Clinical Samples Research Articles

Antimicrobial resistance pattern of Klebsiella species isolated from various clinical specimens

Objective: The present study was carried out to identify Klebsiella species and their antimicrobial resistance pattern from various clinical specimens. Methods: This cross study was conducted at a tertiary care hospital, Dhaka, Bangladesh from July2020 to June 2021. Written consent was taken from the concerned authority. A total 6739 clinical samples including urine, wound swab/ pus, sputum, blood and endotracheal aspirates were collected and processed for isolation of bacteria by following standard guidelines from adult patients. All the urine, wound swab, blood and endotracheal aspirates were inoculated in both blood agar and MacConkey agar media, for blood samples blood culture was done in BacT/ALERT 3D 60 aerobic bottles irrespective to antibiotics administration. After incubation at 370C aerobically for 24 hours incubated plates were examined. Phenotypic identification of the organisms were done by observing colony morphology, hemolytic properties, pigment production, Gram staining and biochemical tests (oxidase test, catalase test and biochemical reactions after inoculation in TSI, MIU, citrate agar media). The Kirby - Bauer disk diffusion method was used for antimicrobial susceptibility testing of the isolated organism using Mueller-Hinton agar and commercially available antibiotic discs (Oxoid Ltd, UK). The data were analyzed using SPSS software Version-20 (SPSS Inc., Chicago, IL, USA). Categorical variables were expressed as numbers (n) and percentages (%). Results: A total 6739 samples were included in the present study. Of which, 170 were urine, 162 were wound swabs, 71 were sputum, 52 were endotracheal aspirates and 45 were blood samples. Culture positivity among the samples is shown in table I. Conclusion: Most of the Klebsiella species are highly resistant to commonly used antibiotics. This higher resistance is the major cause morbidity and mortality related with infections in hospitalized patients. So, a regular surveillance of antibiotic susceptibility pattern is needed to prevent indiscriminate use of antibiotics. CME J 2024; 3(2); 12-15

Novel CRISPR/Cas13-based assay for detection of bovine coronavirus associated with severe diarrhea in calves.

Bovine coronavirus (BCoV) is one of the important causes of diarrhoea in cattle. The virus is responsible for the high fatality rate associated with acute diarrhoea in calves. Rapid and accurate tests need to be conducted to detect the virus and minimise economic losses associated with the disease. Nucleic acid-based detection assays including PCR is an accurate test for detecting pathogens. However, these tests need skilled personnel, time and expensive devices. In this study, we developed a novel assay for the detection of BCoV in clinical cases. This novel assay combined reverse transcription-recombinase polymerase amplification with CRISPR/Cas13 and conducted a rapid visualisation of cleavage activity using a Lateral Flow Device. A conserved sequence of the BCV M gene was used as a target gene and the assays were tested in terms of specificity, sensitivity and time consumption. The result showed the specificity of the assay as 100% with no false positives being detected. Ten copies of the input RNA were enough to detect the virus and perform the assay. It took up to forty minutes for reading the results. Conducted together, the assay should be used as a rapid test to clinically diagnose infectious pathogens including bovine coronavirus. However, the assay needed the RNA to be extracted from the clinical sample in order to detect the virus. Therefore, more studies are needed to optimise the assay to be able to detect the virus in the clinical sample without extracting the RNA.

Investigation of SPOCD1 as A Suitable Diagnostic and Prognostic Biomarker in Various Common Cancer Types: Bioinformatics and Practical Analysis.

The objective of this study was to assess whether Spen paralogue and orthologue C-terminal domain containing 1 (SPOCD1) gene expression could serve as a valuable prognostic and diagnostic biomarker in common cancers, drawing from insights in recent literature. We sought to verify this concept by utilizing data sourced from The Cancer Genome Atlas (TCGA) alongside clinical samples. In this bioinformatics and experimental study, SPOCD1 RNA-seq data from 12 common cancers were collected from TCGA Pan-Cancer Atlas using the R package "TCGA BIOLINKS" and normalized for analysis. Various analytical tools, including receiver operating characteristic (ROC) curves, Kaplan-Meier and Coxregression analyses, and pathway enrichment analysis via the molecular signatures database (MSigDB), were applied. Drug resistance/sensitivity correlations with SPOCD1 expression were explored using the Gene Expression Omnibus (GEO) database. Clinical colorectal cancer (CRC) samples, including both colon and rectal malignant samples, were also evaluated. The results showed elevated SPOCD1 expression in most cancers (9/12), with notable prognostic value in COAD, HNSC, KICH, and LIHC, and a correlation with poor prognosis in COAD for disease-free survival. ROC curve analysis suggested SPOCD1 as a diagnostic biomarker in the majority of cases (7/12), although this pattern was inconsistent in clinical CRC samples. Pathway enrichment analysis revealed a strong correlation between SPOCD1 expression and critical molecular pathways. Unlike former results, we found that SPOCD1 upregulated when interacting with PD-0325901. However, treating with Panobinostat led to downregulation. Both are as anticancer reagents. This study confirms the potential of SPOCD1 as a diagnostic and prognostic biomarker in prevalent cancers. However, extensive clinical data, particularly for CRC, are required to validate its reliability. Different COAD subtypes may exhibit varying correlations with SPOCD1 expression levels, underscoring the need for further investigation to fully understand its diagnostic and prognostic value.

Repurposing Type I-A CRISPR-Cas3 for a robust diagnosis of human papillomavirus (HPV)

R-loop-triggered collateral single-stranded DNA (ssDNA) nuclease activity within Class 1 Type I CRISPR-Cas systems holds immense potential for nucleic acid detection. However, the hyperactive ssDNase activity of Cas3 introduces unwanted noise and false-positive results. In this study, we identified a novel Type I-A Cas3 variant derived from Thermococcus siculi, which remains in an auto-inhibited state until it is triggered by Cascade complex and R-loop formation. This Type I-A CRISPR-Cas3 system not only exhibits an expanded protospacer adjacent motif (PAM) recognition capability but also demonstrates remarkable intolerance towards mismatched sequences. Furthermore, it exhibits dual activation modes—responding to both DNA and RNA targets. The culmination of our research efforts has led to the development of the Hyper-Active-Verification Establishment (HAVE, 惠父). This innovation enables swift and precise human papillomavirus (HPV) diagnosis in clinical samples, providing a robust molecular diagnostic tool based on the Type I-A CRISPR-Cas3 system. Our findings contribute to understanding type I-A CRISPR-Cas3 system regulation and facilitate the creation of advanced diagnostic solutions with broad clinical applicability.

Evaluation of the analytical and clinical performance of the Abbott Alinity m instrument for the quantification of HIV-1 RNA plasma viral load in a reference laboratory in Rio de Janeiro, Brazil.

We sought to evaluate the analytical performance of the Alinity m system (Abbott Molecular) and to compare the clinical performance of HIV-1 assays on the Alinity m and m2000 RealTime platforms (Abbott Molecular). The sensitivity, precision, and accuracy of the Alinity m instrument were determined using a panel of standard samples (n = 46). The carryover effect was assessed by analyzing HIV-negative clinical samples (n = 20). Clinical performance of the Alinity m and m2000 RealTime platforms was compared using surplus HIV-positive patient plasma samples (n = 39). The Alinity m HIV-1 assay demonstrated 100% sensitivity, a high precision (coefficient of variation (s/x̄) × 100 ≤1.5% [SD ≤ 0.05] logarithm to base 10 [log10] copies/mL), and partial accuracy over the quantification range. Analysis of clinical samples suggested that the Alinity m HIV-1 assay does not cause carryover effect and produced a mean bias of 0.209 log10 copies/mL (95% CI, 0.153-0.265) compared with the m2000 RealTime System. The Alinity m instrument's performance correlated to that of the m2000 RealTime platform and showed excellent sensitivity, precision, and accuracy, despite producing overquantification not clinically relevant for disease management. Furthermore, use of the Alinity m platform can reduce turnaround time.

Examining the Stability and Predictive Utility of Across- and Within-Domain Intra-Individual Variability in Mild Cognitive Impairment.

Dispersion is a form of intra-individual variability across neuropsychological tests that has been shown to predict cognitive decline. However, few studies have investigated the stability and predictive utility of both across- and within-domain dispersion. The current study aims to fill these gaps in the literature by examining multiple indices of dispersion in a longitudinal clinical sample of individuals diagnosed with mild cognitive impairment (MCI) at baseline. Two hundred thirty-eight MCI patients from a cognitive disorders clinic underwent testing at baseline and after approximately 1.5 years. Linear regression was used to examine whether baseline across- and within-domain dispersion predicted cognitive decline in individuals whose diagnostic classification progressed to dementia (i.e., MCI-Decline) and those who retained an MCI diagnosis at follow-up (i.e., MCI-Stable). Cognitive decline was operationalized dichotomously using group status and continuously using standardized regression-based (SRB) z-scores. Dispersion variables at baseline and follow-up were positively correlated in both groups, with the exception of within-domain executive functioning and language dispersion in the MCI-Decline group. None of the dispersion variables predicted diagnostic conversion to MCI. Using SRB z-scores, greater across-domain dispersion predicted greater overall cognitive decline at follow-up, but this was not the case for within-domain variables with the exception of visuospatial skills. Results suggest that across- and within-domain dispersion are relatively stable across time, and that across-domain dispersion is predictive of subtle cognitive decline in patients with MCI. However, these results also highlight that findings may differ based on the tests included in dispersion calculations.

Clinical Evaluation of the Accuracy of the Panbio™ COVID-19/Flu A&B Rapid Panel: A Combination Antigen Rapid Diagnostic Test for the Omicron Variant and Influenza A Virus.

It is difficult to differentiate between coronavirus disease 2019 (COVID-19) and influenza based on the symptoms. In the present study, a newly developed antigen rapid diagnostic test (Ag-RDT) called Panbio™ COVID-19/Flu A&B that can simultaneously detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza A/B virus was evaluated. Its accuracy was evaluated using 235 pairs of nasopharyngeal samples collected from patients with respiratory symptoms and fever (>37.5°C). Reverse transcription polymerase chain reaction was used as a reference method to evaluate the accuracy of the SARS-CoV-2 detection. We confirmed the accuracy of the developed Ag-RDT against the Omicron variant where the sensitivity and specificity were 94.8% and 100%, respectively. In addition, to identify the influenza A virus, a noninferiority test was conducted using a commercial Ag-RDT, which has a sensitivity and specificity in comparison with viral culture of 94.8% and 98.4%, respectively. The positive and negative predictive values for influenza A virus were 98.5% and 98.1%, respectively, for the Panbio COVID-19/Flu A&B test. The evaluation of this newly developed Ag-RDT using clinical samples suggests that it has a high efficacy in clinical settings.

The Clinical Impact of Real-Time fMRI Neurofeedback on Emotion Regulation: A Systematic Review.

Emotion dysregulation has long been considered a key symptom in multiple psychiatric disorders. Difficulties in emotion regulation have been associated with neural dysregulation in fronto-limbic circuits. Real-time fMRI-based neurofeedback (rt-fMRI-NFB) has become increasingly popular as a potential treatment for emotional dysregulation in psychiatric disorders, as it is able to directly target the impaired neural circuits. However, the clinical impact of these rt-fMRI-NFB protocols in psychiatric populations is still largely unknown. Here we provide a comprehensive overview of primary studies from 2010 to 2023 that used rt-fMRI-NFB to target emotion regulation. We assessed 41 out of 4001 original studies for methodological quality and risk of bias and synthesised concerning the frequency of significant rt-fMRI-NFB-related effects on the neural and behaviour level. Successful modulation of brain activity was reported in between 25 and 50 percent of study samples, while neural effects in clinical samples were more diverse than in healthy samples. Interestingly, the frequency of rt-fMRI-NFB-related behavioural improvement was over 75 percent in clinical samples, while healthy samples showed behavioural improvements between 0 and 25 percent. Concerning clinical subsamples, rt-fMRI-NFB-related behavioural improvement was observed in up to 100 percent of major depressive disorder (MDD) and post-traumatic stress disorder (PTSD) samples. Substance use samples showed behavioural benefits ranging between 50 and 75 percent. Neural effects appeared to be less frequent than behavioural improvements: most neural outcomes ranged between 25 and 50 percent for MDD and substance use and between 0 and 25 percent for PTSD. Using multiple individualised regions of interest (ROIs) for rt-fMRI-NFB training resulted in more frequent behavioural benefits than rt-fMRI-NFB solely based on the amygdala or the prefrontal cortex. While a significant improvement in behavioural outcomes was reported in most clinical studies, the study protocols were heterogeneous, which limits the current evaluation of rt-fMRI-NFB as a putative treatment for emotional dysregulation.

Corynebacterium jeikeium from exudative pharyngitis: A close mimicker of Corynebacterium diphtheriae

Abstract Coryneforms other than Corynebacterium diphtheriae are part of bacterial flora of human skin and mucous membrane. Corynebacterium jeikeium is a rarely reported species from clinical samples, although reported from oral or pharyngeal samples of healthy individuals. Here, we report a case of exudative pharyngitis due to C. jeikeium.

Safety of embryo cryopreservation: insights from mid-term placental transcriptional changes

BackgroundIn recent years, with benefits from the continuous improvement of clinical technology and the advantage of fertility preservation, the application of embryo cryopreservation has been growing rapidly worldwide. However, amidst this growth, concerns about its safety persist. Numerous studies have highlighted the elevated risk of perinatal complications linked to frozen embryo transfer (FET), such as large for gestational age (LGA) and hypertensive disorders during pregnancy. Thus, it is imperative to explore the potential risk of embryo cryopreservation and its related mechanisms.MethodsGiven the strict ethical constraints on clinical samples, we employed mouse models in this study. Three experimental groups were established: the naturally conceived (NC) group, the fresh embryo transfer (Fresh-ET) group, and the FET group. Blastocyst formation rates and implantation rates were calculated post-embryo cryopreservation. The impact of FET on fetal growth was evaluated upon fetal and placental weight. Placental RNA-seq was conducted, encompassing comprehensive analyses of various comparisons (Fresh-ET vs. NC, FET vs. NC, and FET vs. Fresh-ET).ResultsReduced rates of blastocyst formation and implantation were observed post-embryo cryopreservation. Fresh-ET resulted in a significant decrease in fetal weight compared to NC group, whereas FET reversed this decline. RNA-seq analysis indicated that the majority of the expression changes in FET were inherited from Fresh-ET, and alterations solely attributed to embryo cryopreservation were moderate. Unexpectedly, certain genes that showed alterations in Fresh-ET tended to be restored in FET. Further analysis suggested that this regression may underlie the improvement of fetal growth restriction in FET. The expression of imprinted genes was disrupted in both FET and Fresh-ET groups.ConclusionBased on our experimental data on mouse models, the impact of embryo cryopreservation is less pronounced than other in vitro manipulations in Fresh-ET. However, the impairment of the embryonic developmental potential and the gene alterations in placenta still suggested it to be a risky operation.

Comprehensive genomics reveals novel sequence types of multidrug resistant Klebsiella oxytoca with uncharacterized capsular polysaccharide K- and lipopolysaccharide O-antigen loci from the National Hospital of Uganda

The Klebsiella oxytoca complex comprises diverse opportunistic bacterial pathogens associated with hospital and community-acquired infections with growing alarming antimicrobial resistance. We aimed to uncover the genomic features underlying the virulence and antimicrobial resistance of isolates from Mulago National Hospital in Uganda. We coupled whole genome sequencing with Pathogenwatch multilocus sequence typing (MLST) and downstream bioinformatic analysis to delineate sequence types (STs) capsular polysaccharide K- and O-antigen loci, along with antimicrobial resistance (AMR) profiles of eight clinical isolates from the National Referral Hospital of Uganda. Our findings revealed that only two isolates (RSM6774 and RSM7756) possess a known capsular polysaccharide K-locus (KL74). The rest carry various unknown K-loci (KL115, KL128, KLI52, KL161 and KLI63). We also found that two isolates possess unknown loci for the lipopolysaccharide O-antigen (O1/O2v1 type OL104 and unknown O1). The rest possess known O1 and O3 serotypes. From MLST, we found four novel sequence types (STs), carrying novel alleles for the housekeeping genes glyceraldehyde-6-phosphate dehydrogenase A (gapA), glucose-6-phosphate isomerase (pgi), and RNA polymerase subunit beta (rpoB). Our AMR analysis revealed that all the isolates are resistant to ampicillin and ceftriaxone, with varied resistance to other antibiotics, but all carry genes for extended-spectrum beta-lactamases (ESBLs). Notably, one strain (RSM7756) possesses outstanding chromosomal and plasmid-encoded AMR to beta-lactams, cephalosporins, fluoroquinolones and methoprims. Conclusively, clinical samples from Mulago National Referral Hospital harbor novel STs and multidrug resistant K. oxytoca strains, with significant public health importance, which could have been underrated.

The ANGPTL4-HIF-1α loop: a critical regulator of renal interstitial fibrosis

BackgroundRenal interstitial fibrosis (RIF) is a progressive, irreversible terminal kidney disease with a poor prognosis and high mortality. Angiopoietin-like 4 (ANGPTL4) is known to be associated with fibrosis in various organs, but its impact on the RIF process remains unclear. This study aimed to elucidate the role and underlying mechanisms of ANGPTL4 in the progression of RIF.MethodsIn vivo, a chronic kidney disease (CKD) rat model of renal interstitial fibrosis was established via intragastric administration of adenine at different time points (4 and 6 weeks). Blood and urine samples were collected to assess renal function and 24-h urinary protein levels. Kidney tissues were subjected to HE and Masson staining for pathological observation. Immunohistochemistry and real-time quantitative PCR (qRT‒PCR) were performed to evaluate the expression of ANGPTL4 and hypoxia-inducible factor-1α (HIF-1α), followed by Pearson correlation analysis. Subsequently, kidney biopsy tissues from 11 CKD patients (6 with RIF and 5 without RIF) were subjected to immunohistochemical staining to validate the expression of ANGPTL4. In vitro, a fibrosis model of human renal tubular epithelial cells (HK2) was established through hypoxic stimulation. Subsequently, an HIF-1α inhibitor (2-MeOE2) was used, and ANGPTL4 was manipulated using siRNA or plasmid overexpression. Changes in ANGPTL4 and fibrosis markers were analyzed through Western blotting, qRT‒PCR, and immunofluorescence.ResultsANGPTL4 was significantly upregulated in the CKD rat model and was significantly positively correlated with renal injury markers, the fibrotic area, and HIF-1α. These results were confirmed by clinical samples, which showed a significant increase in the expression level of ANGPTL4 in CKD patients with RIF, which was positively correlated with HIF-1α. Further in vitro studies indicated that the expression of ANGPTL4 is regulated by HIF-1α, which in turn is subject to negative feedback regulation by ANGPTL4. Moreover, modulation of ANGPTL4 expression influences the progression of fibrosis in HK2 cells.ConclusionOur findings indicate that ANGPTL4 is a key regulatory factor in renal fibrosis, forming a loop with HIF-1α, potentially serving as a novel therapeutic target for RIF.

Circ_0000069 promotes the development of hepatocellular carcinoma by regulating CCL25

BackgroundHepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths globally, influenced by aberrant circRNA expression. Investigating circRNA-miRNA-mRNA interactions can unveil underlying mechanisms of HCC and identify potential therapeutic targets.MethodsIn this study, we conducted differential analyses of mRNAs, miRNAs, and circRNAs, and established their relationships using various databases such as miRanda, miRDB, and miTarBase. Additionally, functional enrichment and immune infiltration analyses were performed to evaluate the roles of key genes. We also conducted qPCR assays and western blotting (WB) to examine the expression levels of circRNA, CCL25, and MAP2K1 in both HCC cells and clinical samples. Furthermore, we utilized overexpression and knockdown techniques for circ_0000069 and conducted wound healing, transwell invasion assays, and a tumorigenesis experiment to assess the migratory and invasive abilities of HCC cells.ResultsOur findings revealed significant differential expression of 612 upregulated genes and 1173 downregulated genes in HCC samples compared to normal liver tissue. Additionally, 429 upregulated circRNAs and 453 downregulated circRNAs were identified. Significantly, circ_0000069 exhibited upregulation in HCC tissues and cell lines. The overexpression of circ_0000069 notably increased the invasion and migration capacity of Huh7 cells, whereas the downregulation of circ_0000069 reduced this capability in HepG2 cells. Furthermore, this effect was counteracted by CCL25 silencing or overexpression, separately. Animal studies further confirmed that the overexpression of hsa_circ_0000069 facilitated tumor growth in xenografted nude mice, while the inhibition of CCL25 attenuated this effect.ConclusionCirc_0000069 appears to promote HCC progression by regulating CCL25, suggesting that both circ_0000069 and CCL25 can serve as potential therapeutic targets.

The influence of PER3 VNTR genotypes on the age of onset in a group of bipolar I disorder patients: an exploratory study

BackgroundPER3 is a circadian gene that contains a variable number of tandem repeats (VNTR) which codifies for three genotypes: 4/4; 4/5; and 5/5 and is involved in non-visual response to light, a critical process associated with bipolar disorder onset. Benedetti et al. (Neurosci Lett 445(2):184–7) related this VNTR with bipolar disorder age of onset and linked genotype 5/5 with an earlier onset. In this study, we aimed to investigate these associations of PER3 VNTR genotypes with age of onset in a homogenous sample of German patients with bipolar I disorder through Kaplan-Meier curves.Methods45 patients were enrolled and divided into three groups according to PER3 VNTR genotypes. Recognizing common biological features, we built a combined group of -5 allele carriers (4/5 + 5/5). As a primary outcome, Kaplan-Meier analysis was conducted to delineate the three genotypes’ influence on age of onset. The secondary Kaplan-Meier analysis aimed to evaluate the relation between the 4/4 homozygotes group and the combined group (4/5 + 5/5) with age of onset. Finally, we proceeded to compare groups through a Log Rank Test and performed an analysis of covariance (ANCOVA).ResultsThe Kaplan-Meier analysis with three separate genotypes didn’t replicate the findings of Benedetti’s study. The analysis comparing genotype 4/4 with the combined group showed the influence of PER3 VNTR variants on the age of onset and relates genotype 4/4 to an earlier onset. ANCOVA between the combined and the 4/4 genotype groups, correlated genotype 4/4 with an increased number of depressive episodes.ConclusionThis study showed no significant effect of PER3 VNTR genotypes on the age of onset and in linking genotype 5/5 with an earlier onset age. Contrasting results may arise from intrinsic differences between the two studies but also shed light on hypothetically different levels of functioning of PER3 VNTR genotypes in the context of bipolar pathology. Further studies will require bigger and more homogeneous clinical samples.

The expression and clinical significance of CFAP65 in colon cancer

BackgroundCFAP65 (cilia and flagella associated protein 65) is a fundamental protein in the development and formation of ciliated flagella, but few studies have focused on its role in cancer. This study aimed to investigate the prognostic significance of CFAP65 in colon cancer.MethodsThe functionally enriched genes related to CFAP65 were analyzed through the Gene Ontology (GO) database. Subsequently, CFAP65 expression levels in colon cancer were evaluated by reverse transcription and quantitative polymerase chain reaction (RT-qPCR) and immunoblotting in 20 pairs of frozen samples, including tumors and their matched paratumor tissue. Furthermore, protein expression of CFAP65 in 189 colon cancer patients were assessed via immunohistochemical staining. The correlations between CFAP65 expression and clinical features as well as long-term survival were statistically analyzed.ResultsCFAP65-related genes are significantly enriched on cellular processes of cell motility, ion channels, and GTPase-associated signaling. The expression of CFAP65 was significantly higher in colon cancer tissue compared to paratumor tissue. The proportion of high expression and low expression of CFAP65 in the clinical samples of colon cancer were 61.9% and 38.1%, respectively, and its expression level was not associated with the clinical parameters including gender, age, tumor location, histological differentiation, tumor stage, vascular invasion and mismatch repair deficiency. The five-year disease-free survival rate of the patients with CFAP65 low expression tumors was significantly lower than that those with high expression tumors (56.9% vs. 72.6%, P = 0.03), but the overall survival rate has no significant difference (69% vs. 78.6%, P = 0.171). The cox hazard regression analysis model showed that CFAP65 expression, tumor stage and tumor location were independent prognostic factors.ConclusionsIn conclusion, we demonstrate CFAP65 is a potential predictive marker for tumor progression in colon cancer.

Development and Evaluation of a Noninvasive Microfluidic-Based Paper Analytical Device for Leptospirosis Diagnosis.

Leptospirosis is a re-emerging infectious disease that presents a diagnostic enigma for clinicians with frequent misdiagnosis due to lack of rapid and accurate diagnostic tests, as the current methods are encumbered by inherent limitations. The development of a diagnostic sensor with a sample-in-result-out capability is pivotal for prompt diagnosis. Herein, we developed a microfluidic paper-based analytical device (spin-μPAD) featuring a sample-in-result-out fashion for the detection of Leptospira specific urinary biomarker, sph2 sphingomyelinase, crucial for noninvasive point-of-care testing. Fabrication of paper devices involved precise photolithography techniques, ensuring a high degree of reproducibility and replicability. By optimizing the device's configuration and protein components, a remarkable sensitivity and specificity was achieved for detecting leptospiral sph2 in urine, even at low concentrations down to 1.5 fg/mL, with an assay time of 15 min. Further, the spin-μPAD was validated with 20 clinical samples, suspected of leptospirosis including other febrile illnesses, and compared with gold standard microscopic agglutination test, culture, Lepto IgM ELISA, darkfield microscopy, and Leptocheck WB spot test. In contrast to commercial diagnostic tools, the spin-μPAD was noninvasive, rapid, easy to use, specific, sensitive, and cost-effective. The results highlight the potential of this innovative spin-μPAD for an efficient and dependable approach to noninvasive leptospirosis diagnosis, addressing critical needs in the realms of public health and clinical settings.

Associations between parental bonding and health-related quality of life in a clinical sample of youths aged 14-35 years in Singapore.

Individuals with psychiatric disorders tend to report having poorer bonds with their parents during their early years. These individuals often experience lower quality of life as well. This study investigated the associations between aspects of parental bonding and health-related quality of life (HRQOL) in a clinical sample of youths. It was hypothesised that high parental care and low parental overprotectiveness would be associated with higher levels of HRQOL. Data were obtained from a larger cross-sectional study. The sample consisted of 400 psychiatric outpatients: 191 patients aged 14-21 (mean ± standard deviation 18.1 ± 2.22) years and 209 patients aged 22-35 (28.0 ± 4.33) years. The Parental Bonding Instrument was used to measure parental care and overprotectiveness. Short Form-12 measured physical health (physical component summary [PCS]) and mental health (mental component summary [MCS]) components of HRQOL, and the 8-item Patient Health Questionnaire assessed depressive symptoms. These scales and a sociodemographic form were self-administered. Multivariable linear regression was used for analysis. About half of the sample reported affectionless control for mothers (46.6%) and fathers (45.9%). After controlling for sociodemographic variables, no significant relationship was found between aspects of parental bonding and PCS scores. Maternal care was associated with MCS scores (β = 0.32, P < 0.01) and PHQ-8 scores (β = -0.12, P < 0.05). Our results suggest that youths who experienced quality care from their mothers exhibit better mental health functioning despite their clinical diagnoses, which suggests that early maternal care exerts an overall long-term protective effect. Early parental education that promotes positive parenting practices could improve the overall HRQOL of individuals in adulthood despite their clinical diagnoses.

Multiplex Dual-Target Reverse Transcription PCR for Subtyping Avian Influenza A(H5) Virus.

An increased risk for human infection with avian influenza A(H5N1) viruses is of concern. We developed an internally controlled, dual-target reverse transcription PCR for influenza A(H5) subtyping. This test could be used to detect influenza A(H5) in clinical samples.

Occurrence of multi-carbapenemase-producing Enterobacterales in a tertiary hospital in Madrid (Spain): a new epidemiologic scenario

Introduction: Multi-carbapenemase-producing Enterobacterales (M-CPE) are increasingly described. We characterized the M-CPE isolates prospectively recovered in our hospital (Madrid, Spain) over two years (2021-2022). Methods: We collected 796 carbapenem resistant Enterobacterales (CRE) from clinical and surveillance samples. Carbapenemase production was confirmed with phenotypic (immunochromatographic, disk diffusion) and molecular (PCR, WGS) techniques. Antimicrobial susceptibility was evaluated by a standard broth microdilution method. Clinical and demographic data were collected. Results: Overall, 23 M-CPE (10 Klebsiella pneumoniae, 6 Citrobacter freundii complex, 3 Escherichia coli, 2 Klebsiella oxytoca, and 2 Enterobacter hormaechei) isolates were recovered from 17 patients (3% with CPE, 0.27 cases per 1000 admissions). OXA-48+KPC-3 (7/23) and KPC-3+VIM-1 (5/23) were the most frequent carbapenemase combinations. All patients had prior antibiotics exposure, including carbapenems (8/17). High resistance rates to ceftazidime/avibactam (14/23), imipenem/relebactam (16/23) and meropenem/vaborbactam (7/23) were found. Ceftazidime/avibactam+aztreonam combination was synergistic in all metallo-β-lactamase producers. Clonal and non-clonal related isolates were found, particularly in K. pneumoniae (5 ST29, 3 ST147, 3 ST307) and C. freundii (3 ST8, 2 ST125, 1 ST563). NDM-1+OXA-48 was introduced with the ST147-K. pneumoniae high-risk clone linked to the transfer of an Ukrainian patient. We identified four possible nosocomial clonal transmission events between patients of the same clone with the same combination of carbapenemases (KPC-3+VIM-1-ST29-K. pneumoniae, NDM-1+OXA-48-ST147-K. pneumoniae and KPC-2+VIM-1-ST145-K. oxytoca). Carbapenemase-encoding genes were located on different plasmids, except for VIM-1+KPC-2-ST145-K. oxytoca. Cross-species transmission and a possible acquisition overtime was found, particularly between K. pneumoniae and E. coli producing OXA-48+KPC-3. Conclusion: M-CPE is an emerging threat in our hospital. Co-production of different carbapenemases, including metallo-β-lactamases, limits therapeutic options and depicts the need to reinforce infection control measures.

Identification and bioinformatic characterization of a serum miRNA signature for early detection of laryngeal squamous cell carcinoma

BackgroundThe growing understanding of cancer biology and the establishment of new treatment modalities has not yielded the expected results in terms of survival for Laryngeal Squamous Cell Cancer (LSCC). Early diagnosis, as well as prompt identification of patients with high risk of relapse would ensure greater chance of therapeutic success. However, this goal remains a challenge due to the absence of specific biomarkers for this neoplasm.MethodsSerum samples from 45 LSCC patients and 23 healthy donors were collected for miRNA expression profiling by TaqMan Array analysis. Additional 20 patients and 42 healthy volunteers were included for the validation set, reaching an equal number of clinical samples for each group. The potential diagnostic ability of the such identified three-miRNA signature was confirmed by ROC analysis. Moreover, each miRNA was analyzed for the possible correlation with HNSCC patients’ survival and TNM status by online databases Kaplan–Meier (KM) plotter and OncomiR. In silico analysis of common candidate targets and their network relevance to predict shared biological functions was finally performed by PANTHER and GeneMANIA software.ResultsWe characterized serum miRNA profile of LSCC patients identifying a novel molecular signature, including miR-223, miR-93 and miR-532, as circulating marker endowed with high selectivity and specificity. The oncogenic effect and the prognostic significance of each miRNA was investigated by bioinformatic analysis, denoting significant correlation with OS. To analyse the molecular basis underlying the pro-tumorigenic role of the signature, we focused on the simultaneously regulated gene targets—IL6ST, GTDC1, MAP1B, CPEB3, PRKACB, NFIB, PURB, ATP2B1, ZNF148, PSD3, TBC1D15, PURA, KLF12—found by prediction tools and deepened for their functional role by pathway enrichment analysis. The results showed the involvement of 7 different biological processes, among which inflammation, proliferation, migration, apoptosis and angiogenesis.ConclusionsIn conclusion, we have identified a possible miRNA signature for early LSCC diagnosis and we assumed that miR-93, miR-223 and miR-532 could orchestrate the regulation of multiple cancer-related processes. These findings encourage the possibility to deepen the molecular mechanisms underlying their oncogenic role, for the desirable development of novel therapeutic opportunities based on the use of short single-stranded oligonucleotides acting as non-coding RNA antagonists in cancer.