UNASSIGNED: Tick-borne encephalitis virus (TBEV) infections result in severe central nervous system diseases in humans across Asia and Europe. In China, cases of tick-borne encephalitis are primarily caused by the Far East subtype of TBEV, which exhibits a distinct disease course compared to other extensively studied subtypes. However, there is limited knowledge regarding the nucleic acid and serological diagnostic characteristics of patients infected with the TBEV in China, which is the focus of investigation in the present study.
UNASSIGNED: This study established a TaqMan qPCR approach to detect TBEV RNA in the serum with optimal specificity, sensitivity, and precision. Using TaqMan qPCR and ELISA assay for TBEV IgM detection, serum samples from 63 hospitalized patients bitten by ticks in Northeast China were investigated for diagnostic characteristics.
UNASSIGNED: Twenty-five patients were positive for viral RNA; nineteen patients were positive for IgM, and nine were positive for both viral RNA and IgM. Through comparative analysis, TBEV RNA copies were negatively correlated with the virus incubation period. IgM levels were positively correlated with the clinical symptom scores of patients. The severity of clinical symptoms and the length after the tick bite could be used to predict the IgM occurrence. Furthermore, IgM levels and viral RNA copies were not correlated in double-positive patients.
UNASSIGNED: Both nucleic acid and serological detection methods exhibited distinct windows for detecting TBEV infection, with some overlap, and were associated with specific correlated factors. This study provided novel insights into the diagnosis and course of TBEV-induced tick-borne encephalitis in China.

While waters might be contaminated by oocysts from >40 Cryptosporidium species, only viable oocysts of C. parvum and C. hominis truly pose the main health risk to the immunocompetent population. Oocyst viability is also an important but often neglected risk factor in monitoring waterborne parasites. However, commonly used methods in water monitoring and surveys cannot distinguish species (microscopic observation) or oocyst viability (PCR), as dead oocysts in water could retain gross structure and DNA content for weeks to months. Here, we report new TaqMan qRT-PCR/qPCR assays for quantitative detection of viable C. parvum and C. hominis oocysts. By targeting a hypothetical protein-encoding gene cgd6_3920 that is highly expressed in oocysts and variable between species, the qRT-PCR/qPCR assays achieve excellent analytical specificity and sensitivity (limit of quantification [LOQ] = 0.25 and 1.0 oocyst/reaction). Using calibration curves, the number and ratio of viable oocysts in specimens could be calculated. Additionally, we also establish a TaqMan-18S qPCR for cost-effective screening of pan-Cryptosporidium-positive specimens (LOQ = 0.1 oocyst/reaction). The assay feasibility is validated using field water (N = 43) and soil (79) specimens from 17 locations in Changchun, China, which detects four Cryptosporidium species from seven locations, including three gp60-subtypes (i.e., IIdA19G1, IIdA17G1 and IIdA24G2) of C. parvum oocysts showing varied viability ratios. These new TaqMan q(RT)-PCR assays supplement current methods in the survey of waters and other samples (e.g., surfaces, foods and beverages), and are applicable to assessing the efficiency of oocyst deactivation protocols.

BACKGROUND: To establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion.
RESULTS: Fifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes.
CONCLUSIONS: Microbiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

Background: Breast cancer is the most prevalent cancer among women worldwide. The potential involvement of Epstein-Barr virus (EBV) in breast cancer pathogenesis has been a subject of debate, but its correlation with clinical outcomes remains uncertain. Methods: In this study, we collected 276 pathologically confirmed breast cancer tissue samples from the tissue bank of MacKay Memorial Hospital and the National Health Research Institutes in Taiwan. DNA was extracted from frozen tissue using The QIAamp DNA Mini Kit. The Taqman quantitative PCR method was employed to assess the EBV copy number per cell in these samples, using NAMALWA cells as a reference. We performed statistical analyses, including 2 × 2 contingency tables, Cox regression analysis, and Kaplan-Meier survival curves, to explore the association between clinicopathologic factors and survival outcomes in breast cancer patients. We analyzed both relapse survival, which reflects the period patients remain free from cancer recurrence post-treatment, and overall survival, which encompasses all-cause mortality. Results: Our results revealed a significant association between EBV status and relapse survival (hazard ratio: 2.75, 95% CI: 1.30, 5.86; p = 0.008) in breast cancer patients. However, no significant association was found in overall survival outcomes. Additionally, we observed significant associations between ER status and tumor histologic grade with both overall and relapse survival. Patients with EBV-positive tumors exhibited higher recurrence rates compared to those with EBV-negative tumors. Furthermore, we noted significant correlations between EBV status and HER-2 (p = 0.0005) and histological grade (p = 0.02) in our cohort of breast cancer patients. Conclusions: The presence of EBV in breast cancer tumors appears to exert an impact on patient outcomes, particularly concerning recurrence rates. Our findings highlight the significance of considering EBV status as a potential prognostic marker in breast cancer patients. Nonetheless, further research is essential to elucidate the underlying molecular mechanisms and develop novel therapeutic approaches.

OBJECTIVE: To identify a microRNA signature associated to sarcopenia in community-dwelling older adults form the SarcoPhAge cohort.
METHODS: In a screening phase by next generation sequencing (NGS), we compared the hsa-miRome expression of 18 subjects with sarcopenia (79.6 ± 6.8 years, 9 men) and 19 healthy subjects without sarcopenia (77.1 ± 6 years, 9 men) at baseline. Thereafter, we have selected eight candidate hsa-miRNAs according to the NGS results and after a critical assessment of previous literature. In a validation phase and by real-time qPCR, we then analyzed the expression levels of these 8 hsa-miRNAs at baseline selecting 92 healthy subjects (74.2 ± 10 years) and 92 subjects with sarcopenia (75.3 ± 6.8 years). For both steps, the groups were matched for age and sex.
RESULTS: In the validation phase, serum has-miRNA-133a-3p and has-miRNA-200a-3p were significantly decreased in the group with sarcopenia vs controls [RQ: relative quantification; median (interquartile range)]: -0.16 (-1.26/+0.90) vs +0.34 (-0.73/+1.33) (p < 0.01) and -0.26 (-1.07/+0.68) vs +0.27 (-0.55/+1.10) (p < 0.01) respectively. Has-miRNA-744-5p was decreased and has-miRNA-151a-3p was increased in the group with sarcopenia vs controls, but this barely reached significance: +0.16 (-1.34/+0.79) vs +0.44 (-0.31/+1.00) (p = 0.050) and  +0.35 (-0.22/+0.90) vs  +0.03 (-0.68/+0.75) (p = 0.054).
CONCLUSIONS: In subjects with sarcopenia, serum hsa-miRNA-133a-3p and hsa-miRNA-200a-3p expression were downregulated, consistent with their potential targets inhibiting muscle cells proliferation and differentiation.

Proliferative enteropathy caused by Lawsonia intracellularis is an important economic associated disease to pig industry, but the knowledge about the prevalence of L. intracellularis in pig farms in China is limited. In addition, there is no complete genome sequence available for L. intracellularis isolates from China. In this study, we developed a TaqMan qPCR for the screening of L. intracellularis by targeting the bacterial 16S rDNA gene. Laboratory evaluations revealed a good sensitivity and specificity on detecting L. intracellularis nucleic acid. Using this method, we investigated 891 fecal samples from apparently healthy pigs in 47 farms. The results demonstrated a screening positive rate of 37.3% (95% CI, 34.1-40.5%) for the samples, and a farm screening positive rate of 93.6% (95% CI, 65.3-94.4%). The screening positive rate at herd level ranged from 6.67% (95% CI, 0.2-31.9%) to 40% (95% CI, 38-79.6%), while at animal level, the highest screening positive rate was found in 12-week-old pigs [85.7% (95% CI, 67.3-96.0%)]. Investigation of 705 diarrheal or bloody feces from symptomatic pigs revealed that the highest positive rate was found in replacement gilts which was 37.18% (95% CI, 45.1-89.5%). Secondly, we conducted the complete genome sequence of a L. intracellularis PPE-GX01-2022 from China through PacBio sequencing. The genome of PPE-GX01-2022 consisted of a chromosome of 1,439,110 bp in length and three plasmids of 193,063, 39,799, and 27,067 bp, respectively. This genome encoded 1,428 predicted proteins, 44 tRNAs, and 6 rRNAs. Sequence comparisons demonstrated that the genome sequence of PPE-GX01-2022 was highly homologous to those of two isolates from US, and these three isolates shared 1,378 core genes. The screening results suggest a high prevalence rate of L. intracellularis in Chinese pig farms. In addition, the genome sequence of the Chinese isolate was highly homologous to those of the field isolates from the US.

A highly sensitive quantitative PCR (qPCR) method was developed for detection and quantification of Bacillus velezensis HMB26553 in cotton rhizosphere. The study aimed to develop a quantitative detection method for the strain HMB26553, and explore the relationship between its colonization of the cotton rhizosphere and its control effect. The whole genome sequence of strain HMB26553 was obtained by genome sequencing and a unique specific sequence pB-gene0026 on plasmid plaBV2 was identified by using high-throughput alignment against NCBI. Plasmid plaBV2 could be stably genetically inherited. Based on this sequence, specific primers for amplifying 106 bp and a minor groove binder (MGB) TaqMan probe for enhancing sensitivity were designed. The copy number of plaBV2 in strain HMB26553, which was 2, was confirmed by internal reference primers and the MGB TaqMan probe based on housekeeping gene gyrB. The established detection technique based on these primers and probes had high specificity and sensitivity compared to traditional plate counting method, with a detection limit of 1.5 copy genome. Using this method, the study discovered a likely correlation between the quantity of colonization in cotton rhizosphere and efficacy against cotton damping-off caused by Rhizoctonia after seed soaking and irrigation with strain HMB26553. Thus, this method provides scientific support for the rational application of strain HMB26553 in the future.

Humans are exposed to Toxoplasma gondii infection as pet cats gradually become family members and represent an increasing public health risk worldwide. Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this study, real-time fluorescence quantitative loop-mediated isothermal amplification (qLAMP) and visual LAMP detection technologies were established to conduct tests of T. gondii based on the membrane DNA extraction method, and the optimal detection mix was determined by adding the protective reagent trehalose and screening the concentrations of Mg2+ and dNTPs. Paraffin and lyophilization were used to reduce and even remove aerosol pollution, constructing a detailed anti-contamination protocol. Based on the positive standard plasmid DNA, the LODs of qLAMP and visual LAMP were 92 copies/μL and 92 copies/μL, and the standard curve of qLAMP was Y=2.9503X+20.8992 with R2 = 0.99. The applicability of the qLAMP and visual LAMP assays in disease diagnosis was assessed by evaluating 200 clinical cat faeces samples. The assays showed good diagnostic consistency, with kappa values of 1.0 and 0.99 compared with TaqMan qPCR, respectively. Compared with TaqMan qPCR, the diagnostic specificity/sensitivity of qLAMP and visual LAMP were 100%/100% and 100%/80%, respectively. The qLAMP and visual LAMP assays reported here are rapid and simple tests without extensive sample preparation and have a short turnaround time within 60 min, making them suitable for point-of-care testing.

Apple blotch (AB) is a major disease of apple in Asia and recently emerged in Europe and the United States. It is caused by the fungus Diplocarpon coronariae (formerly Marssonina coronaria; teleomorph: Diplocarpon mali) and leads to severe defoliation of apple trees in late summer, resulting in reduced yield and fruit quality. To develop effective disease management strategies, a sound knowledge of the pathogen\'s biology is crucial. Data on the early phase of disease development are scarce: No data on spore dispersal in Europe are available. We developed a highly sensitive TaqMan qPCR method to quantify D. coronariae conidia in spore trap samples. We monitored temporal and spatial dispersal of conidia of D. coronariae and the progress of AB in spring and early summer in an extensively managed apple orchard in Switzerland in 2019 and 2020. Our results show that D. coronariae overwinters in leaf litter, and spore dispersal and primary infections occur in late April and early May. We provide the first results describing early-season dispersal of conidia of D. coronariae, which, combined with the observed disease progress, helps to understand the disease dynamics and will be a basis for improved disease forecast models. Using the new qPCR method, we detected D. coronariae in buds, on bark, and on fruit mummies, suggesting that several apple tissues might serve as overwintering habitats for the fungus, in addition to fallen leaves. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.