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.

Stability and expression patterns of housekeeping genes in Mediterranean mussels (Mytilus galloprovincialis) under field investigations.

The use of marine mussels as biological models encompasses a broad range of research fields, in which the application of RNA analyses disclosed novel biomarkers of environmental stress and investigated biochemical mechanisms of action. Quantitative real-time PCR (qPCR) is the gold standard for these studies, and despite its wide use and available protocols, it may be affected by technical flaws requiring reference gene data normalization. In this study, stability of housekeeping genes commonly employed as reference genes in qPCR analyses with Mytilus galloprovincialis was explored under field conditions. Mussels were collected from farms in the Northwestern Adriatic Sea. The sampling strategy considered latitudinal gradients of environmental parameters (proxied by location), gender, and their interactions with seasonality. Analyses of gene stability were performed using different algorithms. BestKeeper and geNorm agreed that combination of the ribosomal genes 18S ribosomal RNA (18S) and 28S ribosomal RNA (28S) was the best normalization strategy in the conditions tested, which agrees with available evidence. NormFinder provided different normalization strategies, involving combinations of tubulin (TUB)/28S (Gender/Season effect) or TUB/helicase (HEL) (Location/Season effect). Since NormFinder considers data grouping and computes both intra- and inter-group stability variations, it should work better with complex experimental designs and dataset structuring. Under the selected normalization strategies, expressions of the variable housekeeping genes actin (ACT) and elongation factor-1α (EF1) correlated with seasonal and latitudinal changes of abiotic environmental factors and mussel physiological status. Results point to consider ACT and EF1 expressions as molecular biomarkers of mussel general physiological status in field studies.

Evaluating the quantitative performance of environmental DNA metabarcoding for freshwater zooplankton community: a case study in Lake Biwa, Japan.

Zooplankton monitoring is important for understanding their population dynamics and life history, ecosystem health, and environmental changes. Compared with traditional morphological identification, environmental DNA (eDNA) analysis allows for more sensitive and efficient monitoring of zooplankton diversity. Previous eDNA studies have primarily used metabarcoding approaches to reveal their richness and composition, whereas its performance in predicting zooplankton abundance remains understudied. We conducted water and bulk sampling in Lake Biwa, Japan, showing that the number of sequence reads by metabarcoding moderately correlated with eDNA concentrations estimated by quantitative real-time PCR (qPCR). In addition, the eDNA read number was significantly related to cladoceran and copepod abundance estimated by microscopy sorting, although there remained too much uncertainty in the read-abundance relationship. Moreover, there was a significant difference in species composition between eDNA metabarcoding and sorting. Although our results indicated the potential applicability of eDNA metabarcoding for quantifying multiple zooplankton abundance, several methodological validations in eDNA metabarcoding would also be required to optimize its performance in the future.

Histological and Molecular Biological Changes in Canine Skin Following Acute Radiation Therapy-Induced Skin Injury.

Radiation therapy is a crucial cancer treatment, but it can damage healthy tissues, leading to side effects like skin injuries and molecular alterations. This study aimed to elucidate histological and molecular changes in canine skin post-radiation therapy (post-RT) over nine weeks, focusing on inflammation, stem cell activity, angiogenesis, keratinocyte regeneration, and apoptosis. Four male beagles received a cumulative radiation dose of 48 Gy, followed by clinical observations, histological examinations, and an RT-qPCR analysis of skin biopsies. Histological changes correlated with clinical recovery from inflammation. A post-RT analysis revealed a notable decrease in the mRNA levels of Oct4, Sox2, and Nanog from weeks 1 to 9. VEGF 188 levels initially saw a slight increase at week 1, but they had significantly declined by week 9. Both mRNA and protein levels of COX-2 and Keratin 10 significantly decreased over the 9 weeks following RT, although COX-2 expression surged in the first 2 weeks, and Keratin 10 levels increased at weeks 4 to 5 compared to normal skin. Apoptosis peaked at 2 weeks and diminished, nearing normal by 9 weeks. These findings offer insights into the mechanisms of radiation-induced skin injury and provide guidance for managing side effects in canine radiation therapy.

Human Papillomavirus Genotypes Distribution in High-Grade Cervical Lesions and Invasive Cervical Carcinoma in Women Living in Mauritania: Implications for Cervical Cancer Prevention and HPV Prophylactic Vaccination.

Cervical cancer related to high-risk human papillomavirus (HR-HPV) is the second female cancer in Mauritania (Northwest Sahelian Africa). We assessed the distribution of HPV genotypes in Mauritanian women with high-grade cervical intraepithelial neoplasia (CIN2/3) or invasive cervical cancer (ICC). A prospective study was conducted in the Centre Hospitalier National, Nouakchott, Mauritania, to collect cervical biopsies among women suspected of CIN2/3 or cancer. HPV DNA detection and genotyping were carried out from formalin-fixed, paraffin-embedded biopsies using multiplex PCR (Human Papillomavirus Genotyping Real-Time PCR Kit, Bioperfectus Technologies Co., Taizhou, China). Fifty biopsies were included from women (mean age: 56.7 years) suffering from CIN2/3 (28.0%) and ICC (72.0%) which corresponded to 32 (64.0%) squamous cell carcinomas (SCC) and 4 (8.0%) adenocarcinomas (ADC). HPV DNA detection was successful in 47 (94.0%) samples. The most prevalent HR-HPV genotypes were HPV-45 (40.4%), HPV-16 (38.3%), HPV-39 and HPV-52 (23.4%), HPV-33 (17.0%), HPV-18 (14.9%), HPV-35 (4.2%), and HPV-56 (2.1%). The majority (93.6%) of HPV-positive biopsies contained at least one HPV type covered by the 9-valent Gardasil-9® vaccine, and 40.9% were infected by multiple vaccine HPV genotypes. To eradicate cervical cancer in Mauritania, prophylactic HPV vaccination must be combined with primary molecular screening of cervical HR-HPV infection.

Transcriptomic and Physiological Studies Unveil that Brassinolide Maintains the Balance of Maize's Multiple Metabolisms under Low-Temperature Stress.

Low-temperature (LT) is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Brassinolides (BRs) have been shown to enhance LT tolerance in several plant species; the physiological and molecular mechanisms by which BRs enhance maize tolerance are still unclear. Here, we characterized changes in the physiology and transcriptome of N192 and Ji853 seedlings at the three-leaf stage with or without 2 µM 2,4-epibrassinolide (EBR) application at 25 and 15 °C environments via high-performance liquid chromatography and RNA-Sequencing. Physiological analyses revealed that EBR increased the antioxidant enzyme activities, enhanced the cell membrane stability, decreased the malondialdehyde formation, and inhibited the reactive oxygen species (ROS) accumulation in maize seedlings under 15 °C stress; meanwhile, EBR also maintained hormone balance by increasing indole-3-acetic acid and gibberellin 3 contents and decreasing the abscisic acid level under stress. Transcriptome analysis revealed 332 differentially expressed genes (DEGs) enriched in ROS homeostasis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) cascade. These DEGs exhibited synergistic and antagonistic interactions, forming a complex LT tolerance network in maize. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that 109 hub genes involved in LT stress regulation pathways were discovered from the four modules with the highest correlation with target traits. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous BRs in enhancing LT tolerance of maize at the seedling stage, thus opening up possibilities for a breeding program of maize tolerance to LT stress.

Methodological assessment for efficient collection of Schistosoma mansoni environmental DNA and improved schistosomiasis surveillance in tropical wetlands.

Schistosomiasis, caused by trematodes of genus Schistosoma, is among the most seriously neglected tropical diseases. Although rapid surveillance of risk areas for Schistosoma transmission is vital to control schistosomiasis, the habitat and infection status of this parasite are difficult to assess. Environmental DNA (eDNA) analysis, involving the detection of extra-organismal DNA in water samples, facilitates cost-efficient and sensitive biomonitoring of aquatic environments and is a promising tool to identify Schistosoma habitat and infection risk areas. However, in tropical wetlands, highly turbid water causes filter clogging, thereby decreasing the filtration volume and increasing the risk of false negatives. Therefore, in this study, we aimed to conduct laboratory experiments and field surveys in Lake Victoria, Mbita, to determine the appropriate filter pore size for S. mansoni eDNA collection in terms of particle size and filtration volume. In the laboratory experiment, aquarium water was sequentially filtered using different pore size filters. Targeting >3 µm size fraction was found to be sufficient to capture S. mansoni eDNA particles, regardless of their life cycle stage (egg, miracidia, and cercaria). In the field surveys, GF/D (2.7 µm nominal pore size) filter yielded 2.5-times the filtration volume obtained with a smaller pore size filter and pre-filtration methods under the same time constraints. Moreover, a site-occupancy model was applied to the field detection results to estimate S. mansoni eDNA occurrence and detection probabilities and assess the number of water samples and PCR replicates necessary for efficient eDNA detection. Overall, this study reveals an effective method for S. mansoni eDNA detection in turbid water, facilitating the rapid and sensitive monitoring of its distribution and cost-effective identification of schistosomiasis transmission risk areas.

Monitoring SARS-CoV-2 IgA, IgM and IgG antibodies in dried blood and saliva samples using antibody proximity extension assays (AbPEA).

Using a modified proximity extension assay, total and immunoglobulin (Ig) class-specific anti-SARS-CoV-2 antibodies were sensitively and conveniently detected directly from ø1.2 mm discs cut from dried blood and saliva spots (DBS and DSS) without the need for elution. For total Ig detection, antigen probes were prepared by conjugating recombinant spike protein subunit 1 (S1-RBD) to a pair of oligonucleotides. To detect isotype-specific antibody reactivity, one antigen probe was replaced with oligonucleotide-conjugated antibodies specific for antibody isotypes. Binding of pairs of oligonucleotide-conjugated probes to antibodies in patient samples brings oligonucleotides in proximity. An added DNA polymerase uses a transient hybridization between the oligonucleotides to prime synthesis of a DNA strand, which serves as a DNA amplicon that is quantified by real-time PCR. The S1-RBD-specific IgG, IgM, and IgA antibodies in DBS samples collected over the course of a first and second vaccination exhibited kinetics consistent with previous reports. Both DBS and DSS collected from 42 individuals in the autumn of 2023 showed significant level of total S1-RBD antibodies with a correlation of R = 0.70. However, levels in DSS were generally 10 to 100-fold lower than in DBS. Anti-S1-RBD IgG and IgA in DSS demonstrated a correlation of R = 0.6.

Molecular detection of Coxiella burnetii in ticks infesting wild and domestic animals in the Eastern region of Punjab, Pakistan.

Tick-borne pathogens are significant for human, veterinary, and wildlife health. Coxiella burnetii is an example that is widely distributed across various hosts and can cross species boundaries. In Pakistan, there is a scarcity of data regarding C. burnetii at the intersection of wildlife and livestock. Ticks were collected from ruminants and wildlife from the districts of Kasur, Pakpattan, and Okara in Pakistan. Five tick species totaling 571 ticks were collected, with the following distribution: 56.4% Hyalomma anatolicum, 22.4% Rhipicephalus microplus, 10.5% Hyalomma marginatum, 7.9% Rhipicephalus sanguineus, and 2.8% Rhipicephalus turanicus. Fifty tick pools were screened for C. burnetii to amplify a segment of the IS1111 using real-time PCR assays. Ticks collected from sheep and goats had a greater rate of positivity for C. burnetii (40% and 38%, respectively) compared to Indian long-eared hedgehogs with a prevalence of 2%. Coxiella burnetii was prominent in Rhipicephalus microplus (92.3%) and Hyalomma anatolicum (88.9%), followed by Rhipicephalus turanicus (66.6%), Rhipicephalus sanguineus (33.3%), and Hyalomma marginatum (25.0%). Ticks from Pakpattan district displayed the highest prevalence of C. burnetii (88.9%), whereas the lowest was observed in ticks from Kasur district (77.3%). There was no significant association between tick gender and C. burnetii infection. Female host animals were more likely to harbor ticks containing C. burnetii, with a prevalence rate of 81.8%. The research underscores the urgent need for comprehensive studies on C. burnetii in Pakistan, especially at the interface of wildlife and livestock. The high prevalence rates observed in certain tick species and geographic regions emphasize the importance of targeted public health interventions. Future research should focus on elucidating the transmission dynamics and implementing effective control measures to mitigate the impact of these pathogens on human, veterinary, and wildlife health in the region.

Anti-biofilm and antibacterial effect of Bacteriocin derived from Lactobacillus plantarum on the multidrug-resistant Acinetobacter baumannii.

This research examines the impact of bacteriocin derived from Lactobacillus plantarum PTCC 1745 on the biofilm formations of A. baumannii isolates. Bacteriocin derived from L. plantarum PTCC 1745 was obtained through ammonium sulfate precipitation, cation-exchange chromatography, and reversed-phase high-performance liquid chromatography (RP-HPLC). Testing for bacteriocin susceptibility has been conducted using the broth dilution method. The anti-biofilm activity of bacteriocin was evaluated using a microtiter plate method. Quantitative real-time PCR assay evaluated bap gene expression in bacteriocin-treated cells. According to SDS-PAGE, bacteriocin from L. plantarum has a 25-kDa apparent molecular weight. The MICs of bacteriocin ranged from 30 to 120 µg/mL, while the MBCs varied between 60 to 120 µg/mL. Compared to the non-treated group, strains bacteriocin-treated isolates had 59% less ability to form biofilm. The mean relative expression of the bap gene among the MDR A. baumannii isolates decreased by 52% compared to the untreated control. This study demonstrated that bacteriocin derived from L. plantarum PTCC 1745 had antibacterial and antibiofilm activity against MDR A. baumannii isolates.

Analysis of the Main Checkpoints of the JNK-MAPK Pathway in HTLV-1-Associated Leukemia/Lymphoma via Boolean Network Simulation.

The c-Jun N-terminal kinase (JNK) pathway is a signal transduction pathway that plays a critical role in cell growth and survival. Its dysregulation is related to various cancers, including adult T-cell leukemia/lymphoma (ATLL), an aggressive peripheral T-cell malignancy caused by human T-cell lymphotropic virus type 1 (HTLV-1) infection. There is currently no vaccine or definitive treatment for ATLL. This research aimed to identify the JNK-MAPK pathway checkpoints to identify possible therapeutic targets using Boolean network analysis. First, the genes involved in the JNK pathway and their interactions were identified and mapped. Next, a Boolean network analysis was performed using the R programming language, which suggested protein kinase B (AKT) and MAP kinase phosphatase (MKP) for further evaluation. Finally, to confirm the effect of these two genes, a clinical study was conducted among ATLL patients and healthy individuals. The quantitative real time polymerase chain reaction (qRT‒PCR) results revealed a statistically significant decrease in the expression of AKT and MKP in ATLL patients compared to normal controls. This highlights the potential role of these two genes as potential therapeutic targets in ATLL.

Comparison of toxin gene expression levels and molecular typing of Clostridioides difficile strains isolated from patients with diarrhea.

Aim: This study aimed to evaluate the expression of tcdA, tcdB, and binary toxin genes (cdtA and cdtB) by Real-Time PCR and molecular typing of Clostridioides difficile isolated from patient diarrhea samples from Hamadan Hospitals, west of Iran. Background: The concentration of C. difficile toxins (CDTs) is associated with the severity of the disease and the mortality rate. Measuring CDT levels could provide a reliable and objective means of determining the severity of C. difficile infection (CDI). Methods: From November 2018 to September 2019, 130 diarrhea samples were collected from hospitalized patients in three hospitals in Hamadan. C. difficle isolates were detected by culture and PCR. The presence of the genes encoding the toxin was identified by PCR, whereas the measurement of toxin expression was conducted using a relative Real-Time PCR technique. Genetic linkage of the isolates was also assessed by Ribotyping and Repetitive Extragenic Palindromic (rep-PCR) methods. Results: Among 130 diarrhea samples, 16 (12.3%) were positive for C. difficile. Genes encoding cdtA and tcdB were detected in all isolates, and 8 (50%) and 6 (37.5%) isolates were positive for the cdtA and cdtB genes. Real-time PCR results showed different expression levels of the toxin genes. A significant increase in the expression of the tcdA gene was observed compared with the control strain (P<0.05). Besides, more expression of cdtA gene was observed in the strains compared with cdtB gene. Ribotyping and rep-PCR results showed high genetic diversity of C. difficile among hospitals investigated. Conclusion: We encountered toxigenic C. difficile strains with various toxin expression levels, ribotypes, and rep types based on the findings of this study. This indicated that various clones from various sources circulate in the hospitals and among patients.

Design and implementation of a TaqMan® real-time PCR method for detection and quantification of bovine leukemia virus.

The bovine leukemia virus (BLV) is an important infectious agent transmitted from cattle to humans. It is considered one of the oncogenic viruses in breast cancer, so an accurate detection of this virus is important. The study aimed to design a specific and sensitive method based on TaqMan® real-time polymerase chain reaction (RT-PCR) for BLV detection. Probes and primers were designed using bioinformatics software for a 108 pairs region of the BLV tax gene. Criteria employed for determining analytical sensitivity were prepared using in-vitro RNA transcriptions. The National Center for Biotechnology Information (NCBI), basic local alignment search tool (BLAST) databases various viral panels and genomic samples from healthy individuals (Qom Province, Iran in 2023) were used to verify analytical specificity and clinical specificity, respectively. This method can measure a minimum of 10 copies of DNA and RNA mL-1. Moreover, the assay is linear in the range of 100 - 109 copies mL-1. By testing negative specimens, the method specificity was 100%. The reproducibility results of the reaction were examined at the intra- and inter-assay comparison. In fact, 10 technical replicates of each concentration of the control sample were analyzed in each working reaction. Due to the locally made kit, exact sensitivity and specificity, rapid analysis, and relatively low cost, as compared to commercial kits of other countries, the method introduced in the present study could be suitable for accurate detection of the BLV. Also, the TaqMan® real-time PCR method could be detected in cattle and human and before malignant changes of breast cancer which could reduce infection and breast cancer.

Promoting molecular diagnostic equity: assessing in-house real-time PCR for Neisseria gonorrhoeae in anal samples from MSM recruited in an outpatient setting in Morocco.

Objectives: Gonorrhea is a prevalent sexually transmitted infection among men who have sex with men (MSM). In Morocco, the basic laboratory diagnosis of Neisseria gonorrhoeae (NG) is based on microscopy and, in some settings, on culture. However, no nucleic acid amplification test (NAAT) has been implemented for routine diagnosis of gonorrhoeae.The aim of this study is to assess the effectiveness of an in-house real-time PCR test for detecting N. gonorrhoeae DNA in anal swabs samples collected during an Integrated Behavioral and Biological survey. Patients and methods: Samples from 245 MSM, recruited using a Respondent Driven Sampling, were collected and tested for NG infection using GeneXpert CT/NG assay (Cepheid, USA). An In-House real-time PCR technique targeting the pseudo gene porA was developed and used for a parallel investigation of the same infection. The reliability of the in-house RT-PCR was validated through tests of reproducibility, repeatability, limit of detection, and cross-reactivity with other bacteria. The intrinsic performance characteristics of the qRT-PCR were assessed, namely, the sensitivity, the specificity, the positive predictive value (PPV), and the negative predictive value (NPV). The GeneXpert CT/NG assay was adopted as a reference method. Results: For N. gonorrhoeae detection, the in-house real-time PCR assay showed a sensitivity and specificity of 80% and 100%, respectively. The PPV of the assay was 100% and the NPV was 97.3%. Conclusion: The in-house real-time PCR assay has high specificity and sensitivity, and it emerges as a promising approach for detecting N. gonorrhoeae in clinical specimens, particularly in decentralized settings such as regional laboratories.

Development and evaluation of a duplex real-time multienzyme isothermal rapid amplification assay for the detection of hypervirulent Klebsiella pneumoniae in clinical spiked blood specimens.

Objectives: Our objective was to establish a rapid and precise method for detecting hypervirulent Klebsiella pneumoniae (hvKP) by utilizing a duplex real-time multienzyme isothermal rapid amplification (real-time MIRA) and to evaluate its performance in clinical spiked blood specimens. Methods: The research comprised two phases: an initial pilot study to establish the methodology and a clinical validation study to assess its effectiveness. In the pilot phase, we designed specific primers and probes targeting the hvKP pg344 and incA genes and subsequently developed a duplex real-time MIRA assay to evaluate its detection limits, specificity, and efficiency. In the clinical validation phase, we analyzed thirty-three spiked blood specimens using the duplex real-time MIRA assay. Results: The duplex real-time MIRA assay demonstrated no cross-reactivity with other strains. Sensitivity experiments confirmed that the assay had a detection limit as low as 8 × 102 CFU per reaction for hvKP. The analysis of clinical spiked blood specimens indicated that the sensitivity and specificity of the duplex real-time MIRA assay were on par with those of duplex real-time PCR. Conclusions: These findings confirm that the duplex real-time MIRA assay is a fast, straightforward, and dependable method for detecting hvKP.

Standardization and application of ARMS TaqMan real-time PCR for screening of folate metabolism genes in Han Chinese.

Folate has antioxidant properties, and low concentration in seminal plasma may be associated with increased DNA damage in sperm. Mutations of the methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes, including MTHFR C677T (rs1801133), MTHFR A1298C (rs1801131), and MTRR A66G (rs1801394), can lead to decreased activity of the encoded folate metabolic enzymes, thereby affecting male reproduction. The current SNP detection methods commonly used in clinical practice have some shortcomings, such as long time-consuming, complex detection steps, or high cost. The purpose of this study was to establish a simple, time-saving, sensitive, accurate, and easy to clinical popularization method for folate metabolism gene detection. We combined ARMS-PCR with TaqMan fluorescent probe to establish an ARMS TaqMan real-time PCR detection method. According to the variation of rs1801131, rs1801133, and rs1801394, two specific primers (one wild type and one mutant) were designed. Mismatched nucleotides were introduced at the penultimate or third position to improve the specificity of the primer. Specific TaqMan probe was introduced to detect PCR products to improve the sensitivity of the method. The results showed that the sensitivity of ARMS TaqMan real-time PCR in SNP genotyping was 1 ng, and the accuracy was 100%. A total of 249 clinical samples were detected by the established method, and the correlation between three SNPs and semen quality was analyzed. We found that individuals carrying the AG + GG genotype of rs1801394 had a lower risk of abnormal semen quality. In conclusion, we developed a highly sensitive, accurate, rapid, and easy to be popularized method for detecting SNPs of rs1801394, rs1801131, and rs1801133. ARMS TaqMan real-time PCR is a reliable SNP genotyping method in folate metabolism genes.

Polyvinylpyrrolidone capped silver nanoparticles enhance the autophagic clearance of Acinetobacter baumannii from human pulmonary cells.

Acinetobacter baumannii, an opportunistic pathogen has shown an upsurge in its multi-drug resistant isolates. OmpA of A. baumannii induces incomplete autophagy and apoptosis in host cells. Various therapeutic alternatives are under investigation against A. baumannii. Here, the major emphasis has been laid on comparing the efficacy of AgNP with different capping agents. OmpA targeted lead, Ivermectin capped AgNP (IVM-AgNP) has been compared with the antibacterial polyvinylpyrrolidone capped AgNP (PVP-AgNP) for their role in the modulations of host autophagy. Upregulation of p62 and LC3B confirmed by real-time PCR analysis indicated an increased autophagic flux upon the treatment with AgNPs. The elongation and closure of autophagic vacuoles was also supported by upregulated Atg genes (Atg4, Atg3, Atg5) in A. baumannii infected cells after treatment with AgNP. Autophagic flux increased on treatment with PVP-AgNP as suggested by the rise in mcherryLC3B fluorescence in A549 cells treated with PVP-AgNP as compared to the GFP-LC3B of IVM-AgNP. This suggests that PVP-AgNP treatment more effectively promotes the elongation and maturation stages of autophagy by increasing autophagic flux. These results indicate that capped AgNPs have the efficiency to revert the incomplete autophagy induced by A. baumannii back to normal autophagic levels.

Performance of three multiplex real-time PCR assays for simultaneous detection of 12 infectious pathogens in mice affected with respiratory and digestive diseases.

Introduction: Research quality can be improved with reliable and reproducible experimental results when animal experiments are conducted using laboratory animals with guaranteed microbiological and genetic quality through health monitoring. Therefore, health monitoring requires the rapid and accurate diagnosis of infectious diseases in laboratory animals. Methods: This study presents a performance evaluation of a commercially available multiplex real-time PCR (mRT-PCR) assay for the rapid detection of 12 infectious pathogens (Set 1: Sendai virus [SeV, formally murine respirovirus], Mycoplasma spp., Rodentibacter pneumotropicus, and Rodentibacter heylii; Set 2: Helicobacter spp., Murine norovirus [MNV], Murine hepatitis virus [MHV], and Salmonella spp.; Set 3: Staphylococcus aureus, Streptobacillus moniliformis, Corynebacterium kutscheri, and Pseudomonas aeruginosa). To evaluate the efficacy of the mRT-PCR assay, 102 clinical samples encompassing fecal and cecal specimens were analyzed. The resulting data were then compared with the findings from sequence analysis for validation. Results: The assay's detection limit ranged from 1 to 100 copies per reaction. Specificity testing involving various viruses and bacteria indicated no cross-reactivity between strains. Additionally, the assay exhibited good reproducibility, with mean coefficients of variation for inter- and intra assay variation below 3%. The overall positive rate was 52.9% (n = 54), with the mRT-PCR assay findings matching sequence analysis results (κ = 1). MHV (n = 29, 28.4%) was the most prevalent pathogen, followed by Helicobacter spp. (n = 28, 27.5%), R. heylii (n = 18, 17.6%), Mycoplasma spp. (n = 14, 13.7%), MNV (n = 12, 11.8%), S. aureus (n = 9, 8.8%), P. aeruginosa (n = 4, 3.9%), and R. pneumotropicus (n = 1, 0.9%). Discussion: This assay offers a rapid turnaround time of 100 min, including 30 min for DNA preparation and 70 min for target DNA/RNA amplification. It ensures accuracy, minimizing false positives or negatives, making it a convenient tool for the simultaneous detection of infectious diseases in many samples. Overall, the propose­d assay holds promise for the effective detection of the most important pathogens in laboratory animal health monitoring.

A multiplexed real-time PCR assay for simultaneous quantification of human immunodeficiency virus and Hepatitis B virus for low-and-middle- income countries.

Due to shared routes of transmission, including sexual contact and vertical transmission, HIV-HBV co-infection is common, particularly in sub-Saharan Africa. Measurement of viral load (VL), for both HIV and HBV, plays a critical role for determining their infectious phase and monitoring response to antiviral therapy. Implementation of viral load testing in clinical settings is a significant challenge in resource-limited countries, notably because of cost and availability issues. We designed HIV and HBV primers for conserved regions of the HIV and HBV genomes that were specifically adapted to viral strains circulating in West Africa that are HIV-1 subtype CRF02AG and HBV genotype E. We first validated two monoplex qPCR assays for individual quantification and, then developed a multiplex qPCR for simultaneous quantification of both viruses. HIV RNA and HBV DNA amplification was performed in a single tube using a one-step reverse transcription-PCR reaction with primers and probes targeting both viruses. Performance characteristics such as the quantification range, sensitivity, and specificity of this multiplex qPCR assay were compared to reference qPCR tests for both HIV and HBV viral load quantification. The multiplex assay was validated using clinical samples from co- or mono-infected patients and gave comparable viral load quantification to the HIV and HBV reference test respectively. The multiplex qPCR demonstrated an overall sensitivity of 71.25 % [68.16-74.3] for HBV and 82 % [78.09-85.90] for HIV and an overall specificity of 100 % [94.95-100] for both viruses. Although the overall sensitivities of the HIV and HBV assays were lower than the commercial comparator assays, the sensitivity in the clinical decision range of >1000 copies/mL for HIV was 80 % [71.26-88.73] and >1000 IU/mL for HBV was 100 % [95.51-100] which indicates the test results can be used to guide treatment decisions. This in-house developed multiplex qPCR assay represents a useful diagnostic tool as it can be performed on affordable "open" real-time PCR platforms currently used for HIV or SARS-Cov-2 infection surveillance in Mali.