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ABSTRACT Aims Methods and Results Conclusions Leishmaniasis is an endemic disease in several regions of Brazil, a tropical country that presents specific environmental conditions that contribute to the development of phlebotomine vectors. This study aimed to detect Leishmania species in naturally infected bats from 17 municipalities in the São Paulo state.Spleen and liver samples from 203 bats were analysed by real‐time PCR and confirmed by conventional PCR followed by gene sequencing. Leishmania DNA was amplified by real‐time PCR in 6.4% of the bats and by conventional PCR followed by sequencing in 3.4% of the bats. Positive samples were characterised and included in GenBank. Leishmania species were confirmed in M. molossus, M. nigricans and E. glaucinus bats. Leishmania (Leishmania) amazonensis and L. infantum (syn. L. chagasi) were identified. This is the first detection of Leishmania spp. in bats in the studied areas. All the positive bats came from urban areas. Insectivorous bats were statistically more positive. There was similarity between our sequences and those of a human isolate and a phlebotomine from the region.This result points to bats as important possible reservoir of Leishmania in Brazil and guides the country's health authorities towards epidemiological surveillance, control and prevention actions in endemic areas. [ABSTRACT FROM AUTHOR]

In the United States, the burden of hepatitis C virus (HCV) infection is disproportionately high among young adults including pregnant persons, resulting in increased infections among children as perinatal transmission remains the main route of HCV infection in children. Hence, in 2020, the Centers for Disease Control and Prevention (CDC) recommended universal HCV screening during each pregnancy. HCV infection in infancy is usually asymptomatic, so the diagnosis entirely relies on testing of perinatally exposed infants which, historically, included anti-HCV antibody testing at ≥18 months of age. However, nation-wide perinatal HCV testing rates have been suboptimal with significant loss to follow-up. To address this problem, in 2023, the CDC introduced early single HCV RNA testing at 2–6 months of age with an alternative for HCV RNA testing up to 17 months of age if not previously tested. The high sensitivity and specificity of the HCV real-time PCR laid the grounds for this policy shift. In this review, we highlight how these new CDC recommendations will enhance testing of infants and children and ultimately contribute to overall HCV elimination efforts. We also emphasize the role of all pediatric providers and obstetricians in implementing these new guidelines. Additionally, we offer our perspective and practical advice for testing of perinatally exposed infants and children. Currently, curative oral antivirals for HCV-infection treatment are approved for children ≥3 years of age. As pediatricians, advocating for children's wellness, it is our utmost duty to ensure that every child exposed to perinatal hepatitis C has been tested, diagnosed, linked to care, treated, and achieved cure. [ABSTRACT FROM AUTHOR]

Objective: To do the etiological analysis of ocular herps virus infection, revealing the pathogen species and the distribution of different virus types within the eye. Methods: Samples were collected from 2017 to 2021 at the Department of Ophthalmology, Peking University Third Hospital and tested using real-time PCR for common ocular viruses: herpes simplex virus 1 (HSV-1), cytomegalovirus (CMV), varicella-zoster virus (VZV) and Epstein-Barr virus (EBV). The pathogenesis of the different viruses was classified and analyzed according to the site of infection. Results: Viral PCR detections were performed on 3627 samples collected over the 5-years and 649 (17.89%) samples contained one or more of the viruses tested. The overall detection rate of CMV was highest at 9.93%. Of all sample types, aqueous humor was the most common (1752 cases), of which 340 were positive (19.41% positive rate). Corneal samples were the next most common, with 1481 cases and 250 positive results (16.88% positive rate). CMV positivity was higher in aqueous humor and corneal samples than other viruses; vitreous body had the highest positive rate at 36.36% (20/55), among which 18 cases were VZV positive. Conclusions: Distribution of virus types differed among infection sites, with CMV the most common virus type detected in the cornea and aqueous humor, while VZV was the most common virus detected in the vitreous body. Key message: What is known: • In recent years, ocular viral infections have attracted increasing attention from ophthalmologists, as more diseases with unknown etiology are now confirmed to be related to viral infection. • The Herpesviridae family is the most common cause of ocular viral infection. What is new: • We retrospectively analyzed the results of 3627 specimens from 2017 to 2021. No statistical analysis with large sample size and complete sample types similar to this study currently exists in the published literature. • Distribution of virus types differed among infection sites, with CMV the most common virus type detected in the cornea and aqueous humor, while VZV was the most common virus detected in the vitreous body. [ABSTRACT FROM AUTHOR]

Foodborne pathogens can easily contaminate chicken meat due to its high nutritional content, and these pathogens can infect humans. One of the most important pathogens contaminating chicken meat and causing severe public health problems is Listeria monocytogenes, which would be responsible for Listeriosis. Therefore, rapid and sensitive detection of L. monocytogenes in chicken meat samples is of great significance. In the current study, the presence of L. monocytogenes in chicken meat samples collected from several markets in Erzurum was detected by comparing two different DNA isolation methods with the Real-time PCR. As a result of the analyses, it was determined that 34% of the chicken meat samples collected were positive for L. monocytogenes in both two methods. According to the comparison analyses of the Bland-Altman method, no significant difference was found between the thermal lysis method and the DNA isolation method by commercial kit. As a result of this study, it has been shown that the thermal lysis method can be successfully applied for the detection of foodborne pathogens in chicken meat when evaluated in terms of workload and cost. The current study is the first report on the comparison of thermal lysis method and DNA isolation by commercial kit in the detection of L. monocytogenes from chicken meat by Real-time PCR. [ABSTRACT FROM AUTHOR]

Sheath rot of rice is one of the most devastating diseases of rice due to its ability to reduce the yield significantly in all rice cultivating areas of India. The purpose of this study was to assess the effectiveness of Bacillus cereus strain RBS-57 against sheath rot disease of rice. In addition, it enables us to understand the molecular mechanism of the host–pathogen-bioagent interactions using a proteomic approach. A combination of seed treatment, seedling dip, and foliar spray with RBS-57 liquid formulation has recorded the lowest sheath rot disease index, both under pot experiment (21.33%) and field conditions (15.33% in trial I and 12.42% in trial II, respectively). In addition to that, RBS-57 application enhanced the plant growth and yield attributes. Moreover, a 2D-PAGE study uses protein profiling to illustrate the molecular response of the tripartite interaction between host–pathogen-bioagent. MALDI-TOF mass spectrometry (MS/MS) analysis identified a total of 20 differentially expressed proteins, primarily implicated in plant metabolism and development, defense response, transcription and signalling. Selected genes were validated by quantitative Real-Time PCR (qRT-PCR) analysis. The alterations in protein abundance and transcripts were positively correlated for all the genes. The present study provides initial insights into the molecular mechanism that underlies the tripartite interaction between the host–pathogen-bioagent in rice plants. [ABSTRACT FROM AUTHOR]

Approximately 900 million hectares of farmland are destroyed by soil salinization, reducing global grain crop yield and quality. Although some current rice cultivars exhibit salt tolerance and high production yields, the exact mechanisms of their salt stress responses remain unclear. Hence, this study aims to elucidate the biochemical and biomolecular responses in different rice varieties, namely OM 9577 (salt-tolerant), OC 10 (moderately salt-tolerant), and Dai Thom 8 (salt-sensitive). The research findings indicate that increasing NaCl concentration leads to a decline in the fresh, dry mass of shoots and roots of all examined varieties (from 7.5 to 27% in OM 9577, OC 10; and from 16 to 54% in Dai Thom 8), as well as an increase in Na+ accumulation. OM 9577 and OC 10 accumulated higher levels of Ca2+, protein, proline, glycine betaine, sugars, and some monosaccharides (d-lyxofuranose, D-arabinose, ß-D-glucopyranose, ß-D-mannopyranose, D-galactofuranose) compared to Dai Thom 8. Transcript levels of some genes related to salt-tolerant mechanisms also have determined, including OsSOS1, OsNHX1, OsHKT1;4, OsHKT1;5, OsCaM, OsMAPKKK, OsCDPK, OsWAK, OsCML31, OsSOD, OsAOX1b, Os79, OsDUF26, OsWRKY26, OsWRKY62, OsAP2/ERF–58. The data demonstrated that transcript levels of some target genes were higher in OM 9577 and OC 10 than those in Dai Thom 8. Specifically, transcript levels of OsWRKY62, OsCML31, Os79, OsSOS1, OsHKT1:5, and OsCDPK were highest in OM 9577, while OsDUF26 and OsNHX1 transcript levels were highest in OC 10. These results offer potential biochemical and biomolecular markers for screening salt-tolerant rice cultivars and improving existing cultivars by transgenic or conventional breeding. [ABSTRACT FROM AUTHOR]

Among pets, cats are the most popular in Europe. Despite the fact, the interest in the safety and quality of their food is much lower compared to the interest of caregivers in the nutrition of dogs. In this research, 27 commercial cat foods were analyzed for mislabeled component composition. Cat foods were divided into a control group, a group of fish foods and a group of other foods with alternative sources of animal protein. Chicken and pig DNA detection was performed using real-time PCR. In this research, 100% of the cat foods contained chicken DNA and 96% of the foods – pig DNA, despite the lack of declaration of these ingredients on the product label. The results indicate that cat food appear to be mislabeled to an even greater extent than dog food. Moreover, manufacturers' declarations in terms of ingredient composition do not reflect the actual composition of commercial products available on the market and intended for everyday feeding of animals. Mislabeling of these products also poses a risk for animals suffering from food allergies. [ABSTRACT FROM AUTHOR]

Introduction: Homologous recombination (HR) is a crucial DNA-repair mechanism, and its disruption can lead to the accumulation of mutations that initiate and promote cancer formation. The key HR genes, BRCA1 and BRCA2, are particularly significant as their germline pathogenic variants are associated with a hereditary predisposition to breast and/or ovarian cancer. Materials and methods: The study was performed on 45 FFPE breast cancer tissues obtained from 24 and 21 patients, with and without the germline BRCA1/2 mutation, respectively. The expression of 11 genes: BRCA1, BRCA2, ATM, BARD1, FANCA, FANCB, FANCI, RAD50, RAD51D, BRIP1, and CHEK2 was assessed using Custom RT2 PCR Array (Qiagen), and results were analysed using R. Results: Cancer tissues from patients with BRCA1 or BRCA2 germline mutations displayed no significant differences in the expression of the selected HR genes compared to BRCA1 or BRCA2 wild-type cancer tissues. In BRCA1mut cancer tissues, BRCA1 expression was significantly higher than in BRCA2mut and BRCA wild-type cancer tissues. Conclusions: In cancer tissues harbouring either BRCA1 or BRCA2 germline mutations, no significant differences in expression were observed at the mRNA level of any tested HR genes, except BRCA1. However, the significant differences observed in BRCA1 expression between germline BRCA1mut, germline BRCA2mut and BRCA1/2wt tissues may indicate a compensatory mechanism at the mRNA level to mitigate the loss of BRCA1 function in the cells. [ABSTRACT FROM AUTHOR]

Phthalic acid isomers are the monomers of phthalate molecules, also known as phthalic acid esters, widely employed in the plastics industry. This study aims to investigate the biodegradation of phthalic acid (PA) and terephthalic acid (TPA) by five industry-borne Comamonas testosteroni strains: 3APTOL, 3ABBK, 2B, 3A1, and C8. To assess the ability of C. testosteroni strains to biodegrade phthalic acid isomers in fermentation media, an analytical method was employed, consisting of high-performance liquid chromatography (HPLC) analyses. Subsequently, molecular screening of the genomic and plasmid DNA was conducted to identify the degradative genes responsible for the breakdown of these chemicals. The genes of interest, including ophA2, tphA2, tphA3, pmdA, and pmdB, were screened by real-time PCR. The five C. testosteroni strains effectively degraded 100% of 100 mg/L PA (p = 0.033) and TPA (p = 0.0114). Molecular analyses indicated that all C. testosteroni strains contained the pertinent genes at different levels within their genomes and plasmids, as reflected in the threshold cycle (Ct) values. Additionally, DNA temperature of melting (Tm) analyses uncovered minor differences between groups of genes in genomic and plasmid DNA. C. testosteroni strains could be excellent candidates for the removal of phthalic acid isomers from environmental systems. [ABSTRACT FROM AUTHOR]

Babesia bigemina is a major cause of bovine babesiosis, an economically devastating tick-borne disease that needs timely diagnosis for precise treatment. In present investigation, the detection efficacy of real-time PCR (qPCR) in comparison to conventional PCR and microscopy targeting 18 S ribosomal gene was evaluated on 95 bovines (70 cattle and 25 buffaloes) suspected for babesiosis. Real-time PCR was standardized with the 10-fold serial dilutions in duplication of the given positive control (2 × 106 copy number) ranging from 106 to 100 copy number/µL and mean Ct value of each dilution was taken to extrapolate the curve. The samples with Ct value 36.92 of 10 copy number/µL were considered as positive. Out of 95 samples, 5 (5.26%), 21 (22.10%) and 49 (51.58%) positive by microscopy, conventional PCR and real-time PCR were in corresponding range of > 106-104, 104-103, and 103-<10 copy number/µL, respectively. The concordance of real-time PCR with conventional PCR and microscopy was moderate (Kappa = 0.523) and slight (Kappa = 0.09), respectively. The cows were at four times risk than the buffaloes for B. bigemina infection (Odds ratio:3.85, CI:1.4255–10.4370). This pioneer report from Punjab state (India) of application of real-time PCR to detect B. bigemina in bovines was found to be more sensitive than conventional PCR and microscopy that needs further investigations on a greater number of random samples. [ABSTRACT FROM AUTHOR]

Backgrounds: Toxoplasmosis, a prevalent parasitic infection, is primarily caused by Toxoplasma gondii (T. gondii). This infection poses a significant threat to neonates during pregnancy and individuals with compromised immune systems. Consequently, it is imperative to develop a novel diagnostic approach that combines high sensitivity with low-risk sampling to effectively manage patients. The aim of this study is to utilize serological and molecular techniques for the diagnosis of T. gondii infection in 100 pregnant women who were under the care of a gynecologist and were candidates for amniocentesis. Methods: During the 15-19th weeks of pregnancy, a total of 100 samples each of amniotic fluid, buffy coat, plasma, and urine simultaneously were collected from pregnant women candidates for amniocentesis in Mazandaran province, northern Iran. This study involved various assessments: (1) detecting anti-T. gondii IgM and IgG in plasma through chemiluminescence assay (2) determining IgG avidity in plasma using the Enzyme-linked immunosorbent assay technique (3) identifying of T. gondii DNA in amniotic fluid, buffy coat and urine by nested PCR (nPCR) and quantitative real-time PCR (qPCR) methods targeting the REP-529 gene, as well as genotyping using GRA6 target genes, and (4) assessing the sensitivity and specificity of the nPCR and qPCR tests. Results: Out of 100 pregnant women screened, 70 were between the ages of 31 to 40 years old. Among them, 23 and 44 had one and two previous pregnancies. Additionally, 13 and 8 women had one and two history of abortions, respectively. Following serologic testing, 52% of the individuals were positive for T. gondii antibodies. Of these, 52 samples were positive for IgG antibodies, and one sample was positive for both IgG and IgM antibodies. Notably, all 52 cases with IgG positivity exhibited a high level of IgG avidity. Regarding the molecular testing of amniotic fluid samples, two pregnant women tested positive in the nPCR assay, while three tested positive in the qPCR assay. Furthermore, genotyping revealed that all positive samples belonged to type I of the T. gondii genotype. Moreover, none of the 100 buffy coat and urine samples tested positive for T. gondii using the nPCR and qPCR techniques. Conclusion: The findings of the current study suggest that serological methods alone may not be reliable in diagnosing congenital toxoplasmosis and cannot rule out the diagnosis of toxoplasmosis and must be approved by molecular tests. [ABSTRACT FROM AUTHOR]

The aim of this study is to compare the bacterial load in the guts of honey bees supported and unsupplemented with probiotic supplements. To investigate the effects of a commercial bee probiotic containing different Lactobacillus species and different spice extracts on the composition of the gut microbiota of honey bees, QPCR counts of Lactobacillus spp. and Firmicutes phylum gene copies in gut mixtures from 12 different bee groups with and without probiotic supplementation were performed. There was a significant difference between the levels of Lactobacillus spp. in the guts of both groups. When Lactobacillus spp. levels in the guts of honey bees not given probiotics were compared to the Lactobacillus spp. levels in the guts of honey bees given probiotics, it was determined that there was an approximately 5.5-fold difference. However, it was observed that there was no significant difference in the Firmicutes load in the bee guts of both groups. These findings show that the applied probiotic formulation significantly affects the intestinal microbiome of healthy individuals and provides a proportional change in microbial abundance, especially in terms of Lactobacillus spp. [ABSTRACT FROM AUTHOR]

Background: Entomological data for onchocerciasis surveillance relies on sampling black flies through human landing collectors in the field and laboratory testing of the flies for infection using pooled screening O-150 PCR-ELISA assay. Both techniques require improvements. This study aimed to optimize the Esperanza Window Trap (EWT) for black fly collection. We tested alternative carbon dioxide (CO2) mimics to attract black flies to the traps. Additionally, we evaluated new quantitative PCR (qPCR) methods that target mitochondrial DNA markers and have been proposed to enhance the sensitivity and specificity for detecting Onchocerca volvulus infections in blackflies. Methods: Traps baited with low, medium and high release rates of either 2-butanone or cyclopentanone as CO2 mimics were field tested against traps baited with organically generated CO2 in four ecological zones in Nigeria: Guinea savannah, derived savannah, rainforest and montane forest. The performance of EWTs baited with CO2 or in combination with 2-butanone (low release) were subsequently evaluated against the human landing collection (HLC). Trap scaling was also pilot tested by comparing two EWTs to a single HLC team. Collected black flies were used to test detection of O. volvulus in black flies using Ov ND5 real-time PCR (qPCR) compared to the conventional pool screening O-150 PCR. Results: EWTs baited with 2-butanone caught similar numbers of black flies (Simulium damnosum s.l.) to those baited with CO2, while cyclopentanone collected significantly fewer flies in all locations. The low release of 2-butanone was the most effective overall, although HLCs collected higher numbers of black flies than EWT baited with CO2 either singly or in combination with low-release 2-butanone. The combination of two EWTs baited with CO2 and deployed 100 m apart from each other collected similar numbers of flies as one HLC. More black fly pools were positive for O. volvulus by Ov ND5 qPCR compared with O-150 PCR in derived savannah (31.15 vs. 15.57%), montane forest (11.54 vs. 0%) and rainforest (23.08 vs. 2.56%), with only one positive pool in Guinea savannah detected by both methods. Conclusions: The 2-butanone has potential to be used in xenomonitoring as a standardized replacement for organically generated CO2. Ov ND5 qPCR detected more positive pools than O-150 PCR. The positive pools found in foci hitherto considered to have interrupted/eliminated onchocerciasis highlight the need for more sensitive and specific methods that support programmatic assessments to identify and combat recrudescence. [ABSTRACT FROM AUTHOR]

The causes of diarrhea after 10 years of rotavirus vaccination in Rwanda were investigated with real-time polymerase chain reaction in 496 children with diarrhea and 298 without. Rotavirus was detected in 11% of children with diarrhea (odds ratio, 2.48; P =.002). Comparison of population attributable fractions (PAFs) shows that Shigella (PAF, 11%) and enterotoxigenic Escherichia coli producing labile toxin (PAF, 12%) have replaced rotavirus as the main causative agents. The PAF for rotavirus had declined from 41% prevaccination to 6.5% postvaccination, indicating that rotavirus has become one among several similarly important causes of childhood diarrhea in Rwanda. A rotavirus genotype shift to G3P[8] points at the importance of continued genotype surveillance. [ABSTRACT FROM AUTHOR]

Tuna are economically important as food resources in food markets. However, because tuna is often processed into steaks or fillets, the meat can be difficult to identify through morphological features. For effective fishery management and to protect the rights of consumers, it is necessary to develop a molecular method to accurately identify the species used in tuna products. Herein, we discovered five single-nucleotide polymorphism (SNP) sites via 2b-RAD sequencing and developed five SNP-based real-time polymerase chain reaction assays for the rapid identification of five highly priced tuna species. Three species-specific TaqMan systems were designed to identify albacore tuna (Thunnus alalunga), bigeye tuna (T. obesus), and southern bluefin tuna (T. maccoyii) and two cycling systems were designed to identify yellowfin tuna (T. albacares) and Atlantic bluefin tuna (T. thynnus). The systems showed good specificity and sensitivity (sensitivity of 0.0002 ng μL−1 for albacore tuna, bigeye tuna, and southern bluefin tuna and 0.002 ng μL−1 for yellowfin tuna and Atlantic bluefin tuna). Both systems were able to distinguish the target species from other species in a specific, sensitive, and accurate manner. Thus, these methods can be employed for the identification of species used in tuna products, protecting consumers and producers from economic fraud. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML) has a heterogeneous molecular profile, clinical presentations, and response to treatments and outcomes. DNA methylation is conducted by DNA methyltransferases including DNMT3B. Poly ADP-ribose polymerase 1 belongs to a family of enzymes that mediate important cellular processes including DNA repair, transcription, and cell death/cell proliferation, and it is involved in the development, spread, treatment, and prognosis of some cancers. The objective of this study is to assess the impact of PARP1 and DNMT3B genes expression on laboratory characteristics, response to treatment and survival in Egyptian cytogenetically normal AML patients. Methods: This study included 67 Egyptian CN-AML patients in addition to 8 healthy bone marrow donors. Measurement of DNMT3B and PARP1 gene expression was done on bone marrow samples via real-time semiquantitative polymerase chain reaction. Result: Expression of both DNMT3B and PARP1 genes was significantly upregulated in AML (P =.001, P =.036, respectively). Upregulated DNMT3B was associated with higher total leukocyte count (TLC), PB, and BM blast cell%. Also, upregulated PARP1 correlated with higher TLC, PB, and BM blast cell%. High expression of both DNMT3B and PARP1 correlated with greater frequencies of FLT3-ITD. High DNMT3B expression, and combined upregulation of both PARP1 and DNMT3B genes associated significantly with ELN stratification. But no correlation was found with response (CR), overall survival (OS), disease-free survival (DFS), or event-free survival (EFS). Conclusion: Our findings highlight the importance of considering DNMT3B and PARP1 expression levels as potential prognostic biomarkers for progression and aggressiveness of CN-AML patients in AML. Assessing their expression levels could be an indicator to guide treatment decisions and potentially improve patient outcomes. [ABSTRACT FROM AUTHOR]

The expression of the LYST and SLFN12L genes in peripheral blood leukocytes (PBL) in patients with pulmonary sarcoidosis with a chronic course of the disease and in healthy people was studied. Patients with chronic pulmonary sarcoidosis, on the second stage of the disease (20 people without therapy, average age of whom was 41.00 ± 12.56 years) and 25 healthy people (average age was 45.86 ± 2.13 years) were enrolled in this study. The level of gene expression was determined by real-time PCR. A significant decrease in the transcripts of the SLFN12L gene (p = 0.002) and a low content of mRNA of the LYST gene (p = 0.09) in the PBL of patients with pulmonary sarcoidosis compared to healthy people was established. Differential expression of LYST and SLFN12L may indicate the involvement of these genes in the pathogenesis of this disease, and it is possible they participate in the modulation of immune reactions during the development of inflammatory processes in pulmonary sarcoidosis. [ABSTRACT FROM AUTHOR]

Every year, more and more data demonstrate that microRNA expression levels can be changed significantly after acute radiation exposure, and microRNAs themselves play an important role in the cellular response to ionizing radiation. However, regulation of microRNA expression after chronic radiation exposure within the low and middle dose range is poorly studied. In the present study, the expression of mature miR-29a, miR-30c, and miR-150 microRNAs in whole blood from 81 individuals in the long-term period after chronic low dose-rate radiation exposure was analyzed by real-time PCR method. The mean age of the studied people was 72 years, and the accumulated radiation doses in red bone marrow (RBM), thymus, and peripheral lymphoid organs ranged from 2.13 to 1867.55 mGy and from 0.18 to 488.79 mGy, respectively. More than 70 years after the onset of radiation exposure, a statistically significant dose-dependent decrease in miR-30c microRNA expression was found in exposed individuals in RBM, thymus, and peripheral lymphoid organs. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa (Xf) bacteria form biofilm on the cuticular surfaces of the functional foregut (precibarium and cibarium) of its vectors, xylem fluid-ingesting sharpshooter leafhoppers and spittlebugs. While much is known about Xf biofilm development and maturation in vitro, little is known about these processes in vectors. Real-time (RT)-PCR was used to quantify Xf genomes daily in the functional foreguts of blue-green sharpshooters, Graphocephala atropunctata, over 7 days of exposure to infected grapevines. Scanning electron microscopy (SEM) was used to examine Xf biofilm formation at 4 and 7 days of that time course. PCR showed populations building and reducing over a 4-day cycle. SEM revealed that foreguts at 4 days showed variability in quantity and location of bacterial attachment. Only early-stage biofilm formation occurred in low-turbulence areas of the cibarium, while high-turbulence areas of the cibarium and precibarium had rare but older, more developed macro-colonies. Biofilm was almost absent at 7 days but left behind adhesive material and remnants of prior colonization. Evidence supports the hypothesis that bacterial colonization was repeatedly interrupted and constrained by the vector. Behaviors such as egestion and enzymatic salivation likely can loosen and eject Xf biofilm, perhaps when profuse biofilm interferes with ingestion. Thus, vector acquisition of Xf is a dynamic and stochastic process of interactions between bacteria and insects. We further hypothesize for future testing that the insect can regulate this interaction. A deep understanding of Xf acquisition will aid the ongoing development of grapevine resistance to vector transmission of xylellae diseases. IMPORTANCE Xylella fastidiosa (Xf) is one of the most destructive invasive plant pathogens in the world, able to hijack new vectors when it invades a region; yet the temporal interplay of bacterial colonization and insect behavior is unknown. This paper describes important findings about the process of Xf biofilm formation and maturation in a vector, contrasting similarities and differences with such formation in vitro. Results support the hypothesis that the behavior of the vector constrains and may regulate Xf biofilm formation, in dynamic competition with the bacterium. The data from this paper partly explain why Xf is so successful at invasion. Because the bacterium can be acquired and inoculated very quickly, it can move readily from old to new vectors and host plants in all-new environments. Our findings are relevant to biosecurity decisions because they demonstrate the importance of identifying potential vector species in the Xylella invasion front. [ABSTRACT FROM AUTHOR]

Lymphocystis disease has a broad host range and has been reported to enter Indonesia. However, information regarding its susceptibility and predilection organs in fish is lacking. This study examined the susceptibility of four important fish species in Indonesia, namely, giant gourami (Osphronemus goramy), hybrid tilapia (Oreochromis sp.), Siamese fighting fish (Betta splendens), and hybrid catfish (Clarias sp.). The fish were infected with virus filtrate by intraperitoneal injection and immersion. The postinfection observation period was 60 days. Viral load was quantified by qPCR and expressed as major capsid protein (MCP) copy number/mg tissue. Mortality was observed in all fish species, with the highest recorded in hybrid catfish and the lowest in Siamese fighting fish. All the fish species showed changes in their clinical symptoms, such as anorexia and separation from schools. However, only giant gourami showed internal change seven days after injection (dpi), with white lesion detected in the liver. Viral load quantification showed that LCDV had different predilection organs in the four fish species. The highest viral load of giant gourami (1.7 x 104) was observed in the liver at 7 dpi, hybrid tilapia (7.5 x 103) was observed in the fins at 21 dpi, Siamese fighting fish (8.4 x 103) was observed in the fins at 14 dpi, and hybrid catfish (1.2 x 103) were observed in the fins and gills at 7 and 14 dpi. The findings indicated that giant gourami, hybrid tilapia, Siamese fighting fish, and hybrid catfish were susceptible to LCDV infection with different predilection organs. [ABSTRACT FROM AUTHOR]

Background: Colorectal cancer (CRC) is associated with high incidence and mortality rates globally. The presence of intraperitoneal free cancer cells (IFCCs) is recognized as an independent prognostic factor for CRC patients. However, a clinical gold standard for IFCCs detection is lacking. The GILUPI CellCollector has demonstrated high sensitivity and specificity in detecting free cancer cells, yet its application for CRC IFCCs detection remains unreported. Methods: We selected CRC and normal cell lines to evaluate the CellCollector's ability to detect tumor cells. A total of 70 CRC patients and 17 patients with benign disease undergoing laparoscopic procedures were investigated. Peritoneal lavage fluid was collected pre‐ and post‐operation, and both real‐time PCR (CEA mRNA) and CellCollector detection were performed. We compared the sensitivity and specificity of these two methods. Results: CellCollector can distinguish well between CRC and normal cells in cell line experiments. CellCollector detects IFCCs better than real‐time PCR (CEA) in CRC patients in different TNM Stages. The sensitivity of CellCollector was higher than that of real‐time PCR (84.6% vs. 48.4%), and the specificity of CellCollector was also higher than real‐time PCR (79.1% vs. 60.4%). There was no significant difference in the results of IFCCs detected by CellCollector before and after total mesorectal excision (TME) or complete mesocolic excision (CME) radical colorectomy (p > 0.05), but there was a significant difference in real‐time PCR detection (p < 0.05). Conclusions: The CellCollector demonstrates superior sensitivity and specificity compared to real‐time PCR for detecting IFCCs in CRC patients, suggesting its potential as a clinical tool for IFCCs detection. Trial Registration: ClinicalTrials.gov identifier: NCT01978444 [ABSTRACT FROM AUTHOR]

Through this study, the synergistic behavior of small-molecular-weight, amphiphilic surfactant molecules and the triblock copolymer Pluronic 188 was extensively evaluated based on their ability to formulate nanocarriers with novel properties for the delivery of class II and IV (biopharmaceutical classification system) chemotherapeutic compounds. The combination of four different surfactants at multiple weight ratios and twelve initially formulated nanosystems resulted in four hybrid delivery platforms, which were further studied in terms of multiple physicochemical characteristics, as well as their stability in protein-rich media (fetal bovine serum/phosphate-buffer saline). Finally, we obtained a single final nanoformulation that exhibited a high loading capacity (%EE ≥ 75%) and a sustained drug release profile under physiological conditions (model drug methotrexate), without altering the original physicochemical characteristics of the carrier. With a mean hydrodynamic radius (Rh) of less than 70 nm, a polydispersity index of 0.219, and no protein complexation, the system is a suitable candidate for in vivo, intravenous, and/or intramuscular administration. The cytotoxicity and genotoxicity of both loaded and unloaded carriers were evaluated through the examination of the upregulation or downregulation of apoptosis-related pathways. Multiple conventional 2D and 3D spheroidal conformations were used for these assessments, including HEK293, HCT-116, and MCF-7 cell lines, the results of which stressed the safety and biocompatibility of the empty nanocarrier. Additionally, experiments on Caenorhabditis elegans were conducted to evaluate the system's in vivo toxicity, focusing on developmental stages, egg-laying behavior, and locomotion. Nanosystems studied in terms of chemotherapeutic encapsulation have mostly focused on the physiochemical aspect of the development of such novel delivery platforms, with only few exceptions proceeding step-by-step from cellular 2D to 3D to in vivo experimentation. The present study offers a holistic view of the behavior of such a novel system, advancing our understanding of the capabilities of polymeric/surfactant-based nanodelivery platforms. [ABSTRACT FROM AUTHOR]

One of the most critical goals in healthcare is safe and effective drug therapy, which is directly related to an individual's response to treatment. Precision medicine can improve drug safety in many scenarios, including polypharmacy, and it requires the development of new genetic characterization methods. In this report, we use real-time PCR, microarray techniques, and mass spectrometry (MALDI-TOF), which allows us to compare them and identify the potential benefits of technological improvements, leading to better quality medical care. These comparative studies, as part of our pharmacogenetic Five-Step Precision Medicine (5SPM) approach, reveal the superiority of mass spectrometry over the other methods analyzed and highlight the importance of updating the laboratory's pharmacogenetic methodology to identify new variants with clinical impact. [ABSTRACT FROM AUTHOR]

In 2023, a molecular study was conducted on the Hungarian goat population to determine genotypes and subtypes of small ruminant lentiviruses (SRLV) infecting these herds. Ten goat herds seropositive for SRLV infection according to a serosurvey conducted earlier in Hungary were selected, and 135 adult goats (>1 year old) were blood sampled. The two-stage nested real-time PCR (nRT-PCR) was used to detect proviral DNA of SRLV and distinguish between two main viral genotypes (A and B). PCR products were submitted for Sanger dideoxy sequencing, and phylogenetic and molecular evolutionary analyses were conducted on the 200–250 bp-long proviral DNA sequences from the end of long terminal repeat (LTR) region and beginning of gag gene using the MEGA11 software. Reference strains included strains most identical to Hungarian sequences according to the Standard Nucleotide BLAST and prototypic strains for the relevant genotypes and subtypes. Proviral DNA of SRLV was detected in goats from all ten tested herds. A single SRLV genotype was detected in 6 herds—genotype A in three herds and B also in three herds. In four herds, mixed infection with genotypes A and B was confirmed. In total, 110/135 seropositive goats tested positive in the nRT-PCR (81.5%): 49/110 goats (44.5%) for genotype A, 54/110 goats (49.1%) for genotype B, and 7/110 goats (6.4%) for both genotypes. Hungarian sequences belonged to subtypes A1/A18, A2, and subtype B1. This is the first study which shows that Hungarian goats are infected by SRLV belonging to both genotypes A and B. [ABSTRACT FROM AUTHOR]

Tularemia is an acute infectious disease classified as a natural focal infection, requiring continuous monitoring of both human and animal morbidity, as well as tracking of pathogen circulation in natural reservoirs and vectors. These efforts are essential for a comprehensive prevention and containment strategy. The causative agent, Francisella tularensis, comprises three subspecies—tularensis, holarctica, and mediasiatica—which differ in their geographic distribution and virulence. The ability to directly detect the pathogen and differentiate between subspecies has enhanced diagnostics and allowed a more accurate identification of circulation areas. Real-time PCR protocols for identification of F. tularensis subspecies tularensis and holarctica have been developed, utilizing specific primers and probes that target unique genomic regions. In this study, we present the development of a new real-time PCR assay for the detection of Francisella spp. and differentiation of F. tularensis subsp. mediasiatica. The specificity of the assay was tested on DNA from 86 bacterial species across 31 families unrelated to Francisella spp., as well as on DNA collections of F. tularensis subsp. mediasiatica and F. tularensis subsp. holarctica. The limit of detection (LOD95%) for real-time PCR in detecting Francisella spp. was 0.297 fg (0.145 genomic equivalents, GE) for holarctica DNA and 0.733 fg (0.358 GE) for mediasiatica DNA. The LOD95% for subspecies differential identification of mediasiatica was 8.156 fg (3.979, GE). The high sensitivity and specificity of these developed protocols enable direct detection of pathogens in biological and environmental samples, thereby improving the efficiency of tularemia surveillance in Kazakhstan. [ABSTRACT FROM AUTHOR]

The global rise in Helicobacter pylori (H. pylori)-related gastric complications is largely driven by increasing antimicrobial resistance and treatment failures. As a result, accurate diagnosis followed by effective treatment is crucial. We analyzed 232 gastric biopsy samples from patients undergoing endoscopy during the method validation phase, followed by 502 samples in the routine evaluation phase. Each sample was tested using the Allplex™ H. pylori and ClariR Assay on a CFX96™ real-time PCR (RT-PCR) system, with results processed through Seegene Viewer software. In the validation phase, RT-PCR results were compared to bacterial culture, while in the routine phase, they were compared to histology. The sensitivity and specificity for H. pylori detection were 100% and 96.05% (95% Confidence Interval [CI]: 93.38–98.73), respectively. For clarithromycin resistance detection, the sensitivity and specificity were 100% and 93.33% (95% CI: 84.4–100). Additionally, RT-PCR identified 11 positive samples (10.89%) that histology failed to detect. Incorporating the Allplex™ H. pylori and ClariR Assay into our laboratory workflow improved efficiency, reduced turnaround time (TaT), and proved to be more sensitive than both culture and histology combined. [ABSTRACT FROM AUTHOR]

The silkworm, Bombyx mori, is reared on a large scale, mainly for silk production. The waste from this silk production, like pupae, is underused. As an edible insect, B. mori is a good source of protein in human food and animal feed. In recent years, European legislation on the use of insects has evolved and a multitude of European companies have initiated the rearing of insects specifically for food and feed applications. Regarding animal feed, Commission Regulations (EU) 2021/1372 and 2021/1925 authorize eight insect species, including silkworm, as processed animal proteins for use in fish, pig, and poultry feed. The incorporation of edible insects into the human diet falls within Regulation (EU) No. 2015/2283 concerning novel foods. Implementation of authentication methods is imperative to ensure the conformity of the products. In the present study, we propose a specific real-time PCR method for the detection of silkworm (B. mori). The developed PCR test amplifies a 98 bp fragment of the cadherin gene. This gene is present in a single-copy per haploid genome, as demonstrated by experimental evidence. The qualitative method was successfully evaluated on the performance criteria of specificity, sensitivity, efficiency, robustness, and transferability. The applicability of the test was assessed on samples of B. mori from industry. Light microscopy and DNA metabarcoding approaches were used as a complement to genomic analysis as a means of providing authentication of the samples. [ABSTRACT FROM AUTHOR]

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For decades, the risk of exposure to infectious diseases in recreational beaches has been evaluated through the quantification of fecal indicator bacteria in water samples using culture methods. The analyses of sand samples have recently been developed as a complement to the monitoring of recreational waters in beach quality assessments. The growing use of molecular techniques for environmental monitoring allows for the rapid detection of pathogenic genes, thus providing more accurate information regarding the health risk of exposure to contaminated sand. The aim of this work was to determine the relationship between the fecal indicators abundance in water and sand and the presence of Shiga toxin-producer Escherichia coli (STEC) in sand by analyzing samples from touristic beaches using culture-dependent (fecal coliforms assay) and culture-independent (real-time PCR of stx1, stx2, and eae genes) techniques. We found a high concentration of coliform bacteria in water and sand in several beaches in eastern Uruguay, with different levels of sanitation networks and levels of urbanization. The presence of STEC virulence genes (mainly stx1) was confirmed in 8 out of 20 sand samples. The recreational use of sandy beaches may imply a risk to the health of its users, especially near streams and creek outflows, thus highlighting the need of monitoring sand bacteriological quality and pathogens using molecular tools. [ABSTRACT FROM AUTHOR]

Bipolar disorder (BD) is a psychiatric condition that is frequently misdiagnosed and linked to inadequate treatment. Long non‐coding RNAs (lncRNAs) have lately gained recognition as crucial genetic elements and are now regarded as regulatory mechanisms in the neurological system. Our objective was to measure the quantities of HOXA‐AS2 and MEG3 ncRNA transcripts. HOXA‐AS2 and MEG3 ncRNA levels were checked in the peripheral blood of 50 type I BD and 50 control samples by real‐time PCR. Furthermore, we conducted ROC curve analysis and correlation analysis to examine the association between gene expression and specific clinical characteristics in instances with BD. Additionally, a computational study was performed to investigate the binding sites of miRNAs on the HOXA‐AS2 and MEG3 lncRNAs. BD subjects showed a significant increase in the expression of HOXA‐AS2 and MEG3 compared to controls. The lncRNAs HOXA‐AS2 and MEG3 have an area under the ROC curve (AUC) values of 0.70 and 0.71, respectively. There was a significant correlation between the expression levels of ncRNAs HOXA‐AS2 and MEG3 in the peripheral blood of patients with BD and occupation scores. The data presented indicate a potential correlation between the expression of HOXA‐AS2 and MEG3 lncRNAs with an elevated risk of BD. Furthermore, these lncRNAs may be linked to several molecular pathways. Our findings indicate that the amounts of lncRNAs HOXA‐AS2 and MEG3 in transcripts might be a promising potential biomarker for patients with BD. [ABSTRACT FROM AUTHOR]

New technologies for more effective microbiological assays are being adopted by the food industry to intervene more rapidly in its production chain. The aim of this study was to evaluate the alternative methods of TEMPO® CAM and real-time PCR (rtPCR) Biotecon® in comparison with the ISO 10272-2:2017 reference method for Campylobacter spp. quantification in turkey meat, aiming to validate a quick and easily replicable method in these meat matrices. A total of 416 samples were analyzed over a one-year period. The TEMPO® methodology showed inadequate performance with a significant difference (p < 0.05) compared with the reference methodology; therefore, its use was not recommended for turkey meat matrices. However, the performance of the rtPCR Biotecon® methodology showed adequate performance with no significant difference (p > 0.05), and its use was recommended in turkey meat matrices. The study was limited to exclusive research in turkey meat matrices, and expansion of the research into other matrices is recommended to verify whether the behavior of alternative methodologies is similar. The findings of this study illustrate the necessity for a thorough and comprehensive evaluation during the implementation of alternative methodologies that may potentially supplant conventional approaches. [ABSTRACT FROM AUTHOR]

In the Baltic Sea, the dinoflagellates Apocalathium malmogiense, Biecheleria baltica, and Gymnodinium corollarium are important contributors to the spring bloom. However, their relative contribution to the bloom community cannot be unambiguously determined by conventional light microscopy due to a lack of resolution of distinctive morphological features of the three species. Here, we describe a molecular approach based on a quantitative real-time polymerase chain reaction (qPCR) primer and probe system, targeting the ITS1 and ITS2 regions of the rRNA gene for all three species and enabling their quantification. The specificity of the method was demonstrated using monocultures of A. malmogiense, B. baltica, G. corollarium as well as three other dinoflagellate species co-occurring in the Baltic Sea during spring and validated using field-collected phytoplankton samples. [ABSTRACT FROM AUTHOR]

Introduction: This study aimed to assess the diagnostic values of bronchoalveolar lavage fluid (BALF) real-time polymerase chain reaction (PCR) and BALF metagenomic next-generation sequencing (mNGS) for Pneumocystis jirovecii pneumonia (PJP) in patients infected with human immunodeficiency virus (HIV). Methods: A total of 99 HIV-infected PJP patients and 61 HIV-infected patients diagnosed with non-PJP pneumonia between March 2019 and December 2022 were enrolled. P. jirovecii and multiple other co-pathogens detected in BALF by mNGS were analyzed. The clinical final diagnosis was employed as a benchmark. We compared the diagnostic performance of mNGS in PJP with serum BDG and BALF real-time PCR. The mixed infections detected by mNGS and modifications of antimicrobial treatment were also analyzed. Results: The sensitivity of mNGS test of BALF samples reached 85.86%, which was significantly higher than serum BDG (39.39%, P < 0.001). The sensitivity of BALF P. jirovecii PCR (84.85%) was similar with mNGS (P > 0.05). The specificity of mNGS (100%) was also same as PCR (100.0%), and superior to serum BDG (88.52%, P < 0.001). Besides, mNGS performs remarkably well in identifying co-pathogens of PJP patients infected with HIV. In addition to P. jirovecii, 82 cases (82.83%) of other co-pathogens were identified based on mNGS. Moreover, thirty-four patients (34.34%) increased therapeutic dose of trimethoprim-sulfamethoxazole (TMP-SMZ) based on BALF P. jirovecii PCR. Based on the mNGS results, initial antimicrobial treatment was modified in 86.87% (86/99) of PJP patients. Conclusion: BALF mNGS and real-time PCR are two powerful techniques for rapid diagnosis of PJP with high specificity and sensitivity. Moreover, the benefit of mNGS is that it may identify other organisms besides PJP and it may benefit proper and prompt treatment. [ABSTRACT FROM AUTHOR]

Blood culture-negative endocarditis (BCNE) is a challenging disease because of the significant impact of delayed diagnosis on patients. In this study, excised heart valves and blood serum samples were collected from 50 BCNE patients in two central hospitals in Tehran, Iran. Sera were tested by IFA for the presence of IgG and IgM antibodies against Bartonella quintana and B. henselae. Genomic DNA extracted from the heart valves was examined for Bartonella-specific ssrA gene in a probe-based method real-time PCR assay. Any positive sample was Sanger sequenced. IgG titer higher than 1024 was observed in only one patient and all 50 patients tested negative for Bartonella IgM. By real-time PCR, the ssrA gene was detected in the valve of one patient which was further confirmed to be B. quintana. Bartonella-like structures were observed in transmission electron microscopy images of that patient. We present for the first time the involvement of Bartonella in BCNE in Iran. Future research on at-risk populations, as well as domestic and wild mammals as potential reservoirs, is recommended. [ABSTRACT FROM AUTHOR]

Background: Mycoplasma bovis is a global pathogen of cattle but was detected for the first time in New Zealand in 2017, triggering a response under their Biosecurity Act as an "unwanted organism". Following a lengthy eradication campaign, the Ministry of Primary Industries (MPI) now requires all bovine semen destined for export to New Zealand to be screened with an M. bovis-specific real-time PCR (rtPCR) compliant with amended import health standard (IHS) test requirements aimed at preventing the accidental importation of M. bovis. The standard stipulates that semen samples cannot be centrifuged prior to DNA extraction. To comply with these strict requirements, one of the listed tests was validated together with different DNA preparation steps and compared with existing in-house procedures. DNA was extracted from semen straws using the current in-house semi-automated platform procedures for processing culture, tissue and body fluid sample submissions and was compared with the stipulated test requirements. DNA from centrifuged and unspun semen samples spiked with M. bovis was also compared. Results: The rtPCR had a sensitivity and specificity of 100% (95% confidence interval = 79–100% and 74–100%, respectively) when testing DNA from other Mycoplasma species or bovine semen spiked with the latter, with a high level of repeatability for within- and between- run replicates. The consistent limit of detection was 0.001 pg/µl M. bovis DNA and between 5.3 × 102 and 7.5 × 102 CFU/ml M. bovis when artificially spiked in semen. DNA extracted using the KingFisher Flex was detected with lower Cq values than the Maxwell 16, but the comparable improvements in sensitivity were mainly associated with non-centrifuged samples (p < 0.001). None of the procedures tested impeded the detection sensitivity of M. bovis in the presence of competitor organisms Acholeplasma laidlawii, Mycoplasma bovigenitalium and Ureaplasma diversum, confirming M. bovis specificity of the polC target. Conclusions: Under the experimental conditions applied, this rtPCR test efficiently detected M. bovis in extended bovine semen straw samples from DNA extracted using both semi-automated extraction platforms, irrespective of prior centrifugation of extended semen. [ABSTRACT FROM AUTHOR]

Wastewater-based epidemiology has been used in pathogen surveillance for microorganisms at the community level. This study was conducted to determine the occurrence and trends of infectious pathogens in sewage from Yongin city and the relationships between these pathogens and the incidence of infectious diseases in the community. From December 2022 to November 2023, we collected inflow water from six wastewater treatment plants in Yongin city twice a month. The analyzed microorganisms included 15 respiratory viruses, 7 pneumonia-causing bacteria, 19 acute diarrhea-causing pathogens, SARS-CoV-2, Zika virus, hepatitis A virus, poliovirus, Mpox, and measles. They were detected through real-time PCR and conventional PCR. The concentrations of 9 pathogens among them were additionally analyzed using quantitative real time PCR. The correlation was confirmed through statistical analysis with the rate of detection for pathogens reported by the Korea Disease Control and Prevention Agency. Influenza A virus, human adenovirus, and human rhinovirus were moderately correlated (rho values of 0.45 to 0.58). Campylobacter spp. and sapovirus were strong correlated (rho values of 0.62, 0.63). Enteropathogenic E. coli, human coronavirus, and norovirus GII were very strong correlated (rho values of 0.86 to 0.92). We were able to identify the prevalence of respiratory viral infections, pneumonia, and acute diarrhea-causing pathogens in the community through wastewater-based epidemiology data. This study will be helpful in establishing a system for future surveillance of infectious diseases present in sewage. [ABSTRACT FROM AUTHOR]

The substitution of ingredients with Salmonidae, particularly Salmo salar, has led to widespread reports of financial losses and health risks globally, emphasizing the urgent need for the development of a rapid and precise method for species identification. The aim of the present study was to develop a novel method for the rapid screening of Salmonidae ingredients and the accurate detection of S. salar simultaneously using multiplex real-time PCR coupled with melting curve analysis. Specifically, primer sets specific for S. salar and Salmonidae were cross-confirmed. Moreover, the reaction system and conditions of a real-time duplex PCR were optimized, and the proposed methodology was verified, proving that the assay has good specificity and sensitivity. Clear and distinguishable melting peaks, with expected Tm values of around 80 °C (S. salar) and 84 °C (Salmonidae), were observed for twelve products, proving the presence of S. salar. However, four products were not derived from S. salar, but they could have belonged to another species within the Salmonidae family due to the presence of only one specific melting peak at a Tm value of about 84 °C. Therefore, the novel assay in the present study allows for the fast and accurate screening of Salmonidae ingredients and the detection of S. salar simultaneously. [ABSTRACT FROM AUTHOR]

Bariatric surgery is vital for sustainable weight loss and metabolic improvement in obese individuals, but its effects on gut microbiota and their role in these benefits require further investigation. Investigate the temporal changes in gut microbiota in obese patients undergoing bariatric surgery (gastric sleeve gastrectomy or Roux-en-Y Gastric Bypass (RYGB)) compared to healthy controls, aiming to understand their role in weight loss and metabolic health improvement. A case-control study included 30 obese patients aged 65–95 undergoing bariatric surgery, and 18 matched healthy controls. Selection criteria were based on age, race, BMI, history of antibiotics, probiotics, and prebiotics usage. Stool samples were collected at baseline, three months, and six months post-surgery for DNA extraction and quantitative real-time PCR analysis to assess gut microbiota changes. Physical activity and dietary intake were evaluated using standardized questionnaires. Statistical analyses were performed using R. Post-surgery, patients showed significant reductions in weight and BMI, with changes in dietary habits and physical activity. Quantitative real-time PCR analysis revealed substantial alterations in bacterial groups such as Bacteroides and Fusobacterium. However, some groups showed no significant changes, indicating a complex interaction between gut microbiota and bariatric surgery. Notable correlations were found between body weight, BMI, and specific bacterial groups like the C. cluster IV and Lactobacillus, particularly in RYGB patients. Bariatric surgery significantly alters gut microbiota, aiding weight loss and metabolic regulation in obese patients. Understanding these changes is crucial for developing effective obesity management strategies, requiring further research to optimize outcomes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background: Ovine enzootic abortion (OEA) caused by Chlamyidia abortus is one of the most important abortive disease in small ruminants. Diagnosis of Ovine enzootic abortion depends on the isolation and detection of the agent or its nucleic acid. The aim of the present study was to detect Chlamydia abortus using both isolation method and real-time PCR in Brucella free flocks in Iran. Methods: Twenty-eight vaginal and conjunctival swab samples which were Chlamydia abortus seropositive, were selected from ewes and does with recent abortion. Then the samples were tested by real-time PCR and positive molecular samples were inoculated into McCoy cells. Results: Using real-time PCR, 18 samples (64.3%) were positive and 7 (25%) of them were isolated in cell culture. Conclusion: The present results indicate that Chlamydia can play a relatively significant role in the abortion in does and ewes in Iran. Although the isolation of Chlamydia abortus have 100% specificity, because of low sensitivity, time consuming, cost and high probability of contamination, it is not suitable for routine laboratory diagnosis. Therefore, applying real-time PCR which have high sensitivity and specificity is recommended. [ABSTRACT FROM AUTHOR]

Infectious laryngotracheitis (ILT) is a WOAH-listed respiratory disease in poultry caused by Gallid alphaherpesvirus 1, known as ILT virus (ILTV). We monitored two unvaccinated commercial layer flocks of 46- and 64-weeks old birds, more than 3 months after the onset of ILT. For this purpose, tracheal swabs, cloacal swabs, and blood samples were collected. Molecular and serology results were compared with the mortality data. The increased mortality in flocks 1 and 2 lasted 9 and 15 days, reaching 13.0% and 11.3%, respectively. We isolated the virus by inoculation on chicken embryo's chorioallantoic membrane. Tracheal swabs were positive at each sampling point, but cloacal swabs were negative. Based on the molecular and phylogenetic analysis of the ICP4 gene, the ILTV closely matched vaccine strains. In flock 1, seroconversion was evident at the second sampling (day 15). Thereafter, an increase in antibody titer was observed, eventually achieving levels that were nearly identical to those on day 15 and on 109. During the acute period of the outbreak, seroconversion was already visible in flock 2, and a similar pattern was then seen as in flock 1. Three months after the outbreak, the virus DNA was still persistently detected in tracheal swabs. [ABSTRACT FROM AUTHOR]

In the present study, we investigated to what extent soybean was used in industrial and homemade sausage produced in Kosovo, as well as its potential impact on the fatty acid profile. In total, 63 samples, 42 industrial and 21 traditional sausages, were collected either from the market or the production site. All samples were tested by means of real-time PCR for the detection and quantification of soy content, as well as the GC-FID to determine the potential reflection in the fatty acid profile. The presence of soy DNA was detected in 54 out of 63 samples. In total, 41 out of 42 industrial sausage samples were positive for soy DNA, with an average ct value of 22.60 and a standard deviation (SD) of 4.28, whereas 13 out of 21 traditional sausage samples were positive for soy DNA, with a mean ct value of 23.36 and a standard deviation (SD) of 4.56. There was a statistically significant difference in means between industrial sausage and traditional sausage, with p ≤ 0.001 based on CT values (ANOVA test). We investigated the correlation between ct values in real-time PCR for soy DNA with each fatty acid content. There is a moderate correlation of soy DNA ct values with C16:0 palmitin (decrease), C18:0 stearic acid (decrease), C18:1 oleic acid (increase), and overall saturated fatty acids (decrease). With the exception of C14:0 Myristin, C18:1 oleic, and C20:0 Arachin acids and monosaturated fats, the ANOVA test reveals a significant difference in means between groups for the majority of the fatty acids between industrial sausages and traditional sausages. The current study demonstrates that the fatty acid composition of sausages is influenced by the amount of soy present in them. The extent to which other components affect the fatty acid profile is unknown. However, an increase in oleic acid and a decrease in stearic and overall saturated fatty acids are expected. [ABSTRACT FROM AUTHOR]

The African swine fever virus (ASFV) causes severe disease in wild and domestic pigs, with high mortality rates, extensive spread, and significant economic losses globally. Despite ongoing efforts, an effective vaccine remains elusive. Therefore, effective diagnostic methods are needed to rapidly detect and prevent the further spread of ASF. This study assessed nine commercial kits based on real-time polymerase chain reaction (PCR) approved in the Republic of Korea using the synthesized ASFV plasmid, 20 food waste samples, and artificially spiked samples (ASSs). The kits were evaluated for their diagnostic sensitivity, specificity, cost per reaction, and reaction running time. In addition, the results were compared with those of the World Organization for Animal Health (WOAH) standard methods. Three commercial kits (VDx® ASFV qPCR Kit, Palm PCR™ ASFV Fast PCR Kit, and PowerChek™ ASFV Real-time PCR Detection Kit Ver.1.0) demonstrated the highest sensitivity (100 ag/μL), cost-effectiveness (less than KRW 10,000), and shortest running time (less than 70 min). These kits are suitable for the monitoring, early diagnosis, and prevention of the spread of ASF. This is the first report on the performance comparison of ASFV diagnostic kits approved in the Republic of Korea, providing valuable information for selecting kits for testing with food waste samples. [ABSTRACT FROM AUTHOR]

The ascomycete Hymenoscyphus fraxineus causes the devastating ash dieback disease of European ash (Fraxinus excelsior L.). Spore traps are often used to measure the amount of ascospores in the environment, but the pathogen-load of the soil in ash stands has not been recorded so far. This is of particular interest with regard to the occurrence of ash stem necrosis, a decisive factor for the severe course of the disease. In order to gain a more differentiated insight into the pathogen-load in ash stands, we analysed soil samples from four ash tree sites in southern Germany, covering a clone plantation, two seed orchards and a forest. The pathogen-load was determined using a quantitative TaqMan real-time PCR assay for ten to twenty plots per stand. Results obtained by the species-specific assay highlighted that the pathogen-load is heterogeneously distributed in the ash stands. H. fraxineus DNA targets were detected in 17% of the soil samples. The pathogen-load differed according to soil depth, with the highest pathogen abundance in the top 5 cm, followed by 5–10 cm and finally 10–15 cm. Pathogen-load and thereby infection pressure were found to be highly variable for the individual trees in one stand. Overall, the study discovered detectable levels of H. fraxineus in the soil of all four study sites, which supports the hypothesis that H. fraxineus can be found in the soil of ash stands. The qPCR approach was found to be an effective method for monitoring the load of H. fraxineus in soil and for demonstrating the successful application of the method on the sample type of custom-made spore traps. Results suggest the implication of site-specific pathogen-load determination in future H. fraxineus-monitoring and selection of less susceptible ash trees for breeding and seed production. [ABSTRACT FROM AUTHOR]

Waiteacircinata var. zeae, a pathogen with a relatively narrow host range, has recently been detected in cabbage and oilseed rape in Europe and worldwide. In this study, we developed specific conventional and real-time PCR protocols for direct detection of W.circinata var. zeae from mycelium and diseased plant tissue. The newly developed primer pair zeaefor1/zeaerew1, used in PCR protocols, specifically amplified only target isolates of W.circinata var. zeae when tested against isolates of 11 different binucleate and multinucleate anastomosis groups of Rhizoctonia spp. including AG-A, AG-G, AG-F, AG-U, AG-2-1, AG-2-2, AG-3, AG-4 HGI, AG-4 HGII, AG-4 HGIII, and AG-6 and common soil-borne pathogens. Total of nine previously published primer pairs designed for the detection of various Rhizoctonia spp. were also tested and did not amplify target isolates of W.circinata var. zeae. The detection limit of conventional and real-time PCR protocols was 10–2 and 10–5 (with starting concentration 9.5 ng/µl), respectively, and both methods are the first available tools for direct detection and identification of W.circinata var. zeae from mycelium and diseased oilseed rape seedlings. Both conventional and SYBR-Green-based real-time PCR protocols are cost-effective and provide a solid basis for further investigations of W.circinata var. zeae, particularly in relation to distribution, host range, and epidemiology. [ABSTRACT FROM AUTHOR]

Context: Alpine pastures are seminatural grasslands which play a crucial role in biodiversity conservation, service provisioning, and mountain livestock systems. The soil microbial communities of pasture are fundamental in ecosystem nutrient cycles, but they are relatively underexplored in European Alpine pastures. Aims: We explored the many soil microbial genes encoding key functions in the nitrogen cycle in three historical alpine pastures grazed by dairy cattle, considering different soils, temporal dynamics, and exclusion of cattle grazing for one summer. Methods: 216 samples were collected across four sampling times. The abundance of genetic determinants involved in nitrogen fixation (nifH), nitrification (amoA bacterial and archaeal), and denitrification (nirK and nosZ) were quantified using real-time polymerase chain reaction. Key results: The terminal denitrification nosZ gene was the most sensitive indicator and responded significantly to soil chemical composition and animal grazing. Sampling time affected nitrogen fixation nifH and intermediate denitrification nirK in relation to rainfall cumulation dynamics. The amoA nitrification genes showed high variability but no significant effects from the tested factors. Conclusions: In spite of a general homeostatic trend occurring in these habitats and of the short term analysis, some genes acted as sensitive reporters of soil compositional differences, intraseasonal climatic variations, and grazing disturbance. Implications: A stocking rate of >0.6 livestock units per hectare can be recommended, to combine animal production with conditions that favour complete denitrification, thus potentially reducing the nitrous oxide greenhouse gas emissions. Higher livestock grazing intensity can be withstood by the ecosystem without denitrification-related drawbacks when the preceding 10 days display a cumulated rainfall lower than 22 mm. Soil microbial communities are fundamental for ecosystem nutrient cycles, but they are relatively underexplored in alpine pastures – seminatural grasslands important for mountain livestock systems. We investigated microbial functions related to the nitrogen cycle in three historical alpine pastures for one summer. Microbial nitrogen functions showed a general inertia in respect to soil variation and grazing disturbance. This has ramifications for future improvement of pasture management in mountain livestock systems. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Porcine adulteration in food products is unacceptable to consumers who avoid pork consumption due to religious or health reasons; hence, detecting pork and its derivatives in food products is vital. The present work focused on assessing the DNA extraction efficiency of salt method as compared to the DNeasy mericon Food Kit to detect porcine DNA via quantitative PCR (qPCR), and comparing these qPCR findings with the food product labelling. The study selected food products which lacked JAKIM (Jabatan Kemajuan Islam Malaysia) certified halal logo, and those bearing foreign or counterfeit halal logos in Sibu, Sarawak. Twenty-four (n = 24) commercial food products, three (n = 3) pork-based products (positive control), and three (n = 3) JAKIM halal-certified products (negative control) were included. DNA was isolated and used as a template in a qPCR assay to target cytochrome b (cytb). Positive samples were sent for DNA sequencing. The experimental output was compared with the food ingredient and presence of a halal logo on product labelling. Out of 30 samples extracted using the DNeasy mericon Food Kit, DNA from all samples (100%) fell within the optimal DNA purity ratio which ranged from 1.7 to 2.0. The DNA extracted using this method was further used as a template in the qPCR. The qPCR assay demonstrated presence of porcine DNA in two food samples which lacked product labelling, with mean Ct values ± SD of 19.05 ± 0.72 and 28.07 ± 1.67 as compared to the positive control (mean Ct values ± SD of 13.44 ± 0.37 to 14.78 ± 1.10). Basic Local Alignment Search Tool (BLAST) analysis revealed a high percentage identity (94.74 - 100%) to Sus scrofa domesticus (pig) as compared to sequences in the National Centre for Biotechnology Information (NCBI) database. The present work demonstrated a significant halal status of various food items for Muslims and individuals with pork allergies in the studied area. [ABSTRACT FROM AUTHOR]

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/µL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1. [ABSTRACT FROM AUTHOR]