 96.54%) and specificity was 91.43% (95% CI 76.94%-98.20%). The Positive Predictive Value was 95.16% while the Negative Predictive Value was 84.21%. However the Cohen’s Kappa Index Value was recorded as 0.806. Conclusion: The clinical performance of SD Biosensor Roche kits was excellent. On the basis of sensitivity and specificity it was concluded that RAT have comparable results with RT PCR. Keywords: Antigen, COVID-19, diagnosis, immunoassay, PCR, SARS-CoV-2.

Gene-Environment Interaction of Interleukin 10 Gene Polymorphism, rs1800896, with Lifestyle on Cardiovascular Risk in Type 2 Diabetes

Abstract Background: Gene–environment interactions play a major role in the phenotypic expression of complex disease traits such as those for cardiovascular diseases. Aims: This study aimed to determine the gene–environment interactions that underpin the relationship between interleukin-10 (IL-10) single-nucleotide polymorphism (1082 G/A [rs1800896]) with lifestyle on cardiovascular disease risk in adult Nigerians with type 2 diabetes mellitus (DM). Setting and Design: This case–control study involved patients with type 2 DM with high cardiovascular risk, determined by the Framingham’s classification, and age, sex, and diabetes-duration matched subjects with low and intermediate cardiovascular risks. Subjects and Methods: The genotypes were detected by polymerase chain reaction (PCR) followed by allelic discrimination using the Applied Biosystems 7900HT Fast Real-Time PCR System. One-way analysis of variance and logistic regression were employed for analysis. Odds ratios (ORs) with 95% confidence intervals (CI) were calculated to estimate the risk caused by the polymorphism. P &lt;0.05 was considered significant. Results: The odds for cardiovascular risk decreased progressively in individuals with the GG, GA, and AA genotypes (OR = 0.80, 95% CI = 0.49–1.28, P = 0.345 and OR = 0.46, 95% CI = 0.24–0.88, P = 0.018 for GA and AA genotypes, respectively. A significantly higher proportion of homozygous (AA) individuals were in the low cardiovascular risk group (54.2%, P = 0.018). Compared with the whole study population, individuals with the AA genotype had consistently lower odds for cardiovascular risk in subpopulations like alcohol users (OR = 0.25 [0.11–0.55], P = 0.001), but the odds were higher among smokers (OR = 1.80 [1.14–2.90], P = 0.017) and those with sedentary lifestyles (OR = 2.46, 95% CI = 1.14–5.33, P = 0.024). Conclusions: The homozygous mutant genotype AA of the IL-10 gene 1082 G/A had a protective effect on cardiovascular risk in type 2 DM. However, this protection was absent in those leading a sedentary lifestyle.

A-297 Usefulness of Non-Skin Samples in the PCR Diagnosis of Monkeypox

Abstract Background During 2022, human mpox cases were reported in several European countries. Until November 2022, in Spain, 7404 cases were detected, most of them linked to sexual intercourse. In that context, fast identification of cases and sources of infection were essential for an optimal management. Polymerase chain reaction (PCR) was the preferred laboratory test. Our objective was to evaluate the usefulness of sera and nasopharyngeal samples for MPXV detection, and its application in contact studies without skin or mucosal injuries. Methods In the Hospital Clínico Universitario of Zaragoza (Spain), 106 samples from 50 patients, 38 male and 12 female, aged between 11 to 62 years, with skin lesions suspected MPXV infection were analysed. Samples were collected between May and September 2022 in inactivating universal viral transport medium (Biocomma). Samples were obtained between 1 and 19 days of evolution. To perform the real-time PCR, the VIASURE Monkeypox Virus Real Time PCR Detection Kit and the VIASURE 48—Real Time PCR System were used. Nucleic acid extraction was performed with the automated nucleic acid extraction system MagLEAD12gC using the MagDEA®Dx SV reagents. Results Table 1 shows the results expressed in Ct values. Skin, anogenital and serum samples were the most efficient for MPXV diagnosis. Nevertheless, these data are influenced by the sample collection date. Conclusion Real-time PCR was positive in samples from all locations in 71,4% of the infected patients. Respiratory samples (pharyngeal and nasopharyngeal) were the ones that showed the highest number of false negative results, whereas there was only one skin sample and one serum sample with a negative result. This study shows that sera samples can be used in the diagnosis of MPXV infection because of ease of collection, applicability to contacts without lesions and the option of serology testing for other STI agents (HBV, HCV, HIV, Treponema pallidum…).

Quantification of Pork, Chicken, Beef, and Sheep Contents in Meat Products Using Duplex Real-Time PCR.

Accurate methods for meat speciation and quantification are essential for ensuring the supply of safe and wholesome meat and composite products with animal origins to negate the potential associated hazards, aid classification of consignments at the import control system, and thwart food fraud committed for financial gain. To better enhance meat safety control and combat food fraud, this study developed two duplex real-time polymerase chain reaction (real-time PCR) systems specifically designed for chicken, pork, sheep, and beef, using single-copy, chromosomally encoded, species-specific gene sequences to accurately measure the content of each meat type in meat products. DNA extracted from the raw and boiled reference materials prepared in varying proportions (ranging from 1% to 75%) were used in the development of the duplex assay to derive calibration factors to determine the meat content in different meat products. The method was further validated using proficiency test samples and market monitoring samples. Our findings showed that this method exhibits high specificity and sensitivity, with a significant accuracy range of 0.14% to 24.07% in quantifying the four meat types in both raw and processed meat products. Validation results further confirmed the effectiveness of our method in accurately quantifying meat content. Thus, we have demonstrated the duplex qPCR assays as promising approaches for implementation in routine analysis to strengthen meat safety control systems and combat meat fraud, thereby safeguarding consumer health and trust in the meat industry.

Low-Cost, Real-Time Polymerase Chain Reaction System with Integrated RNA Extraction.

Rapid, easy-to-use, and low-cost systems for biological sample testing are important for point-of-care diagnostics and various other health applications. The recent pandemic of Coronavirus Disease 2019 (COVID-19) caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) showed an urgent need to rapidly and accurately identify the genetic material of SARS-CoV-2, an enveloped ribonucleic acid (RNA) virus, in upper respiratory specimens from people. In general, sensitive testing methods require genetic material extraction from the specimen. Unfortunately, current commercially available extraction kits are expensive and involve time-consuming and laborious extraction procedures. To overcome the difficulties associated with common extraction methods, we propose a simple enzymatic assay for the nucleic acid extraction step using heat mediation to improve the polymerase chain reaction (PCR) reaction sensitivity. Our protocol was tested on Human Coronavirus 229E (HCoV-229E) as an example, which comes from the large coronaviridae family of viruses that affect birds, amphibians, and mammals, of which SARS-CoV-2 is a member. The proposed assay was performed using a low-cost, custom-made, real-time PCR system that incorporates thermal cycling and fluorescence detection. It had fully customizable reaction settings to allow versatile biological sample testing for various applications, including point-of-care medical diagnosis, food and water quality testing, and emergency health situations. Our results show that heat-mediated RNA extraction is a viable extraction method when compared to commercial extraction kits. Further, our study showed that extraction has a direct impact on purified laboratory samples of HCoV-229E, but no direct impact on infected human cells. This is clinically relevant, as it allows us to circumvent the extraction step on clinical samples when using PCR.

Rapid detection and diagnosis of herpetic keratitis using quantitative microfluidic polymerase chain reaction system for herpes simplex and varicella-zoster virus DNA: a case series

BackgroundA microfluidic real-time polymerase chain reaction (PCR) system can rapidly detect the viral DNA in specimens. Detection of herpes simplex virus (HSV) and varicella-zoster virus (VZV) DNA in tears is a useful diagnostic tool for herpes simplex virus keratitis (HSK) and herpes zoster ophthalmicus (HZO).MethodsIn total, 20 patients were included in this cross-sectional study. Among them, 8 patients with infectious epithelial HSK and 12 patients with HZO were included in HSK and HZO groups, respectively. In addition, 8 patients with non-herpetic keratitis and 4 healthy individuals without keratitis were included in the control group. Numbers of HSV and VZV DNA copies in tears of all patients and individuals were evaluated using a microfluidic real-time PCR system. Regarding HSV/VZV DNA test, tear specimens were collected by filter paper method using Schirmer’s test paper, and subsequently, DNA was extracted from the filter paper using an automated nucleic acid extractor. Afterward, quantitative PCR was performed using a microfluidic real-time PCR system.ResultsFrom tear collection to real-time PCR result determination, the HSV/VZV DNA test took approximately 40 min. In the HSK group, the sensitivity and specificity of the HSV DNA tests were 100% each. The median value (range) of number of HSV DNA copies for affected eyes was 3.4 × 105 copies/μL (under a lower detection limit of 7.6). In the HZO group, the sensitivity and specificity of the VZV DNA tests were 100% each. The median value (range) of number of VZV DNA copies for affected eyes was 5.3 × 105 copies/μL (under a lower detection limit of 5.6 × 10–2).ConclusionIn conclusion, quantitative PCR for HSV and VZV DNA in tears using a microfluidic real-time PCR system is useful for diagnosing and monitoring HSK and HZO.

LBA01-09 CLINICAL UTILITY OF POLYMERASE CHAIN REACTION IN EVALUATION OF PATIENTS WITH SYMPTOMATIC RECURRENT AND/OR COMPLICATED URINARY TRACT INFECTION

You have accessJournal of UrologyCME1 Apr 2023LBA01-09 CLINICAL UTILITY OF POLYMERASE CHAIN REACTION IN EVALUATION OF PATIENTS WITH SYMPTOMATIC RECURRENT AND/OR COMPLICATED URINARY TRACT INFECTION Deepak Kapoor, Mara Holton, Jason Hafron, Rima Aljundi, Bernadette Zwaans, and Mitchell Hollander Deepak KapoorDeepak Kapoor More articles by this author , Mara HoltonMara Holton More articles by this author , Jason HafronJason Hafron More articles by this author , Rima AljundiRima Aljundi More articles by this author , Bernadette ZwaansBernadette Zwaans More articles by this author , and Mitchell HollanderMitchell Hollander More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003360.09AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Patients with recurrent and/or complicated urinary tract infection (RCUTI) have higher risk of complications and present with broader microbial spectrum, and may present with polymicrobial UTI and/or fastidious organisms (pmUTI/FO). Urine culture (UC) has substantial false negative (neg) rates and poorly detects pmUTI/FO. Data suggests that polymerase chain reaction (PCR) enhances urinary pathogen detection and identification of pmUTI/FO. We sought to ascertain if there was a difference in overall pathogen and pmUTI/FO detection between UC and PCR in our patients seen with symptomatic RCUTI. METHODS: Our clinical guidelines recommend PCR testing only in RCUTI; in 2022 3,395 patients such patients who received concomitant UC and PCR were reviewed. Tests were performed by P4 Diagnostix (Pine Brook, NJ). UC plates were inspected after 18-42 hours incubation for colony growth, quantity, and morphology. DNA extraction and amplification for 36-organism PCR analysis was performed using KingFisher™ Flex and QuantStudio™ 12K Flex Real Time PCR System. For our RCUTI population positive (pos) UC was defined as single organism growth of>10k CFU/ml while pos PCR were organisms detected at a cycle threshold <29. Statistical analysis was performed using Chi square and two-way ANOVA test. This IRB-exempt study was conducted in accordance with the Declaration of Helsinki. RESULTS: Patient mean age was 69.6 yrs (range 6-99) with 1,808 males (53%) and 1,587 females (47%). Pathogen and pmUTI/FO detection was greater for PCR than UC (Table 1). 1490/1569 (95%) of pos UC had pos PCR; PCR revealed pmUTI/FO in 1003/1490 (67%) of these. PCR showed pathogens in 641/1826 (35%) of neg UC while UC revealed pathogens in 79/1264 (6%) of neg PCR (Δ 29%, p<.01); pmUTI/FO were seen in 501/641 (78%) in patients with pos PCR/neg UC. 33/79 (42%) of pos UC/neg PCR results were organisms not on the PCR panel. CONCLUSIONS: Organism detection was significantly greater for PCR than UC in patients with RCUTI. Detection of pmUTI/FO was augmented by PCR in both positive and negative UC but 33 potentially clinically relevant pathogens were outside even a 36 organism panel. UC and PCR provided complementary data that may help guide treatment of RCUTI and improve clinical outcomes for this subset of patients. Source of Funding: None © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1179 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Deepak Kapoor More articles by this author Mara Holton More articles by this author Jason Hafron More articles by this author Rima Aljundi More articles by this author Bernadette Zwaans More articles by this author Mitchell Hollander More articles by this author Expand All Advertisement PDF downloadLoading ...

Detection of extended-spectrum beta-lactamase and scarbapenemase-producing Enterobacteriaceae in Tunisia

The emergence of dramatic urinary tract infections (UTIs) caused by the members of the Enterobacteriales is an important public health problem in the community as well as in Tunisian hospitals. This study aims to investigate the prevalence of extended-spectrum β-lactamase (ESBL) and carbapenemase-producing uropathogenic isolates of Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae). Based on decreased susceptibility to β-lactams antibiotics and analyzed for the presence of ESBL and carbapenemase genes by Real Time- polymerase chain reaction (RT-PCR), 56 uropathogenic isolates of E. coli (n = 36) and K. pneumoniae (n = 20) were confirmed positive for ESBLs. The CTX-M-type β-lactamases were mostly detected in E. coli isolates (21 strains, 58.33% [95% CI 38.09% - 72.06%]) followed by blaSHV-like (18 strains, 50% [95% CI 32.92% - 67.07%]), blaTEM-like and blaCMY-2-like simultaneously (15 strains, 41.67% [95% CI 25.51% - 59.24%]). Furthermore, the RT-PCR system on the K. pneumoniae strains demonstrated that blaSHV-12-like was the most predominant (16 strains, 80% [95% CI 56.33% - 94.26%]) followed by blaTEM-like (14 strains, 70% [95% CI 45.72% - 88.10%]), blaCTX-M belonging to groups 9 and 1 (11 strains, 55% [95% CI 31.52% - 76.94%]) and finally blaCMY-2-like (10 strains, 50% [95% CI 27.19% - 72.80%]). In addition, E. coli and K. pneumoniae strains harbored a carbapenemase gene blaOXA-48-like with 22.2% [95% CI 10.11% - 39.15%]; 20% [95% CI 12.83% - 43.66%], respectively. Our results confirm the need to monitor the resistance to extended-spectrum β-lactams and to carbapenems among enterobacteria in Tunisia.

Rapid detection of the MYD88 L265P mutation for pre- and intra-operative diagnosis of primary central nervous system lymphoma.

The myeloid differentiation primary response gene 88 (MYD88) L265P mutation is a disease-specific mutation of primary central nervous system lymphoma (PCNSL) among the central nervous system tumors. Accordingly, this mutation is considered a reliable diagnostic molecular marker of PCNSL. As the intra-operative diagnosis of PCNSL is sometimes difficult to achieve using histological examinations alone, intra-operative detection of the MYD88 L265P mutation could be effective for the accurate diagnosis of PCNSL. Herein, we aimed to develop a novel rapid genotyping system (GeneSoC) using real-time polymerase chain reaction (PCR) based on microfluidic thermal cycling technology. This real-time PCR system shortened the analysis time, which enabled the detection of the MYD88 L265P mutation within 15 min. Rapid detection of the MYD88 L265P mutation was performed intra-operatively using GeneSoC in 24 consecutive cases with suspected malignant brain tumors, including 10 cases with suspected PCNSL before surgery. The MYD88 L265P mutation was detected in eight cases in which tumors were pathologically diagnosed as PCNSL after the operation, while wild-type MYD88 was detected in 16 cases. Although two of the 16 cases with wild-type MYD88 were pathologically diagnosed as PCNSL after the operation, MYD88 L265P could be detected in all eight PCNSL cases harboring MYD88 L265P. The MYD88 L265P mutation could also be detected using cell-free DNA derived from the cerebrospinal fluid of two PCNSL cases. Detection of the MYD88 L265P mutation using GeneSoC might not only improve the accuracy of intra-operative diagnosis of PCNSL but also help the future pre-operative diagnosis through liquid biopsy of cerebrospinal fluid.

Ultra-fast, sensitive and low-cost real-time PCR system for nucleic acid detection.

Nucleic acid detection directly identifies the presence of pathogenic microorganisms and has various advantages, such as high sensitivity, commendable specificity and a short window period, and has been widely used in many fields, such as early tumor screening, prenatal diagnosis and infectious disease detection. Real-time PCR (polymerase chain reaction) is the most commonly used method for nucleic acid detection in clinical practice, but it always takes about 1-3 hours, severely limiting its application in particular scenarios such as emergency testing, large-scale testing and on-site testing. To solve the time-consuming problem, a real-time PCR system based on multiple temperature zones was proposed, which realized the speed of temperature change of biological reagents from 2-4 °C s-1 to 13.33 °C s-1. The system integrates the advantages of fixed microchamber-type and microchannel-type amplification systems, including a microfluidic chip capable of fast heat transfer and a real-time PCR device with a temperature control strategy based on the temperature difference. The detection of HCMV biological samples using the real-time PCR system in this research took only 15 min, which was 75% shorter compared to the commercial qPCR instrument such as BIO-RAD, and the detection sensitivity remained essentially the same. The system could complete nucleic acid detection within 9 min under extreme conditions, characterized by fast detection speed and high sensitivity, providing a promising solution for ultra-fast nucleic acid detection.

Single nucleotide polymorphisms in odontogenesis-related genes associated with tooth-size discrepancy

Abstract Introduction: The present study aimed to determine the association between single nucleotide polymorphisms (SNPs) in RUNX2, SMAD6, BMP2, and BMP4 genes in relation to tooth-size discrepancy (TSD). Methods: A cross-sectional study of patients undergoing orthodontic treatment measured the mesiodistal width of permanent teeth from pretreatment dental casts. Sixty-two patients were included in the study and TSD was assessed according to the Bolton analysis. The patients were allocated into a control group (without a TSD), an anterior excess group and an overall excess group. Genomic DNA was extracted from saliva samples, and SNPs previously associated with tooth size were evaluated using a real-time polymerase chain reaction (PCR) system. The Fisher exact test was performed to compare genotype and allele frequencies at an α = 0.05. An Odds Ratio (OR) and 95% Confidence Interval (95% CI) were calculated. Results: The rs59983488 SNP in the RUNX2 gene was significantly related to the presence of anterior mandibular tooth-size excess in allele (T allele: p&lt;0.001; OR = 11.74; 95% CI =2.61–55.80), and genotype models (GT genotype: p = 0.002; OR = 12.69; 95% CI = 2.47–64.83). The rs3934908 SNP in the SMAD6 gene was significantly associated with the presence of an overall maxillary tooth-size excess in allele (T allele: p &lt; 0.001) and genotype models (TT genotype: p = 0.010). Conclusion: The present results suggest that SNPs in RUNX2 (rs59983488) and SMAD6 (rs3934908) genes may be associated with the presence of tooth-size excess.

Dearth of influenza among older adults admitted with respiratory symptoms in Malaysia during the coronavirus disease 2019 pandemic in 2021.

IntroductionInfluenza is a common respiratory virus which leads to over 400,000 annual deaths globally. Mortality from influenza is highest among those aged 75 years and over living in Africa and Southeast Asia.ObjectiveTo determine the burden of influenza among older adults presenting to public hospitals with severe acute respiratory infection (SARI) during the coronavirus disease 2019 (COVID-19) pandemic.MethodsThis multi-center, prospective, observational study recruited individuals aged 65 years and over who presented to four Malaysian hospitals with SARI from 1 January to 31 December 2021. Those with prior confirmed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection were excluded. SARS-CoV-2 was detected through real-time polymerase chain reaction (PCR) with routine diagnostic kits. Influenza A, influenza B and respiratory syncytial virus (RSV) viruses were detected with Xpress Flu/RSV kits using the GeneXpert rapid real-time PCR system (Cepheid, USA).ResultsSamples were obtained from 512 participants, comprising 296 (57.8%) men and 216 (42.2%) women, with a mean age (SD) of 74.0 (7.1) years. Inpatient death occurred in 48 (9.6%) individuals. Significant differences existed in age, ethnicity, and comorbidities across study sites. One (0.2%) case of influenza A, two (0.4%) cases of RSV and 63 (12.5%) cases of SARS-CoV-2 infection were detected over the 1-year period. Cases of COVID-19 mirrored national trends derived from open source data, while the dearth of influenza cases mirrored national and global Flunet figures.ConclusionOur observational study conducted during the COVID-19 pandemic detected only one case of influenza, alongside a high SARS-CoV-2 positivity rate. The poor uptake of influenza vaccination nationally, worsened by the recent pandemic restrictions, could lead to waning immunity from the absence of seasonal exposure. Potentially deadly outbreaks may then occur when lockdown and infection control measures are eventually removed.

Blood-Based Biomarkers in Afp Normal/Stable Hepatocellular Carcinoma: Diagnostic and Prognostic Relevance of Mir-10b for Patients on Liver Transplant List

As a diagnostic criteria of hepatocellular carcinoma (HCC), the exact threshold of alpha-fetoprotein (AFP) is controversial. In additional, not all HCC tumors are AFP positive or secrete elevated amounts of AFP into the serum. However, the diagnosis of HCC is quite important on the liver transplant list. Therefore, the purpose of this study was to investigate the expression of circulating micro RNAs (miRNAs) in AFP-stable HCC patients. Thus, we aimed to determine a diagnostic biomarker in these patients. Sixteen miRNAs were evaluated using a real-time quantitative reverse transcription polymerase chain reaction system in AFP-stable HCC and AFP-trending HCC patients. In our study, 46.7% (n=28) of the patients diagnosed with HCC had stable/normal AFP levels. We detected that high expression of miR-24, miR-10b and the low expression of miR-143 were independently and significantly associated with HCC in AFP-stable compared with AFP trending (P < .05). Additionally, we demonstrated that the overexpression of miR-10b was associated with poor disease-free survival in HCC (P=0.001). Although more clinical validations are needed for the diagnosis of HCC, our current results indicate that the coexistence of high expression of miR-10b and miR-24 may help clinicians adjust in the diagnosis of HCC in patients who are on the liver transplant list but awaiting biopsy for the diagnosis of HCC.

Aptamer-Based Triple Serum Fluorescence Intensity Assay: A Novel and Feasible Method for the Clinical Diagnosis of Primary Hepatic Carcinoma

Background and AimsAptamers are artificial ligands that bind to biological targets with high specificity and affinity. We previously selected a group of aptamers against the serum of primary hepatic carcinoma (PHC) via systematic evolution of ligands by exponential and enrichment (SELEX) method, and some of the aptamers were valuable for PHC diagnosis in polyacrylamide gel electrophoresis (PAGE) analysis. Here, we used aptamers to develop a novel method suitable for the clinical diagnosis of PHC.MethodsThe intensities of serum autofluorescence, cell-free DNA (cfDNA)-related fluorescence and aptamer-related fluorescence, named the aptamer-based triple serum fluorescence intensity (ATSFI), were sequentially measured at 8 °C and 37 °C in one tube by using a real-time polymerase chain reaction (PCR) system as a fluorimeter in patients with PHC (n=346) or liver cirrhosis (n=321). The diagnostic performances of ATSFI indicators alone and in combination were evaluated by area under the receiver operator characteristic curve (AUROC), and the underlying clinical mechanisms were analyzed by bivariate correlation.ResultsThe measurement of ATSFI was high throughput, rapid, convenient, and low cost. The aptamer-related fluorescence indicator SEA-SE37 was the most valuable for PHC diagnosis among all fluorescence indicators and superior to alpha-fetoprotein (AFP) (AUROC 0.879 vs. 0.836). The logistic model of ATSFI indicators exhibited excellent diagnostic performance for PHC, including AFP-negative, early and small PHCs, with AUROCs of 0.935-0.950 and accuracies of 86.8-88.3%. The diagnostic performance was further improved when ATSFI indicators were combined with AFP, with AUROCs of approximately 0.95 and accuracies of approximately 90%, suggesting ATSFI was independent of but complementary to AFP in PHC diagnosis. ATSFI models were highly valuable in clinical decision-making. The aptamer-related fluorescence intensity was generally independent of the clinicopathological characteristics of PHC but correlated with laboratory characteristics of PHC serum.ConclusionsThe ATSFI assay is a novel, robust and feasible method for the clinical diagnosis of PHC.

An Interdisciplinary Design Project for Undergraduate Engineering Training – Portable Real-Time Polymerase Chain Reaction System

Our world has been permanently changed by the pandemic outbreak of COVID-19 starts around the end of 2019. In the first few months of 2020, the whole world was in urgent need of an effective, easy, and quick method for the identification of the infection of the new virus. Polymerase Chain Reaction (PCR) machine, which can test DNA samples by rapidly making millions of copies of a specific DNA sample through the PCR process, including the COVID-19 virus, can perfectly fit this demand. In this study, a design project on PCR is introduced for undergraduate education in electrical and mechanical engineering. The objective of this project is to develop a low-cost, ease-of-use, wallet-size, portable real-time PCR (RT-PCR) machine for accurate testing of various bacteria or viruses. The key function of the PT-PCR system is to precisely control and maintain the temperature of the biosample solution within a range between 55°C and 95°C. This project provides students opportunities in studying and practicing a wide range of engineering technics and skills, including mechanical design, electronics design, microcomputer programming, data acquisition and processing, etc. Students can gain comprehensive understanding of the design of multiphysics system after they overcome various challenges emerging in the project. From the view of engineering education, the process of this project development has demonstrated the importance and benefits of adopting complex interdisciplinary engineering problems for student teams to solve, especially those involve contemporary issues.