Analysis of Cytokine-Inducible SH2-Containing Protein (CISH) in the Study of Immunosenescence.

The aging immune system undergoes significant changes, leading to a state known as immunosenescence. Understanding the molecular mechanisms underlying immunosenescence is crucial for developing targeted interventions to enhance immune functions in older individuals. This bio-protocol review focuses on the application of quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for the mRNA quantification of cytokine-inducible SH2-containing protein (CISH), an immune regulator overexpressed in T-cell responses from older adults. We outline a comprehensive protocol for the quantitative assessment of CISH mRNA expression, providing a valuable tool for researchers investigating immunosenescence.

Validation of Golden Gate Assemblies Using Highly Multiplexed Nanopore Amplicon Sequencing.

Golden Gate cloning has revolutionized synthetic biology. Its concept of modular, highly characterized libraries of parts that can be combined into higher order assemblies allows engineering principles to be applied to biological systems. The basic parts, typically stored in Level 0 plasmids, are sequence validated by the method of choice and can be combined into higher order assemblies on demand. Higher order assemblies are typically transcriptional units, and multiple transcriptional units can be assembled into multi-gene constructs. Higher order Golden Gate assembly based on defined and validated parts usually does not introduce sequence changes. Therefore, simple validation of the assemblies, e.g., by colony polymerase chain reaction (PCR) or restriction digest pattern analysis is sufficient. However, in many experimental setups, researchers do not use defined parts, but rather part libraries, resulting in assemblies of high combinatorial complexity where sequencing again becomes mandatory. Here, we present a detailed protocol for the use of a highly multiplexed dual barcode amplicon sequencing using the Nanopore sequencing platform for in-house sequence validation. The workflow, called DuBA.flow, is a start-to-finish procedure that provides all necessary steps from a single colony to the final easy-to-interpret sequencing report.

Biofoundry-Assisted Golden Gate Cloning with AssemblyTron.

Golden Gate assembly is a requisite method in synthetic biology that facilitates critical conventions such as genetic part abstraction and rapid prototyping. However, compared to robotic implementation, manual Golden Gate implementation is cumbersome, error-prone, and inconsistent for complex assembly designs. AssemblyTron is an open-source python package that provides an affordable automation solution using open-source OpenTrons OT-2 lab robots. Automating Golden Gate assembly with AssemblyTron can reduce failure-rate, resource consumption, and training requirements for building complex DNA constructs, as well as indexed and combinatorial libraries. Here, we dissect a panel of upgrades to AssemblyTron's Golden Gate assembly capabilities, which include Golden Gate assembly into modular cloning part vectors, error-prone polymerase chain reaction (PCR) combinatorial mutant library assembly, and modular cloning indexed plasmid library assembly. These upgrades enable a broad pool of users with varying levels of experience to readily implement advanced Golden Gate applications using low-cost, open-source lab robotics.

The Evolution of Nucleic Acid-Based Diagnosis Methods from the (pre-)CRISPR to CRISPR era and the Associated Machine/Deep Learning Approaches in Relevant RNA Design.

Nucleic acid tests (NATs) are considered as gold standard in molecular diagnosis. To meet the demand for onsite, point-of-care, specific and sensitive, trace and genotype detection of pathogens and pathogenic variants, various types of NATs have been developed since the discovery of PCR. As alternatives to traditional NATs (e.g., PCR), isothermal nucleic acid amplification techniques (INAATs) such as LAMP, RPA, SDA, HDR, NASBA, and HCA were invented gradually. PCR and most of these techniques highly depend on efficient and optimal primer and probe design to deliver accurate and specific results. This chapter starts with a discussion of traditional NATs and INAATs in concert with the description of computational tools available to aid the process of primer/probe design for NATs and INAATs. Besides briefly covering nanoparticles-assisted NATs, a more comprehensive presentation is given on the role CRISPR-based technologies have played in molecular diagnosis. Here we provide examples of a few groundbreaking CRISPR assays that have been developed to counter epidemics and pandemics and outline CRISPR biology, highlighting the role of CRISPR guide RNA and its design in any successful CRISPR-based application. In this respect, we tabularize computational tools that are available to aid the design of guide RNAs in CRISPR-based applications. In the second part of our chapter, we discuss machine learning (ML)- and deep learning (DL)-based computational approaches that facilitate the design of efficient primer and probe for NATs/INAATs and guide RNAs for CRISPR-based applications. Given the role of microRNA (miRNAs) as potential future biomarkers of disease diagnosis, we have also discussed ML/DL-based computational approaches for miRNA-target predictions. Our chapter presents the evolution of nucleic acid-based diagnosis techniques from PCR and INAATs to more advanced CRISPR/Cas-based methodologies in concert with the evolution of deep learning (DL)- and machine learning (ml)-based computational tools in the most relevant application domains.

Prevalência de Influenza A, vírus sincicial respiratório eSARS-CoV-2 em pacientes com síndrome respiratóriaaguda grave em Passo Fundo, Rio Grande do Sul / Prevalence of Influenza A, respiratory syncytial virus andSARS-CoV-2 in patients with severe acute respiratorysyndrome in Passo Fundo, Rio Grande do Sul

A síndrome respiratória aguda grave (SRAG) é caracterizada por sintomas de febre alta, tosse e dispneia, e, na maioria dos casos, relacionada a uma quantidade reduzida de agentes infecciosos. O objetivo foi avaliar a prevalência dos vírus respiratórios Influenza A (FluA), vírus sincicial respiratório (RSV) e do novo coronavírus (SARS-CoV-2) em pacientes com internação hospitalar por SRAG. Estudo transversal, com pacientes em internação hospitalar com SRAG entre novembro de 2021 e maio de 2022. Dados sociodemográficos e clínicos e amostras da nasofaringe foram coletados/as, as quais foram submetidas à extração de RNA e testadas quanto à positividade para Influenza A, RSV e SARS-CoV-2 por meio da técnica de PCR em tempo real pelo método SYBR Green. Foram incluídos 42 pacientes, sendo 59,5% do sexo feminino, 57,1% idosos, 54,8% com ensino fundamental. A maior parte dos pacientes reportou hábito tabagista prévio ou atual (54,8%), não etilista (73,8%) e 83,3% deles apresentavam alguma comorbidade, sendo hipertensão arterial sistêmica e diabetes mellitus tipo 2 as mais prevalentes. Um total de 10,5% dos pacientes testou positivo para FluA, nenhuma amostra positiva para RSV e 76,3% positivos para SARS-CoV-2. Na população estudada, SRAG com agravo hospitalar foi observado em maior proporção, em mulheres, idosos e pessoas com comorbidades, embora sem significância estatística, sendo o novo coronavírus o agente etiológico mais relacionado, o que evidencia a patogenicidade desse agente e suas consequências ainda são evidentes após quase 2 anos de período pandêmico.

Severe acute respiratory syndrome (SARS) is characterized by symptoms of high fever, cough and dyspnea, and is in most cases related to a reduced amount of infectious agents. The objective was to assess the prevalence of respiratory viruses Influenza A (FluA), respiratory syncytial virus (RSV) and the new coronavirus (SARS-CoV-2) in patients hospitalized for SARS. Cross-sectional study, with patients hospitalized with SARS between November 2021 and May 2022. Sociodemographic and clinical data and nasopharyngeal samples were collected, which were subjected to RNA extraction and tested for positivity for Influenza A, RSV and SARS-CoV-2 using the real-time PCR technique using the SYBR Green method. 42 patients were included, 59.5% female, 57.1% elderly, 54.8% with primary education. Most patients reported previous or current smoking habits (54.8%), non-drinkers (73.8) and 83.3% of them had some comorbidity, with systemic arterial hypertension and type 2 diabetes mellitus being the most prevalent. A total of 10.5% of patients tested positive for FluA, no samples positive for RSV, and 76.3% positive for SARS-CoV-2. In the studied population, SARS with hospital injury was observed more frequently in women and the elderly, with associated comorbidities, with the new coronavirus being the most related etiological agent, which shows, although not statistically significant, that the pathogenicity of this agent and its consequences are still evident after almost 2 years of period pandemic.

Parasitological and molecular investigation of Trypanosoma evansi in dromedaries from Greater Cairo, Egypt.

In Egypt, camel trypanosomiasis is widespread. From October 2021 to March 2022, we collected 181 blood samples from apparently healthy one-humped camels (Camelus dromedarius) in Cairo and Giza Governates. The objective of this study was to assess infection rates of trypanosomes using blood smear examination and PCR-sequencing assays. Trypanosomes were detected in 8.3% (15/181) of camels by blood smear and in 23.8% (43/181) by PCR targeting the internal transcribed spacer (ITS). Based on blood smear and ITS-PCR results, and the absence of tsetse flies in the study area, we hypothesized that the Trypanosoma species was likely T. evansi. Validation using PCR based on the variant surface glycoprotein (VSG) of T. evansi Rode Trypanozoon antigen type (RoTat) 1.2 (RoTat 1.2 VSG gene) on ITS-PCR-positive samples (n=43) confirmed that 88.4% (38/43) were RoTat 1.2 T. evansi, while 11.6% (5/43) were non-RoTat 1.2 T. evansi. This marks the second report of non-RoTat 1.2 T. evansi in dromedary camels in Egypt. Considering the underestimated zoonotic risk of T. evansi in Egypt, there is a potential threat to humans, underscoring the need for a "One Health" approach to safeguard animal and human health.

Clinical evaluation of a multiplex PCR-based test for joint infection: a prospective diagnostic accuracy study of forty-nine patients.

PURPOSE: The purpose of this study was to evaluate the diagnostic accuracy (sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV)) of the PCR-based BioFire® Joint Infection Panel (BJI Panel) against microbiological culture growth for patients suspected of having a native or prosthetic joint infection. METHODS: Synovial fluid and tissue biopsies were prospectively collected from patients from June 2022 to June 2023. The results of the BJI Panel were compared with those of culture growth. RESULTS: 51 samples were included. Including all pathogens, the sensitivity was 69%, the specificity 89%, the PPV 73% and the NPV 86%. Including only pathogens in the BJI Panel, the sensitivity was 100%, the specificity 90%, the PPV 73% and the NPV 100%. CONCLUSION: The BJI Panel has a high accuracy for detecting the pathogens in its panel, but the absence of important common pathogens from the panel reduces its sensitivity and NPV. With a short turnaround time and precise pathogen detection, the BJI Panel has the potential to add value as a complementary diagnostic method.

The incidence of donor white blood cell survival (transfusion-associated microchimerism) in Australian pediatric patients.

INTRODUCTION: Donor leucocyte survival following red blood cell (RBC) transfusion, known as transfusion-associated microchimerism (TAM), can occur in some patients. In Australia, despite the introduction of leucocyte filtration (leucodepletion) during RBC manufacture, TAM has been detected in adult trauma patients. However, the incidence of TAM in Australian pediatric patients has not been analyzed. METHODS: Patients aged 0-16 years were recruited across two cohorts. Retrospective participants had RBC transfusion between January 1, 2002 and November 15, 2017 and prospective participants received RBC transfusion between December 1, 2016 and November 25, 2020. Twelve bi-allelic insertion/deletion (InDel) polymorphisms were used to detect microchimerism amplification patterns using real-time PCR (RT-PCR) and droplet digital PCR (ddPCR). RESULTS: Of the retrospective cohort (n = 40), six patients showed amplification of InDel sequences indicating potential microchimerism. For three patients, minor InDel sequences were detected using RT-PCR only, two patients had minor InDel amplification using ddPCR only, and one patient had minor InDel amplification that was confirmed using both techniques. Amplification of minor sequences occurred in three patients who had received a bone marrow transplant in addition to RBC transfusion. In the prospective cohort (n = 25), no InDel amplification indicating potential microchimerism was detected using RT-PCR. DISCUSSION: Cell-based therapies had been administered in three patients where microchimerism amplification patterns were detected. Three patients have microchimerism that may be attributed to RBC transfusion. In prospective patients, who received leucodepleted and gamma-irradiated RBC units, no potential microchimerism amplification were detected. ddPCR may be a suitable technique for TAM analysis but requires further evaluation.

Multiplex molecular assays for the laboratory-based and point-of-care diagnosis of infections caused by seasonal influenza, COVID-19, and RSV.

INTRODUCTION: SARS-CoV-2, seasonal influenza, and respiratory syncytial virus (RSV) are major causes of acute respiratory infections in all age groups and responsible for an enormous socio-economic burden. The recently coined term 'tripledemic' describes co-circulation of these three viruses, a novel epidemiological paradigm that poses profound public health implications. AREAS COVERED: Real-time reverse transcription polymerase chain reaction (RT-PCR) is now considered the reference method for the diagnosis of SARS-CoV-2, influenza, and RSV infections. Syndromic-based multiplex RT-PCR panels that simultaneously detect several respiratory viruses have become increasingly common. This review explores available molecular diagnostics (MDx) platforms for the diagnosis of SARS-CoV-2, influenza, and RSV in the same biological sample. Within some limitations of the published validation and diagnostic accuracy studies, both laboratory-based and point-of-care multiplex panels proved highly performant in identifying SARS-CoV-2, influenza A, influenza B, and RSV. Improved operational efficiency and faster turnaround times make these assays potentially cost-effective or even cost-saving. EXPERT OPINION: The adoption of multiplex MDx assays for the contemporary detection of SARS-CoV-2, influenza, RSV, and other respiratory pathogens will likely increase in the next few years. To maximize the clinical usefulness and cost-effectiveness of these assays, locally issued guidelines and protocols on their implementation should be adopted.

Stability assessment of housekeeping genes for qRT-PCR in Yersinia enterocolitica cultured at 22°C and 37°C.

Yersinia enterocolitica, a species within the genus Yersinia, thrives optimally at 22-25°C but can also grow at the mammalian core body temperature of 37°C. This dual temperature adaptability necessitates establishing both temperature conditions in research to examine the effects on various biological processes. In quantitative real-time PCR (qRT-PCR) assays, the selection of appropriate housekeeping genes is vital for data accuracy. Nevertheless, the lack of alternatives and information often leads to the default use of the 16S rRNA gene despite potential limitations. This investigation sourced 16 potential reference genes through a comprehensive review of the literature and transcriptome sequencing data analysis. We validated the expression stability of these genes via qRT-PCR across 12 Y. enterocolitica strains, representing the four prevalent serotypes O:3, O:5,27, O:8, and O:9, isolated from diarrheal patient stool samples. This approach aimed to minimize the impact of serotype heterogeneity. After acquiring Cq values, gene stability was evaluated using four established algorithms-ΔCq, geNorm, NormFinder, and BestKeeper-and subsequently synthesized into a consolidated ranking through the Robust Rank Aggregation (RRA) method. Our study suggests that the genes glnS, nuoB, glmS, gyrB, dnaK, and thrS maintain consistent expression across varying culture temperatures, supporting their candidacy as robust housekeeping genes. We advise against the exclusive use of 16S rRNA for this purpose. Should tradition prevail in its utilization, it must be employed with discernment, preferably alongside one or two of the housekeeping genes identified in this study as internal controls.IMPORTANCEIn our study, we focused on identifying stable reference genes for quantitative real-time PCR (qRT-PCR) experiments on Y. enterocolitica cultured at different temperatures (22°C and 37°C). After thoroughly evaluating 16 candidate genes, we identified six genes-glnS, nuoB, glmS, gyrB, dnaK, and thrS-as exhibiting stable expression across these temperature conditions, making them ideal reference genes for Y. enterocolitica studies. This discovery is crucial for ensuring the accuracy and reliability of qRT-PCR data, as the choice of appropriate reference genes is key to normalizing expression data and minimizing experimental variability. Importantly, our research extended beyond bioinformatics analysis by incorporating validation with clinical strains, bridging the gap between theoretical predictions and practical application. This approach not only underscores the robustness and reliability of our findings but also directly addresses the critical need for experimental validation in the field. By providing a set of validated, stably expressed reference genes, our work offers valuable guidance for designing experiments involving Y. enterocolitica, enhancing the reliability of research outcomes, and advancing our understanding of this significant pathogen.

Frequency and genotyping of group A rotavirus among Egyptian children with acute gastroenteritis: a hospital-based cross-sectional study.

BACKGROUND: This hospital-based cross-sectional study aims to investigate the epidemiologic and clinical characteristics of rotavirus group A (RVA) infection among children with acute gastroenteritis and to detect the most common G and P genotypes in Egypt. METHODS: A total of 92 stool samples were collected from children under five who were diagnosed with acute gastroenteritis. RVA in stool samples was identified using ELISA and nested RT-PCR. Common G and P genotypes were identified utilizing multiplex nested RT-PCR assays. RESULTS: RVA was detected at a rate of 24% (22 /92) using ELISA and 26.1% (24 /92) using VP6 nested RT-PCR. The ELISA test demonstrated diagnostic sensitivity, specificity, and accuracy of 91.7%, 100%, and 97.8%, respectively. G3 was the most prevalent G type (37.5%), followed by G1 (12.5%), whereas the most commonly detected P type were P[8] (41.7%) and P[6] (8.2%). RVA-positive samples were significantly associated with younger aged children (p = 0.026), and bottle-fed (p = 0.033) children. In addition, RVA-positive samples were more common during cooler seasons (p = 0.0001). Children with rotaviral gastroenteritis had significantly more frequent episodes of diarrhea (10.87 ± 3.63 times/day) and vomiting (8.79 ± 3.57 times/day) per day (p = 0.013 and p = 0.011, respectively). Moreover, they had a more severe Vesikari clinical score (p = 0.049). CONCLUSION: RVA is a prevalent cause of acute gastroenteritis among Egyptian children in our locality. The discovery of various RVA genotypes in the local population, as well as the identification of common G and P untypeable strains, highlights the significance of implementing the rotavirus vaccine in Egyptian national immunization programs accompanied by continuous monitoring of strains.

Optimization and application of digital droplet PCR for the detection of SARS-CoV-2 in saliva specimen using commercially available kit.

The coronavirus disease-19 pandemic has resulted in a significant global health crisis, causing hundreds of millions of cases and millions of deaths. Despite being declared endemic, SARS-CoV-2 infection continues to pose a significant risk, particularly for immunocompromised individuals, highlighting the need for a more sensitive and specific detection. Reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) possesses a sensitive and absolute quantification compared to the gold standard. This study is the first to optimize RT-ddPCR for detecting SARS-CoV-2 in saliva specimens using a commercially available RT-qPCR kit. Optimization involved the assessment of the RT-ddPCR reaction mixture, annealing temperature adjustments, and validation using 40 stored saliva specimens. RT-qPCR was used as a reference method in this study. Compatibility assessment revealed that ddPCR Supermix for Probes (no dUTP) was preferable with an optimal annealing temperature of 57.6°C. Although a 25% higher primer/probe concentration provides a higher amplitude in droplet separation of positive control, the number of copy numbers decreased. An inverse correlation between Ct value and copy number concentration was displayed, presenting that the lower the Ct value, the higher the concentration, for the N and E genes with r2 values of 0.98 and 0.85, respectively. However, ORF1ab was poorly correlated (r2 of 0.34). The sensitivity of targeted and E genes was 100% and 93.3%, respectively; as for the specificity, the percentage ranged from 80.8% to 91.3%. This study implicates the applicability of a modified method in the ddPCR platform for similar types of pathogens using saliva specimens.

Elucidation of the mechanisms of fluconazole resistance and repurposing treatment options against urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt.

BACKGROUND: The incidence of fungal urinary tract infections (UTIs) has dramatically increased in the past decades, with Candida arising as the predominant etiological agent. Managing these infections poses a serious challenge to clinicians, especially with the emergence of fluconazole-resistant (FLC-R) Candida species. In this study, we aimed to determine the mechanisms of fluconazole resistance in urinary Candida spp. isolated from hospitalized patients in Alexandria, Egypt, assess the correlation between fluconazole resistance and virulence, and explore potential treatment options for UTIs caused by FLC-R Candida strains. RESULTS: Fluconazole susceptibility testing of 34 urinary Candida isolates indicated that 76.5% were FLC-R, with a higher prevalence of resistance recorded in non-albicans Candida spp. (88.9%) than in Candida albicans (62.5%). The calculated Spearman's correlation coefficients implied significant positive correlations between fluconazole minimum inhibitory concentrations and both biofilm formation and phospholipase production. Real-time PCR results revealed that most FLC-R isolates (60%) significantly overexpressed at least one efflux pump gene, while 42.3% significantly upregulated the ERG11 gene. The most prevalent mutation detected upon ERG11 sequencing was G464S, which is conclusively linked to fluconazole resistance. The five repurposed agents: amikacin, colistin, dexamethasone, ketorolac, and sulfamethoxazole demonstrated variable fluconazole-sensitizing activities in vitro, with amikacin, dexamethasone, and colistin being the most effective. However, the fluconazole/colistin combination produced a notable reduction (49.1%) in bladder bioburden, a 50% decrease in the inflammatory response, and tripled the median survival span relative to the untreated murine models. CONCLUSIONS: The fluconazole/colistin combination offers a promising treatment option for UTIs caused by FLC-R Candida, providing an alternative to the high-cost, tedious process of novel antifungal drug discovery in the battle against antifungal resistance.

Sequencing vs. amplification for the estimation of allele dosages in sugarcane (Saccharum spp.).

Premise: Detecting single-nucleotide polymorphisms (SNPs) in a cost-effective way is fundamental in any plant breeding pipeline. Here, we compare three genotyping techniques for their ability to reproduce the allele dosage of SNPs of interest in sugarcane (Saccharum spp.). Methods: To identify a reproducible technique to estimate allele dosage for the validation of SNP markers, the correlation between Flex-Seq, kompetitive allele-specific PCR (KASP), and genotyping-by-sequencing and restriction site-associated DNA sequencing (GBS+RADseq) was determined for a set of 76 SNPs. To find alternative methodologies for allele dosage estimation, the KASP and Flex-Seq techniques were compared for the same set of SNPs. For the three techniques, a population of 53 genotypes from the diverse sugarcane panel of the Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia, was selected. Results: The average Pearson correlation coefficients between GBS+RADseq and Flex-Seq, GBS+RADseq and KASP, and Flex-Seq and KASP were 0.62 ± 0.27, 0.38 ± 0.27, and 0.38 ± 0.30, respectively. Discussion: Flex-Seq reproduced the allele dosages determined using GBS+RADseq with good levels of precision because of its depth of sequencing and ability to target specific positions in the genome. Additionally, Flex-Seq outperformed KASP by allowing the conversion of a higher number of SNPs and a more accurate estimation of the allele dosage. Flex-Seq has therefore become the genotyping methodology of choice for marker validation at Cenicaña.

Premisa: Detectar polimorfismos de un único nucleótido (SNP) de forma costo­efectiva es fundamental en cualquier programa de mejoramiento genético. En este artículo nosotros comparamos tres técnicas de genotipado para medir su habilidad en reproducir las dosis alélicas de SNPs de interés en caña de azúcar (Saccharum spp.). Métodos: Para identificar una técnica reproducible para la estimación de dosis alélicas durante los pasos de validación de marcadores, la correlación entre Flex­Seq, kompetitive allele­specific PCR (KASP), y genotyping­by­sequencing and restriction site­associated DNA sequencing (GBS+RADseq) fue determinada para un set de 76 SNPs. Para identificar metodologías alternativas en la estimación de las dosis alélicas, las tecnologías KASP y Flex­Seq fueron comparadas para el mismo grupo de SNPs. Para las tres técnicas, una población de 53 genotipos fue seleccionados de la población diversa de caña de azúcar del Centro de Investigación de la Caña de Azúcar (Cenicaña), Colombia. Resultados: El promedio del coeficiente de correlación de Pearson entre GBS+RADseq y Flex­Seq, GBS+RADseq y KASP, y Flex­Seq y KASP fue de 0.62 ± 0.27, 0.38 ± 0.27, y 0.38 ± 0.30, respectivamente. Discusión: Flex­Seq reprodujo las dosis alélicas determinadas usando GBS+RADseq con buenos niveles de precisión debido a su profundidad de secuenciación y habilidad de secuenciar posiciones especificas en el genoma. Adicionalmente, Flex­Seq superó a KASP al permitir la conversión de un número mayor de SNPs y al estimar las dosis alélicas de forma más precisa. Flex­Seq por tanto se convierte en la metodología de genotipado de elección para la validación de marcadores en Cenicaña.

Acute Q fever in individuals with acute febrile illness & exposure to farm animals: Clinical manifestations & diagnostic approaches.

Background & objectives Q fever is an important zoonotic disease affecting humans as well as animals. The objective of this study was to assess the burden of Q fever in individuals with acute febrile illness, particularly those in close contact with animals. Various diagnostic methods were also evaluated in addition to clinical examination analysis and associated risk factors. Methods Individuals presenting with acute febrile illness who had animal exposure were enrolled (n=92) in this study. Serum samples were tested using IgG and IgM phase 2 enzyme linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA). The PCR targeting the com1 and IS1111 genes was performed on blood samples. PCR amplicons were sequenced and phylogenetically analysed. Demographic data, symptoms, and risk factors were collected through a structured questionnaire. Results Among individuals with acute febrile illness, 34.7 per cent (32 out of 92) were found to be infected with Coxiella burnetii. PCR exhibited the highest sensitivity among the diagnostic methods employed. The most common clinical manifestations included headache, chills, arthralgia, and fatigue. Individuals engaged in daily livestock-rearing activities were found to be at an increased risk of infection. Interpretation & conclusions Q fever is underdiagnosed due to its varied clinical presentations, diagnostic complexities, and lack of awareness. This study underscores the importance of regular screening for Q fever in individuals with acute febrile illness, particularly those with animal exposure. Early diagnosis and increased awareness among healthcare professionals are essential for the timely management and prevention of chronic complications associated with Q fever.

Expression profiling & functional characterization of candidate miRNAs in serum exosomes among Indians with & without HIV-tuberculosis coinfection.

Background & objectives Despite the evidence of population differences in miRNA expression, limited information is available about the expression profile of miRNAs in Indian tuberculosis (TB) patients. The present study aimed to investigate the expression profile of candidate serum exosomal microRNAs in Indian patients with and without HIV-TB coinfection. Methods The pool samples of serum exosomes of study participants (HIV-TB coinfection, extra-pulmonary TB, HIV mono-infection, pulmonary TB) and healthy humans were processed for the isolation of total RNA followed by miRNA analysis using miRCURY LNA human focus PCR panel by real-time PCR. The significantly altered miRNAs were identified using differential expression analysis. The target genes prediction and potential functional analysis of exclusively differentially expressed miRNAs were performed using bioinformatics tools. Results The expression profile of 57, 58, 49 and 11 miRNAs was significantly altered in exosome samples of HIV-TB coinfected, extra-pulmonary TB, HIV mono-infected and pulmonary TB patients compared to healthy controls, respectively. The set of three (hsa-let-7i-5p, hsa-miR-24-3p, hsa-miR-92a-3p), three (hsa-miR-20a-5p, hsa-let-7e-5p, hsa-miR-26a-5p) and four (hsa-miR-21-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, hsa-miR-146a-5p) miRNAs were exclusively significantly differentially expressed in study participants with HIV-TB coinfection, extra-pulmonary TB and pulmonary TB, respectively. Most of the target genes of exclusively differentially expressed miRNAs were enriched in pathways in cancer, MAPK signalling pathway and Ras signalling pathway. Interpretation & conclusions The present study demonstrates a distinct expression profile of miRNAs in serum exosomes of the study participants and identified crucial miRNAs which may have a significant impact on the biomarker analysis and pathogenesis of TB in Indian patients.

MicroRNA expression signature in gastrointestinal stromal tumour & their molecular & histological features.

Background & objectives In gastrointestinal stromal tumour (GIST), not only genetic abnormalities are responsible for adverse clinical events, but epigenetic modifications also play a crucial role. MicroRNA (miRNA) dysregulation plays a significant role in carcinogenesis as miRNAs serve as natural silencer for their targets. Our study aimed to explore the miRNAs expression and its association with molecular and histopathological characteristics of GIST. Methods Fifty GIST samples, including 45 formalin fixed paraffin embedded (FFPE) and fresh tissues were included. Peripheral non-tumour tissues were used as controls. All the cases were confirmed using immunohistochemistry. RNA was extracted using miRNA-specific kit, and the expression was performed using RT-qPCR. The data were evaluated using AriaMx software version 1.5 (Agilent, US). MiRNAs expression was analyzed by using the relative quantification method (ΔΔCT). Results miR-221, miR-222, miR-494 and miR-34a showed significant down-regulation in tumours relative to non-tumour tissues. The expression levels of these miRNAs were significantly down-regulated in c-KIT (proto-oncogene encoding the tyrosine kinase transmembrane receptor)-positive tumours compared to c-KIT-negative. Further analysis revealed that reduced expression was associated with spindle subtypes and gastric localization. However, there was no significant correlation with other histological features. Additionally, miR-221/222, and miR-494 were down-regulated in most of the KIT exon 11 mutant subtypes, while miRNA-34a was associated with platelet derived growth factor receptor alpha (PDGFRA) mutations. Interpretation & conclusions The present study showed that the down-regulation of these miRNAs may help better molecular classification and characterization of GISTs. Our results offer new insight into the association between miRNAs and histological features, enabling a more thorough understanding of GISTs at the molecular level.

Performance of post-mortem diagnostic tests for tuberculosis in wild ungulates at low and high prevalence assessed using Bayesian latent class models.

Animal tuberculosis (TB) is often maintained by multi-host communities, including livestock and wildlife. Quantitative studies of such communities require estimating the true prevalence of TB, correcting the apparent prevalence by the diagnostic sensitivity (Se) and specificity (Sp) of the test. The goal of this study was to lay the foundations for estimating the true prevalence of TB in wild ungulate populations (wild boar and two cervids: red deer and fallow deer). We used Bayesian latent class models to assess the Se and Sp of gross pathology, IS6110 real-time PCR in tissues, bacteriological culture, and P22 indirect ELISA. We analyzed 308 harvested wild ungulates (211 wild boar and 97 cervids: 92 red deer and 5 fallow deer). The Se of bacteriological culture (80.4%, CI95 61.0-96.3%) and gross pathology (87.9%, CI95 69.5-99.9%) was reasonably good in wild boar. These tests showed lower Se in cervids: 60.2% (CI95 38.3-82.3%) for bacteriological culture and 81.5% (CI95 63.6-96.2%) for gross pathology. The Se of the real-time PCR was low (50.7% in wild boar and 53.0% in cervids). These tests showed Sp between 95.2 and 99.1% in both taxa. The P22 ELISA performed reasonably well in wild boar (Se = 71.9%, CI95 59.2-83.4%; Sp = 98.8%, CI95 96.9-99.9%) but lacked Sp in cervids (Se = 77.1%, CI95 62.9-89.7%; Sp = 74.5%, CI95 65.7-83.3%). The real-time PCR in wild boar and cervids and bacteriological culture in cervids tended to show higher Se in low-prevalence populations, possibly due to a higher proportion of early-stage TB lesions. In cervids, the parallel interpretation of gross pathology and bacteriological culture significantly improved the diagnostic performance (Se = 93.1%, CI95 84.7-98.9%; Sp = 92.9%, CI95 86.0-98.3%). Our results allow the estimation of true prevalence from the results of a single diagnostic test applied to harvested wild boar, red deer, and fallow deer, paving the way for more precise quantitative ecological studies of the multi-host TB maintenance community.

A droplet digital PCR method for the detection of scale drop disease virus in yellowfin seabream (Acanthopagrus latus).

In this study, a ddPCR method for the detection of scale drop disease virus (SDDV) in yellowfin seabream (Acanthopagrus latus) was established based on Real-time fluorescence quantitative PCR detection methods and principles. The reaction conditions were optimized, and the sensitivity, specificity, accuracy, and reproducibility were assessed. The results showed that threshold line position was determined to be 1900 by the ddPCR method; the optimum annealing temperature for SDDV detection by the ddPCR method was 60°C; the limit of detection was 1.4-1.7 copies/µL; the results of specific detection of other common viruses, except for SDDV specific amplification, were all negative; and the relative standard deviation (RSD) for the reproducibility validation was 0.77%. The samples of yellowfin seabream (Acanthopagrus latus) liver, spleen, kidney, heart, intestine, brain, blood, muscle, skin and ascites with three replicates, respectively, were tested using the ddPCR method, and the results were consistent with clinical findings. The ddPCR method established in this study has the advantages of high sensitivity, high specificity, good reproducibility and simple steps for the quantitative detection of SDDV, which could be used for the nucleic acid detection of clinical SDDV samples, and provided a new quantitative method for the diagnosis of yellowfin seabream SDDV in the early stage of pathogenesis.

Characterization of the myostatin gene in the neotropical species Piaractus mesopotamicus and the possibility of its use in genetic improvement programs.

BACKGROUND: The myostatin gene has played an important role in the genetic improvement of the main species of economic importance; however, it has not yet been described for some Neotropical fish essential for aquaculture. This study aimed to characterize the myostatin gene of pacu, Piaractus mesopotamicus, and investigate the association of a microsatellite marker in this gene with the weight of fish. METHODS AND RESULTS: The myostatin gene sequence was obtained after following a RACE-PCR strategy based on a partial mRNA sequence available in the GenBank database and the alignment of myostatin sequences from other fish species. The obtained sequence for the P. mesopotamicus gene was analyzed for short tandem repeats, and one dinucleotide was observed at the 3´untranslated region. A short tandem repeat polymorphism was verified in a wild population. Subsequently, the STR was evaluated in a test population of 232 animals in two 220 m² concrete tanks at the Aquaculture Center of Unesp. Eight alleles and 22 genotype combinations were identified. A significant association was observed between microsatellite marker polymorphisms and the weight traits (WEIGHT1 and WEIGHT2). Alleles 210, 222, 226, and 230 were found to favor weight gain. CONCLUSIONS: In summary, this study contributes to the characterization of the myostatin gene in pacu fish and identifies an association between a STR and weight traits. Thus, this gene could be used as a target for genetic breeding using molecular strategies such as CRISPR and quantitative strategies such as marker-assisted selection, which would contribute to improving the production of the species.