Solving the Myxidium rhodei (Myxozoa) puzzle: insights into its phylogeny and host specificity in Cypriniformes.

Myxidium rhodei Léger, 1905 (Cnidaria: Myxozoa) is a kidney-infecting myxosporean that was originally described from the European bitterling Rhodeus amarus. Subsequently, it has been documented based on spore morphology in more than 40 other cypriniform species, with the roach Rutilus rutilus being the most commonly reported host. This study introduces the first comprehensive data assessment of M. rhodei, conducted through morphological, ecological and molecular methods. The morphological and phylogenetic analyses of SSU rDNA sequences of Myxidium isolates obtained from European bitterling and roach did not support parasite conspecificity from these fish. In fact, the roach-infecting isolates represent three distinct parasite species. The first two, M. rutili n. sp. and M. rutilusi n. sp., are closely related cryptic species clustering with other myxosporeans in the freshwater urinary clade, sharing the same tissue tropism. The third one, M. batuevae n. sp., previously assigned to M. cf. rhodei, clustered in the hepatic biliary clade sister to bitterling-infecting M. rhodei. Our examination of diverse cypriniform fishes, coupled with molecular and morphological analyses, allowed us to untangle the cryptic species nature of M. rhodei and discover the existence of novel species. This underscores the largely undiscovered range of myxozoan diversity and highlights the need to incorporate sequence data in diagnosing novel species.

Title: Résoudre le casse-tête de Myxidium rhodei (Myxozoa) : aperçu de sa phylogénie et de sa spécificité d'hôte chez les Cypriniformes. Abstract: Myxidium rhodei Léger, 1905 (Cnidaria : Myxozoa) est un Myxosporea infectant les reins qui a été décrit à l'origine chez la bouvière, Rhodeus amarus. Par la suite, il a été documenté, sur la base de la morphologie des spores, chez plus de 40 autres espèces de cypriniformes, le gardon Rutilus rutilus étant l'hôte le plus fréquemment signalé. Cette étude présente la première évaluation complète des données sur M. rhodei, réalisée par des méthodes morphologiques, écologiques et moléculaires. Les analyse morphologiques et phylogénétiques des séquences d'ADNr SSU des isolats de Myxidium obtenus à partir de bouvières et de gardons européens n'ont pas confirmé la conspécificité du parasite de ces poissons. En fait, les isolats infectant les gardons représentent trois espèces distinctes de parasites. Les deux premières, M. rutili n. sp. et M. rutilusi n. sp., sont des espèces cryptiques étroitement apparentées, regroupées avec d'autres Myxosporea du clade urinaire d'eau douce, partageant le même tropisme tissulaire. La troisième, M. batuevae n. sp., précédemment attribuée à M. cf. rhodei, appartient au clade biliaire hépatique, groupe-frère de M. rhodei infectant la bouvière. Notre examen de divers poissons cypriniformes, couplé à des analyses moléculaires et morphologiques, nous a permis de démêler la nature cryptique des espèces de M. rhodei et de découvrir l'existence de nouvelles espèces. Cela souligne la diversité largement méconnue des Myxozoaires et souligne la nécessité d'incorporer des données de séquence dans le diagnostic de nouvelles espèces.

Study on the relationship between Methylation of Neuregulin (NRG) Gene and Cervical Carcinoma.

Objective: To investigate the relationship between the DNA methylation state of NRG1 promoter and its expression changes, and to analyze the clinical significance of its regulatory mechanism of DNA methylation in cervical carcinoma. Methods: This was a retrospective study. One-hundred and twenty patients from the Department of Gynecology of Cangzhou People's Hospital from September 2017 to September 2019 were selected, including 40 cases of cervical SCC, 40 cases of high grade squamous intraepithelial lesions(HSIL) and 40 cases of control cervical tissues. RT-qPCR, immunohistochemistry and DNA methylation-specific PCR(MSP) were used to detect the mRNA and protein expression of NRG1 and DNA methylation status in different tissue types. Results: Immunohistochemical results showed that the positive protein expression rate of NRG1 gene in the SCC group was lower than that in both HSIL and Control groups. RT-qPCR results showed that the mRNA gene of NRG1 gradually decreased in expression with the increase of cervical tissue lesions, with a statistically significant difference. Similarly, it also found that the mRNA expression level of NRG1 in the SCC group was independent of patients' age (p>0.05), but significantly correlated with tumor pathological staging, surgical pathology staging and lymphatic metastasis (p<0.05). Furthermore, methylation-specific PCR results revealed a significantly higher DNA methylation rate of NRG1 gene in the SCC group than in both HSIL and Control groups, with a statistically significant difference. Moreover, the methylation degree of NRG1 gene in SCC tissues was negatively correlated with its mRNA expression (p<0.05). Conclusions: Abnormal DNA hypermethylation of NRG1 gene inhibits the expression of mRNA and protein in the progression of cervical tissue from normal to cancerous state, which is involved in the occurrence and development of cervical carcinoma.

Integrons and multidrug resistance across phylogenetic groups of clinical isolates of Escherichia coli.

Objective: This study was aimed to investigate the multidrug resistance patterns in clinical isolates of Escherichia coli and their correlation with integrons and phylogenetic groupings. Methods: A total of 37 clinical E. coli isolates were evaluated for drug resistance patterns by disk diffusion method. Phylogenetic groupings and the presence of integrons among E. coli were determined by multiplex PCR assays. Results: Multidrug resistance was identified in 84% of the clinical isolates of E. coli with higher resistance found against cephalosporins (94.6%) and fluoroquinolones (83.8%), while lower resistance was observed against polymyxins (24.3%) and carbapenems (29.7%). Metallo-ß-lactamases were found in all carbapenem resistant isolates. The phylogenetic group B2 was the most dominant (40.5%), followed by groups A (35.1%), D (13.5%) and B1 (10.8%). Integrons were detected in 25 (67.6%) isolates and intI1, intI2, and intI3 genes were found in 62.2%, 18.9% and 10.8% of isolates respectively. Conclusion: Our results show that phylogenetic classification of E. coli is not relevant with antimicrobial resistance. However, there was strong association between the integron classes and resistance against ß-lactam and fluoroquinolones antimicrobials. Additionally, this study highlighted that the presence of integrons plays a crucial role in the development of multidrug resistance in clinical isolates of E. coli. Most significantly, this is the first report of detection of three classes of integron among clinical isolates of E. coli in Pakistan.

Polymerase Chain Reaction (PCR) Profiling of Extensively Drug-Resistant (XDR) Pathogenic Bacteria in Pulmonary Tuberculosis Patients.

Introduction Pulmonary tuberculosis (TB) remains a global health concern, exacerbated by the emergence of extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis. This study employs advanced molecular techniques, specifically polymerase chain reaction (PCR) profiling, to comprehensively characterize the genetic landscape of XDR pathogenic bacteria in patients diagnosed with pulmonary TB. The objective of the study is to elucidate the genes that are associated with drug resistance in pulmonary TB strains through the application of PCR and analyze specific genetic loci that contribute to the development of resistance against multiple drugs. Materials and methods A total of 116 clinical samples suspected of TB were collected from the tertiary healthcare setting of Saveetha Medical College and Hospitals for the identification of MTB, which includes sputum (n = 35), nasal swabs (n = 17), blood (n = 44), and bronchoalveolar lavage (BAL) (n = 20). The collected specimens were processed and subjected to DNA extraction. As per the protocol, reconstitution of the DNA pellet was carried out. The reconstituted DNA was stored at -20 °C for the PCR assay. From the obtained positive sample specimens, XDR pulmonary TB specimens were focused on the targeted genes, specifically the rpoB gene for rifampicin resistance, inhA, and katG gene for thepromoter region for isoniazid resistance. Results Out of a total of 116 samples obtained, 53 tested positive for pulmonary TB, indicative of a mycobacterial infection. Among these positive cases, 43 patients underwent treatment at a tertiary healthcare facility. Subsequently, a PCR assay was performed with the extracted DNA for the target genes rpoB, inhA, and katG. Specifically, 22 sputum samples exhibited gene expression for rpoB, inhA, and katG, while nine nasal swabs showed expression of the rpoB and inhA genes. Additionally, rpoB gene expression was detected in seven blood specimens, and both rpoB and inhA genes were expressed in five BAL samples. Conclusion The swift diagnosis and efficient treatment of XDR-TB can be facilitated by employing advanced and rapid molecular tests and oral medication regimens. Utilizing both newly developed and repurposed anti-TB drugs like pretomanid, bedaquiline, linezolid, and ethionamide. Adhering to these current recommendations holds promise for managing XDR-TB effectively. Nevertheless, it is significant to conduct well-designed clinical trials and studies to further evaluate the efficacy of new agents and shorter treatment regimens, thus ensuring continuous improvement in the management of this challenging condition.

The Utility and Limitations of Universal Polymerase Chain Reaction Screening for SARS-CoV-2 During Hospital Admission.

OBJECTIVE: Universal polymerase chain reaction (PCR) screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on hospital admission is an effective approach to preventing coronavirus disease 2019 (COVID-19) outbreaks in medical facilities. However, false-positive test results due to a recent infection are a concern. We investigated the usefulness and limitations of universal PCR screening for SARS-CoV-2 on hospital admission in a real-world setting. METHODS: We retrospectively analyzed 1320 attempted hospital admissions for 775 patients at the Department of Respiratory Medicine, Kyushu University Hospital, between January 1, 2022, and May 2, 2023. RESULTS: Thirty-nine out of 1201 PCR tests (3.2%) yielded a positive result, with 22 of these results being considered false positives on the basis of a recent infection. We found that 39% of cases showed a positive PCR result between 31 and 60 days after the onset of COVID-19, although the threshold cycle (Ct) for target 1 (ORF1ab gene) of the Cobas SARS-CoV-2 test (Roche Diagnostics, Basel, Switzerland) was >30 in most instances. CONCLUSION: Hospital admission based on the results of PCR screening for SARS-CoV-2 should take into account not only PCR positivity but also the Ct value and recent COVID-19 history.

The influence of P2Y12 gene polymorphisms on clopidogrel therapy in patients after percutaneous coronary intervention.

Aim: We aimed to define the influence of P2Y12 polymorphisms (rs6801273, rs2046934, and rs6809699), diabetes, hypertension, obesity, hypercholesterolemia, statins intake, and smoking habit on clopidogrel therapy in patients undergoing percutaneous coronary intervention. Materials & methods: We used PCR-RFLP and PCR-ASO for P2Y12 genotype analysis. The effectiveness of the therapy was measured with the VerifyNow method and defined in platelet reactivity units. Results: Studied polymorphisms had no statistically significant influence on PRU before (PRU0) and 6 months (PRU6) after the procedure. H1/H1 diabetic carriers had significantly higher PRU6 values than patients without diabetes. Obese H1/H2 subjects had significantly lower PRU6 values than H1/H2 non-obese carriers. Conclusion: We found that obesity and diabetes may influence the long-term outcome of antiplatelet therapy.

Clopidogrel is a medicine that prevents platelets in the blood from clumping and blocking arteries. When the structure of the protein (e.g., P2Y12), responsible for response to clopidogrel is changed, we can observe less efficient therapy. Said changes can be caused for example by genetic polymorphisms, which are two or more variants of the same gene. This is why we wanted to check the impact of P2Y12 polymorphisms. We also wanted to check the impact of diabetes, high blood pressure, being overweight, high cholesterol blood level, cholesterol-reducing drugs, and smoking habits on clopidogrel treatment in patients after a procedure that unblocks blood vessels of the heart to restore its blood supply (percutaneous coronary intervention). We measured the efficacy of the treatment with platelet reactivity units (PRU). Studying polymorphisms had no impact on treatment efficacy before (PRU0) and 6 months (PRU6) after the medical procedure. We found that diabetes can cause higher platelet reactivity after 6 months of therapy. We noticed that being overweight may also be important, as obese patients had lower platelet reactivity values.

Using clotted, pelleted blood samples for direct molecular detection of Bartonella spp. in small mammal wildlife surveillance studies.

OBJECTIVE: Bartonella are emerging bacterial zoonotic pathogens. Utilization of clotted blood samples for surveillance of these bacteria in wildlife has begun to supersede the use of tissues; however, the efficacy of these samples has not been fully investigated. Our objective was to compare the efficacy of spleen and blood samples for DNA extraction and direct detection of Bartonella spp. via qPCR. In addition, we present a protocol for improved DNA extraction from clotted, pelleted (i.e., centrifuged) blood samples obtained from wild small mammals. RESULTS: DNA concentrations from kit-extracted blood clot samples were low and A260/A280 absorbance ratios indicated high impurity. Kit-based DNA extraction of spleen samples was efficient and produced ample DNA concentrations of good quality. We developed an in-house extraction method for the blood clots which resulted in apposite DNA quality when compared to spleen samples extracted via MagMAX DNA Ultra 2.0 kit. We detected Bartonella in 9/30 (30.0%) kit-extracted spleen DNA samples and 11/30 (36.7%) in-house-extracted blood clot samples using PCR. Our results suggest that kit-based methods may be less suitable for DNA extraction from blood clots, and that blood clot samples may be superior to tissues for Bartonella detection.

Emergence of Capnocytophaga canimorsus and Capnocytophaga cynodegmi in oral cavities of newborn puppies, a pilot study.

Capnocytophaga canimorsus and Capnocytophaga cynodegmi are commensal bacteria in the oral cavities of dogs. Both are zoonotic pathogens that could infect humans via dog bites. C. canimorsus may cause life-threatening infections in humans, whereas C. cynodegmi infections tend to be milder and more localized. Capsular serovars A-C of C. canimorsus seem to be virulence-associated. Some of the C. canimorsus serovars described to date can also be detected in other Capnocytophaga species, including C. cynodegmi. The objective of this pilot study was to investigate the emergence of C. canimorsus and C. cynodegmi after birth in oral cavities of puppies and to evaluate the impact of the dam's Capnocytophaga spp. carrier status on the emergence. Ten litters, altogether 59 puppies, were included in the study. The puppies and their dams were sampled at five time points over seven weeks after whelping. Oral swab samples taken were investigated for the presence of C. canimorsus and C. cynodegmi by species-specific polymerase chain reaction (PCR), the specificity of which was verified by sequencing a selection of the PCR products. Samples that were positive in Capnocytophaga PCR reactions were also capsular-typed by PCR to gain more knowledge about the Capnocytophaga spp. present in the samples. Altogether 10.2% and 11.9% of puppies, or 20.0% and 30.0% of litters tested PCR-positive for C. canimorsus and C. cynodegmi, respectively. Capnocytophaga PCR-positive puppy samples were always positive for only C. cynodegmi or C. canimorsus, not both. Most Capnocytophaga PCR-positive puppies became positive at the age of 5 to 7 weeks. Only a minority (5/16) of the C. cynodegmi PCR-positive dog samples were positive in capsular typing PCR, whereas all C. canimorsus PCR-positive dog samples were negative in capsular typing PCR. For all Capnocytophaga PCR-positive puppies, their dam was positive for the same Capnocytophaga species. These results suggest that puppies become colonized by C. cynodegmi or C. canimorsus from their dams at the time of deciduous teeth eruption.

Development of a colloidal gold immunochromatographic strip for the simultaneous detection of porcine epidemic diarrhea virus and transmissible gastroenteritis virus.

In recent years, porcine diarrhea-associated viruses have caused significant economic losses globally. These viruses present similar clinical symptoms, such as watery diarrhea, dehydration, and vomiting. Co-infections with porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV) are common. For the rapid and on-site preliminary diagnosis on the pig farms, this study aimed to develop a colloidal gold immunochromatography assay (GICA) strip for the detection of PEDV and TGEV simultaneously. The GICA kit showed that there was no cross-reactivity with the other five common porcine viruses. With visual observation, the lower limits were approximately 104 TCID50/mL and 104 TCID50/mL for PEDV and TGEV, respectively. The GICA strip could be stored at 4°C or 25°C for 12 months without affecting its efficacy. To validate the GICA strip, 121 clinical samples were tested. The positive rates of PEDV and TGEV were 42.9 and 9.9%, respectively, and the co-infection rate of the two viruses was 5.8% based on the duplex GICA strip. Thus, the established GICA strip is a rapid, specific, and stable tool for on-site preliminary diagnosis of PEDV- and TGEV-associated diarrhea.

Swab Testing to Optimize Pneumonia treatment with empiric Vancomycin (STOP-Vanc): study protocol for a randomized controlled trial.

Background: Vancomycin, an antibiotic with activity against Methicillin-resistant Staphylococcus aureus (MRSA), is frequently included in empiric treatment for community-acquired pneumonia (CAP) despite the fact that MRSA is rarely implicated in CAP. Conducting polymerase chain reaction (PCR) testing on nasal swabs to identify the presence of MRSA colonization has been proposed as an antimicrobial stewardship intervention to reduce the use of vancomycin. Observational studies have shown reductions in vancomycin use after implementation of MRSA colonization testing, and this approach has been adopted by CAP guidelines. However, the ability of this intervention to safely reduce vancomycin use has yet to be tested in a randomized controlled trial. Methods: STOP-Vanc is a pragmatic, prospective, single center, non-blinded randomized trial. Adult patients with suspicion for CAP who are receiving vancomycin and admitted to the Medical Intensive Care Unit at Vanderbilt University Medical Center will be screened for eligibility. Eligible patients will be enrolled and randomized in a 1:1 ratio to either receive MRSA nasal swab PCR testing in addition to usual care (intervention group), or usual care alone (control group). PCR testing results will be transmitted through the electronic health record to the treating clinicians. Primary providers of intervention group patients with negative swab results will also receive a page providing clinical guidance recommending discontinuation of vancomycin. The primary outcome will be vancomycin-free hours alive, defined as the number of hours alive and free of the use of vancomycin within the first seven days following trial enrollment estimated using a proportional odds ratio model. Secondary outcomes include 30-day all-cause mortality and time alive off vancomycin. Discussion: STOP-Vanc will provide the first randomized controlled trial data regarding the use of MRSA nasal swab PCR testing to guide antibiotic de-escalation. This study will provide important information regarding the effect of MRSA PCR testing and antimicrobial stewardship guidance on clinical outcomes in an intensive care unit setting. Trial registration: This trial was registered on ClinicalTrials.gov on February 22, 2024. (ClinicalTrials.gov identifier: NCT06272994).

Molecular Characterization of Lineage-IV Peste Des Petits Ruminants Virus and the Development of In-House Indirect Enzyme-Linked Immunosorbent Assay (IELISA) for its Rapid Detection".

Peste des petits ruminants (PPRV), a highly contagious viral disease, causes significant economic losses concerning sheep and goats. Recently, PPR viruses (PPRVs), have adopted new hosts and lineage IV of PPRVs represents genetic diversity within the same lineage. 350 samples, including blood, swabs, and tissues from sheep/goats, were collected during the 2020-2021 disease outbreaks in Pakistan. These samples were analysed through RT-PCR and three isolates of PPRV with accession numbers, MW600920, MW600921, and MW600922, were submitted to GenBank, based on the partial N-gene sequencing. This analysis provides a better understanding of genetic characterizations and a targeted RT-PCR approach for rapid PPRV diagnosis. An IELISA test was developed using the semi-purified antigen MW600922 isolate grown in Vero cells. The PPRV isolates currently present high divergence with the Turkish strain; conversely, similarities equivalent to 99.73% were observed for isolates collected from Pakistan. The developed indirect ELISA (IELISA) test demonstrated antibody detection rates at dilutions of 1:200 for antibodies (serum) and 1:32 for antigens. In comparison to cELISA, high specificity (85.23%) and sensitivity (90.60%) rates were observed. In contrast to the virus neutralization test (VNT), IELISA was observed to be 100% specific and 82.14% sensitive in its results. Based on these results, serological surveys conducted for PPR antibodies using IELISA can be a more effective strategy on a larger scale. Furthermore, our results demonstrate a significant breakthrough in the research in terms of cost-effectiveness and storage efficiency, and the developed IELISA test is highly recommended for use in developing countries.

Peste des petits ruminants (PPRV) is a transboundary, highly contagious, and economically significant viral disease affecting small ruminants and wildlife. PPRV, a disease that only targets animals, is the focus of the Global Eradication Programme (PPRV GEP), which aims to eradicate the disease by 2030. Following the completion of the first phase of the GEP (2017­2021), Pakistan has initiated the second phase: PPRV presence and the implementation of a control strategy. Rapid and accurate laboratory diagnosis is vital to the disease's effective control and eradication. In the present study, we have improved diagnosis by reverse transcriptase polymerase chain reaction (RT-PCR), which not only can detect low viral concentrations but also contributes to the genetic analysis of lineage-IV viruses. However, the development of cost-effective indirect ELISA (iELISA) may allow for the analysis of serum samples obtained from larger populations of small ruminants.

Microplastics in granular sequencing batch reactors: Effects on pollutant removal dynamics and the microbial community.

This study investigated the relationship between microplastic (MP) presence and pollutant removal in granular sludge sequencing batch reactors (GSBRs). Two types of MPs, polyethylene (PE) and polyethylene terephthalate (PET), were introduced in varying concentrations to assess their effects on microbial community dynamics and rates of nitrogen, phosphorus, and organic compound removal. The study revealed type-dependent variations in the deposition of MPs within the biomass, with PET-MPs exhibiting a stronger affinity for accumulation in biomass. A 50 mg/L dose of PET-MP decreased COD removal efficiency by approximately 4 % while increasing P-PO4 removal efficiency by around 7 % compared to the control reactor. The rate of nitrogen compounds removal decreased with higher PET-MP dosages but increased with higher PE-MP dosages. An analysis of microbial activity and gene abundance highlighted the influence of MPs on the expression of the nosZ and ppk1 genes, which code enzymes responsible for nitrogen and phosphorus transformations. The study also explored shifts in microbial community structure, revealing alterations with changes in MP dose and type. This research contributes valuable insights into the complex interactions between MP, microbial communities, and pollutant removal processes in GSBR systems, with implications for the sustainable management of wastewater treatment in the presence of MP.

Exosomal mRNA in plasma serves as a predictive marker for microvascular invasion in hepatocellular carcinoma.

BACKGROUND AND AIM: There is a pressing need for non-invasive preoperative prediction of microvascular invasion (MVI) in hepatocellular carcinoma (HCC). This study investigates the potential of exosome-derived mRNA in plasma as a biomarker for diagnosing MVI. METHODS: Patients with suspected HCC undergoing hepatectomy were prospectively recruited for preoperative peripheral blood collection. Exosomal RNA profiling was conducted using RNA sequencing in the discovery cohort, followed by differential expression analysis to identify candidate targets. We employed multiplexed droplet digital PCR technology to efficiently validate them in a larger sample size cohort. RESULTS: A total of 131 HCC patients were ultimately enrolled, with 37 in the discovery cohort and 94 in the validation cohort. In the validation cohort, the expression levels of RSAD2, PRPSAP1, and HOXA2 were slightly elevated while CHMP4A showed a slight decrease in patients with MVI compared with those without MVI. These trends were consistent with the findings in the discovery cohort, although they did not reach statistical significance (P > 0.05). Notably, the expression level of exosomal PRPSAP1 in plasma was significantly higher in patients with more than 5 MVI than in those without MVI (0.147 vs 0.070, P = 0.035). CONCLUSION: This study unveils the potential of exosome-derived PRPSAP1 in plasma as a promising indicator for predicting MVI status preoperatively.

Development of triplex assay for simultaneous detection of Escherichia coli, methicillin resistant and sensitive Staphylococcus aureus in raw pork samples of retail markets.

Escherichia coli and Staphylococcus aureus are the most important food borne pathogen transmitting from animal meat and meat products. Therefore, it is vital to design an accurate and specific diagnostic tool for identifying those food-borne pathogens in animal meat and meat products. In the current study, E. coli, methicillin-resistant and sensitive S. aureus (MRSA and MSSA) were simultaneously detected using a developed triplex PCR-based technique. To obtain an optimal reaction parameter, the multiplex assay was optimised by changing just one parameter while holding the others constant. Specificity of the assay was assessed using several porcine bacterial template DNA. The plasmid DNA was used to test the multiplex PCR assay's sensitivity and interference in spiked pork samples. E. coli, MRSA, and MSSA each have PCR amplified products with sizes of 335, 533, and 209 bp, respectively. The assay detects a minimum microbial load of 102 CFU/µl for all the three pathogens and can identify bacterial DNA as low as 10-2 ng/µl. The assay was validated employing 210 pork samples obtained from retail meat shops and slaughter houses, with MRSA, E. coli, and MSSA with the occurrence rate of 1.9%, 42.38%, and 18.1%, respectively. The rate of mixed bacterial contamination in pork meat samples examined with the developed method was 6.19%, 1.43%, 1.90%, and 1.43% for MSSA & E. coli, MRSA & E. coli, MSSA & MRSA, and E. coli, MSSA & MRSA, respectively. The developed multiplex PCR assay is quick and efficient, and it can distinguish between different bacterial pathogens in a single reaction tube.

Lobar pneumonia due to human metapneumovirus: a case report.

Human metapneumovirus (hMPV) is a respiratory pathogen that can cause lower respiratory tract infections and pneumonia in immunocompetent adults. Pneumonia caused by hMPV is reportedly more likely to cause bronchial wall thickening and ground-glass opacity (GGO). A 44-year-old woman with no significant medical history developed fever, cough, and nausea. Computed tomography of the chest showed scattered GGOs in the right upper lobe and infiltrating shadows with air bronchograms in the left lingual and bilateral lower lobes. The patient was admitted to our hospital for further evaluation. Atypical pneumonia was suspected and lascufloxacin (LSFX) was started. Multiplex polymerase chain reaction (PCR) detected hMPV on hospital day 2 using the FilmArray Respiratory Panel 2.1. Pneumonia due to hMPV was suspected and LSFX was discontinued. The patient subsequently showed spontaneous improvement and was discharged on hospital day 6 after admission. After discharge, pneumonia continued to improve. Early detection of respiratory pathogens using multiplex PCR can help determine the appropriate treatment strategy. As hMPV can also cause lobar pneumonia, we should consider pneumonia due to hMPV in the differential diagnosis of lobar pneumonia.

Prognostic value of oxidative phosphorylation-related genes in hepatocellular carcinoma.

PURPOSE: Hepatocellular carcinoma (HCC) is the most prevalent malignancies worldwide. Recently, oxidative phosphorylation (OXPHOS) has received extensive concern as an emerging target in antitumor therapy. However, the OXPHOS-involved underlying genes and clinical utilization in HCC remain worth exploring. The present research aimed to create an OXPHOS-relevant signature in HCC. PATIENTS AND METHODS: In this study, the prognostic signature genes linked with OXPHOS were identified, and prognostic models were built using least absolute shrinkage and selection operator (LASSO) cox regression analysis. Furthermore, the combination study of immune microenvironment and signature genes looked into the involvement of immune cells in signature-based genes in HCC. Following that, chemotherapeutic drug sensitivity and immunotherapy analysis was implemented to predict clinical efficacy in HCC patients. Finally, clinical samples were collected to measure the expression of OXPHOS-related signature genes. RESULTS: Following a series of screens, six prognostic signature genes related with OXPHOS were identified: MRPS23, MPV17, MAPK3, IGF2BP2, CDK5, and IDH2, on which a risk model was built. The findings revealed a significant drop in the survival rate of HCC patients as their risk score increased. Meanwhile, independent prognostic study demonstrated that the risk score could accurately identify HCC patients. Immuno-microenvironmental correlation research suggested that the prognostic characteristics could serve as a reference index for both immunotherapy and chemotherapy. Finally, RT-qPCR exhibited a trend in signature gene expression that was consistent with the results. CONCLUSION: In this study, a total of six prognostic genes associated with OXPHOS were selected and a prognostic model was constructed, providing an essential reference for the study of OXPHOS in HCC.

Development of pre-implantation genetic testing protocol for monogenic disorders (PGT-M) of Hb H disease.

Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α0-thalassemia- SEA deletion/α+-thalassemia- 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease- SEA/-3.7kb using multiplex fluorescent PCR. A novel set of primers for α+-thalassemia- 3.7kb deletion was developed and tested. The PCR protocol for α0-thalassemia- SEA deletion was combined for Hb H disease- SEA/-3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart's. The results were compared and discussed. The results showed three PCR products from α+-thalassemia- 3.7kb primer set, and three from α0thalassemiaSEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease- SEA/-3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease- SEA/-3.7kb which is optimal for PGT-M.

Prevalence of Dichelobacter nodosus in western Austrian sheep flocks: Comparison of bacterial cultures, clinical foot rot and lameness with PCR and analysis of sample pooling for PCR diagnosis.

INTRODUCTION: Ovine foot rot is a highly contagious and multifactorial claw disease, caused by Dichelobacter nodosus (D. nodosus) and is the main cause of lameness in sheep. The aim of this cross-sectional study was to determine the prevalence of D. nodosus in western Austria both at animal and farm levels. Real-time PCR was evaluated in comparison with clinical and bacteriological investigations from interdigital foot swabs to detect D. nodosus-infected animals. In addition, the use of pooled four-foot swabs to detect foot rot was determined. In course of the study a total of 3156 sheep from 124 farms were examined for lameness and clinical signs of foot rot. The found flock prevalence of D. nodosus was 30,65 % with bacterial culture showing a sensitivity of 75,0 % and a specificity of 100,0 % (p < 0,001) respectively, compared with PCR. Furthermore, clinical foot rot scores (Ckorr = 0,87; p < 0,001) and lameness scores (Ckorr = 0,71; p < 0,001) highly correlated with the detection of D. nodosus by PCR. The result showed that the clinical examination can be used to identify animals infected with D. nodosus in flocks, but PCR must be used to confirm the diagnosis. D. nodosus could be detected equally well with risk-based pools-of-five samples as with undiluted samples (p < 0,001), suggesting that a pool-of-five samples might be a suitable and cost-effective method for detecting D. nodosus in sheep flocks. This study provides an overview of foot rot in Tyrolean sheep flocks and outlines the possibilities and limitations of the various diagnostic tools for D. nodosus. Further studies to investigate possible influencing factors, including alpine pasturing, management factors and biosecurity predisposing to foot rot are necessary for the design of effective future control programs in alpine regions.

INTRODUCTION: Le piétin ovin est une maladie des onglons hautement contagieuse et multifactorielle, causée par Dichelobacter nodosus (D. nodosus) qui constitue la principale cause de boiterie chez les ovins. L'objectif de cette étude transversale était de déterminer la prévalence de D. nodosus dans l'ouest de l'Autriche, tant au niveau de l'animal que de l'exploitation. La PCR en temps réel a été évaluée en comparaison avec les examens cliniques et bactériologiques effectués à partir d'écouvillons des espaces interdigités pour détecter les animaux infectés par D. nodosus. En outre, l'utilisation d'un pool d'écouvillons des quatre membres pour détecter le piétin a été déterminée. Au cours de l'étude, un total de 3156 moutons provenant de 124 fermes ont été examinés pour détecter des boiteries et des signes cliniques de piétin. La prévalence de D. nodosus dans les troupeaux était de 30,65 %, la culture bactérienne montrant une sensibilité de 75 % et une spécificité de 100 % (p < 0,001), respectivement, par rapport à la PCR. En outre, les scores cliniques de piétin (Ckorr = 0,87; p < 0,001) et les scores de boiterie (Ckorr = 0,71; p < 0,001) étaient fortement corrélés avec la détection de D. nodosus par PCR. Les résultats montrent que l'examen clinique peut être utilisé pour identifier les animaux infectés par D. nodosus dans les troupeaux mais que la PCR doit être utilisée pour confirmer le diagnostic. D. nodosus a pu être détecté aussi bien avec des pools de cinq échantillons basés sur le risque qu'avec des échantillons non dilués (p < 0,001), ce qui suggère qu'un pool de cinq échantillons pourrait être une méthode appropriée et rentable pour détecter D. nodosus dans les troupeaux de moutons. Cette étude donne un aperçu du piétin dans les troupeaux de moutons tyroliens et souligne les possibilités et les limites des différents outils de diagnostic pour D. nodosus. D'autres études visant à examiner les facteurs d'influence possibles, y compris les pâturages alpins, les facteurs de gestion et la biosécurité prédisposant au piétin, sont nécessaires pour la conception de futurs programmes de contrôle efficaces dans les régions alpines.

Quantitative detection and survival analysis of VBNC Salmonella Typhimurium in flour using droplet digital PCR and DNA-intercalating dyes.

The difficulty in detecting viable but non-culturable (VBNC) Salmonella by culture-dependent methods poses a risk to food safety. In our study, we applied a viability test to Salmonella following a lethal treatment and to flour samples inoculated with Salmonella to evaluate the effectiveness of viability polymerase chain reaction (PCR). Our findings revealed that the combination of both ddPCR and qPCR with those DNA-intercalating dyes could quantify viable cells at low concentrations when the plate counting method failed to detect them post-inactivation. Prolonged UV exposure did not induce cell membrane disruption, as confirmed with PMA-ddPCR, with insignificant differences in gene copies. However, samples exposed to DyeTox13 and DyeTox13 + EMA showed lower gene copy numbers, implying that enzymatic activity was decreased by UV exposure duration. In addition, temperature-dependent survival in flour revealed uniform decay rates and D values (time required for a 1 log reduction) of DNA in untreated samples across various temperatures. By contrast, different decay rates were observed with DNA-intercalating dyes (DyeTox13 and DyeTox13 + EMA), showing faster metabolic activity loss at higher temperatures in flour. The decay rates and D values, determined through plate counting and those DNA-intercalating dyes, indicated the potential presence of VBNC Salmonella. A strong correlation between DyeTox13 dyes and the plate counting method suggested DyeTox13 as a rapid alternative for detecting Salmonella in flour. The ddPCR with DNA-intercalating dyes could effectively evaluate Salmonella viability, facilitating more precise monitoring of VBNC in food. IMPORTANCE: Salmonella, a major foodborne pathogen, poses significant risks, particularly to vulnerable groups like infants, older people, and the immunocompromised. Accurate detection is vital for public health and food safety, given its potential to cause severe and life-threatening symptoms. Our study demonstrated digital polymerase chain reaction (ddPCR) with DNA-intercalating dyes for identifying the different physiological statuses of Salmonella. Also, the application of ddPCR with DNA-intercalating dyes offers quantification of viable cells post-disinfection as an alternative method in food. Utilizing ddPCR and DNA-intercalating dyes, we enhanced the detection of VBNC Salmonella, a form often undetectable by conventional methods. This innovative approach could significantly improve the precision and efficiency of detection for viable Salmonella. By providing deeper insights into its transmission potential, our method is a critical tool in preventing outbreaks and ensuring the safety of food products. This research contributes substantially to global efforts in controlling foodborne illnesses and safeguarding public health.