Search Results (31)

Column-based RNA extraction is time-consuming, involves cumulative losses during transfer, wash, and elution steps, and has reduced recovery from sub-microliter inputs. These limitations restrict gene-expression analysis in small cell populations and low-titre pathogen detection in clinical specimens. We developed the Direct Cell-to-PCR Lysis Buffer, a defined non-ionic detergent formulation containing Tween 20 (0.3%), Triton X-100 (0.1%), and NP-40 (0.1%), for RNA-extraction-free one-step RT-qPCR. Lysates are added directly to reactions without pretreatment, heating, or column purification. Non-ionic detergents produced Ct values comparable to detergent-free controls, whereas SDS, Sarkosyl, and sodium deoxycholate abolished amplification. The method was benchmarked against the standard column-based PureLink RNA Mini Kit using SaOS-2 cells, MCF7 cells, peripheral blood mononuclear cells, and nasal and throat swabs. Direct lysates showed Ct offsets of 0.6 to 2.85 cycles compared with purified RNA, depending on sample type. Ubiquitin-C mRNA was detected down to a single-cell-equivalent input, and plasmid templates were detected to approximately 100 copies per reaction. The buffer was compatible with multiplex ScriptTaq COVID PCR, with unchanged RdRP, N, and RPP30 Ct values, and tolerated sodium azide up to 0.1%, supporting rapid direct RT-qPCR workflows. Full article

Abortion in cattle entails substantial economic loss, and rapid identification of abortigenic pathogens is critical for timely on-farm response and reduction in human exposure risk. In 2024, two Holstein cows from a small farm in Inner Mongolia aborted in close succession without an obvious cause. Vulvar swabs from both cows, one afterbirth sample, and whole blood from one aborted fetus were collected. Shotgun metagenomic sequencing was performed, followed by host-read removal, taxonomic profiling with Kraken2, de novo assembly of Brucella-aligned reads, and whole-genome comparison. Serological tests, Gram-stained smears, and Brucella genus- and species-specific qPCR assays were used as orthogonal verification. Putative resistance and virulence determinants were screened against CARD and VFDB. Brucella reads were detected in all samples, with the highest relative abundance in the 138-afterbirth (96%). qPCR assays detected Brucella DNA and B. abortus-specific signals in all four samples. A draft Brucella genome was assembled from the 138-afterbirth sample and was phylogenetically placed within B. abortus, showing relatedness to previously circulating Chinese lineages. Cows 138 and 198 were RBT-positive with SAT titres of 1:100 (++). No acquired Brucella resistance genes were identified in CARD. Within 72 h of sample receipt, B. abortus was reported to the farm and local authorities and emergency biosecurity measures were implemented. This field investigation shows that metagenomic sequencing, when combined with conventional serology, microscopy, and targeted qPCR, can support rapid etiological investigation when culture is delayed, hazardous, or biosafety level 3 facilities are unavailable. Full article

Background and Clinical Significance: Waldenström macroglobulinemia (WM) is a rare, indolent B-cell non-Hodgkin lymphoma, characterised by the presence of monoclonal immunoglobulin M (IgM) and lymphoplasmacytic infiltration of the bone marrow. It is often associated with various haematological and systemic disorders, including previous cold agglutinin disease (CAD), a condition where cold-sensitive antibodies lead to haemolysis. Case Presentation: A 55-year-old male patient was admitted to the Internal Diseases Ward with symptoms of weakness, reduced effort tolerance, and weight loss, along with life-threatening normoblastic anaemia (haemoglobin [Hb]: 3.90 g/dL). Initial blood tests raised suspicion of CAD due to the presence of multiple blood clots, as well as a decrease in lymphocyte and neutrophil counts. CAD was then confirmed by a cold agglutinin titre of 1:2000 and direct antiglobulin test ([DAT] 4+). Two weeks later, upon transfer to the Haematological Diseases Ward, further investigation revealed elevated IgM levels (up to 31.55 g/L). Additional diagnostic tests, including serum protein electrophoresis, imaging, multiparametric flow cytometry, and bone marrow biopsy, confirmed the diagnosis of WM. The L265P MYD88 mutation test was positive. Treatment with intravenous rituximab was initiated, followed by bendamustine/rituximab (BR) therapy protocol as first-line treatment. After two cycles, the patient’s clinical condition and laboratory results significantly improved, with a marked reduction in IgM (<0.4 g/L). Hb levels steadily rose to 12.60 g/dL, eliminating the need for further blood transfusions. Conclusions: This case highlights the importance of recognising the coexistence of CAD and WM, which may present with overlapping clinical features, including life-threatening anaemia. Extensive diagnostics and prompt treatment with combination therapy can lead to effective clinical improvement. Full article

Background: IgM antiphospholipid antibodies (aPL) were de-emphasised in the 2023 ACR/EULAR criteria, yet their precise clinical significance remains uncertain. Methods: A rapid scoping review of PubMed (January 2000–June 2025) identified original human studies reporting IgM aCL, aβ₂GPI, or aPS/PT prevalence or outcomes; 40 studies met the eligibility criteria. Prevalence and odds ratios (ORs) of clinical associations were extracted. Results: IgM aPL are common across APS phenotypes. Obstetric cohorts showed aCL-IgM prevalences of 3–82%, often equal to or exceeding those of IgG, while aβ₂GPI-IgM reached a prevalence of 2–63%. In mixed thrombotic–obstetric cohorts, aPS/PT-IgM was the most frequent isotype (31–79%). Purely thrombotic studies still reported 0–59% aβ₂GPI-IgM, with PS/PT-IgM at 55% and 62% in two large series. Significant outcome signals from clinical associations of IgM aPL were inconsistent but noteworthy in (i) pregnancy loss for high-titre aCL, aβ₂GPI, and aPS/PT, (ii) thrombosis driven by aPS/PT and (iii) organ-specific arterial events (retinal thrombosis and stroke) in isolated IgM phenotypes. Conclusions: The role of aPL-IgM remains uncertain. The findings advocate for a nuanced approach to IgM interpretation, supporting its reconsideration in specific clinical settings and emphasising the significance of ongoing research into the mechanistic and prognostic utility of IgM aPL. Full article

Myelin oligodendrocyte glycoprotein antibody–associated disease (MOGAD) is an inflammatory demyelinating disorder of the central nervous system characterised by heterogeneous clinical and radiological presentations. Accurate interpretation of serum anti–myelin oligodendrocyte glycoprotein (anti-MOG) antibody titres is critical to improve diagnostic precision and prognostic assessment. This single-centre retrospective study evaluated 19 patients diagnosed with MOGAD in 2023, all of whom were seropositive for anti-MOG IgG, as confirmed by live cell-based assays (CBAs) using full-length human MOG and IgG1-specific secondary antibodies. Antibody quantification combined a ratiometric semi-quantitative fluorescence index with classical endpoint dilution titres, enabling classification into low, medium, and high titre groups. Stratification revealed titre-dependent phenotypic heterogeneity: high-titre patients were older at onset and predominantly presented with optic neuritis, often bilateral, and encephalic involvement, whereas low-titre patients more frequently exhibited spinal cord syndromes, cerebellar or brainstem symptoms, and a higher prevalence of cerebrospinal fluid-restricted oligoclonal bands. Semi-quantitative fluorescence ratios correlated consistently with endpoint titres, and exponential decay analysis demonstrated slower signal loss in high-titre sera, confirming assay reliability. No significant association emerged between titre level and monophasic versus relapsing disease course. Anti-MOG antibody titres could serve not only as a diagnostic biomarker but also to capture clinically relevant immunopathological diversity, supporting a titre-stratified approach to diagnosis and early prognostication. Incorporating semi-quantitative metrics alongside clinical and imaging features may refine the diagnostic algorithm and prevent misclassification of atypical presentations. Full article

The Peste des Petits Ruminants (PPR) live attenuated vaccines, the PPR virus (PPRV) Nigeria 75/1 strain (lineage II) and PPRV India Sungry 96 strain (lineage IV), currently used for control and eradication programme are very efficient vaccines as they provide the host, sheep and goats, a lifelong immunity after a single minimum recommended dose of 10^2.5 TCID₅₀/mL. Unfortunately, both live attenuated vaccines are thermolabile and their use requires maintaining the cold chain from the manufactory premises to the field as most PPR-infected regions are facing of hot climate, with poor infrastructure, and the maintenance of an effective cold chain remains a challenge. To address this challenge, efforts have focused on developing thermotolerant (ThT) PPR vaccines using different stabilisers and improving the freeze-drying process. This study aimed to define the criteria for the evaluation of the stability of ThT PPR vaccines. A total of 37 batches of freeze-dried PPR vaccines using the PPRV Nigeria 75/1 strain, including eight (8) and twenty-nine (29) vaccines labelled as ThT and conventional formulations, respectively, were tested to evaluate the stability at temperatures of 40 °C to simulate the field conditions in some hot climate regions. All the vaccine batches included in this study initially showed acceptable levels of residual moisture, below 3%, and titres above the minimum WOAH standard requirement of 10^2.5 TCID₅₀/mL. Following the incubation at 40 °C, 56.7% and 46% of the 37 vaccine batches tested retained titres above 10^2.5 TCID₅₀/mL on day 3 and day 5, respectively. These vaccines use stabilisers such as skimmed milk, lactalbumin–sucrose, trehalose and one unnamed product (which may be protected for patent). The mean of titre loss among the PPR vaccines maintaining titres above 10^2.5 TCID₅₀/mL was 0.78 log10 at day 3 and 0.99 log10 at day 5, suggesting a significant early degradation during the first 3 days. Based on these data, it is proposed that thermotolerant PPR vaccines should maintain a minimum titre of 10^2.5 TCID₅₀/mL for vaccine dose on day 5 post-incubation at 40 °C with a titre loss below 1 log10 per mL. Preliminary immunogenicity test results showed that the PPR ThT vaccine meeting this criterion could be used in the field without maintaining a cold chain for up to 3 weeks, offering a practical solution for vaccination in remote areas. Full article

Peste des Petits Ruminants (PPR) disease is widely distributed in Africa. Live attenuated PPR vaccines are produced using approved Nigeria 75/1 and Sungri/96 strains by the World Organisation of Animal Health (WOAH) to control the disease. These PPR vaccines are very efficacious; however, the main challenge is the maintaining of the cold chain during vaccine distribution and delivery. This study evaluated the thermotolerance of freeze-dried and reconstituted PPR Nigeria 75/1 vaccines from vaccine manufacturers using eight stabilizer formulations (lactalbumin hydrolysate and sucrose, sucrose and peptone, Weybridge medium, trehalose, Lactose and N-Z Amine, lactalbumin hydrolysate, sucrose and L glutamine, skimmed milk, and lactalbumin hydrolysate, maltose and gelatine). Aliquots of the reconstituted PPR vaccine batches were titrated after 2, 4, and 6 h of storage at 4 °C and 40 °C. The PPR vaccines were also titrated after storage at 40 °C and 45 °C for 3 and 5 days. The results showed that reconstituted PPR vaccine stabilized with lactalbumin hydrolysate–sucrose promoted tolerance at 40 °C for 6 h. It was also noted that all reconstituted PPR vaccine formulations except the formulation stabilized with lactalbumin hydrolysate–maltose–gelatine maintained the titre above a 10^2.5 TCID₅₀/dose after 4 h of storage at 4 °C. Furthermore, the results showed that the PPR vaccine formulation containing lactalbumin hydrolysate sucrose was as the only one that maintained the titres above 10^2.5 TCID₅₀/dose after storage at 45 °C for 5 days, with a titre loss of 10^0.95 TCID₅₀/dose. Therefore, vaccine manufacturers producing PPR vaccines for use in tropical field regions could preferably use lactalbumin hydrolysate–sucrose stabilizer in vaccine formulation. Full article

Cat-scratch disease (CSD) is an infectious disease caused by Bartonella henselae, presenting with fever and lymphadenopathy following contact with felines. The ocular manifestations include neuroretinitis, characterised by optic nerve swelling and a macular star. Case Presentation: We discuss a case of neuroretinitis that presented atypically, without a macular star. There was an initial suspicion of Bartonella, but the serology was negative. Our patient was eventually empirically treated for infective neuroretinitis based on a positive contact history (recently scratched by one of his three pet cats). There was progression to a macular star upon serial dilated fundus examination, and the repeated serology one week after symptom onset showed rising titres, supporting a diagnosis of CSD. Conclusions: A judicious review of systems, repeat assays, serial dilated fundus examination, and early ophthalmic evaluation are useful in cases of suspected neuroretinitis, remaining an important differential in the evaluation of sudden-onset painless vision loss and unilateral disc swelling. Full article

Middle East respiratory syndrome coronavirus (MERS-CoV) causes fatal infections, with about 36% mortality in humans, and is endemic to the Middle East. MERS-CoV uses human dipeptidyl peptidase 4 (hDPP4) as a receptor for infection. Despite continued research efforts, no licensed vaccine is available for protection against this disease in humans. Therefore, this study sought to develop an inactivated fragmented MERS-CoV vaccine grown in Vero cells in an hDPP4-transgenic mouse model. Two-dose immunisation in mice with 15, 20, or 25 μg of spike proteins of inactivated split MERS-CoV antigens induced neutralising antibodies, with titres ranging from NT 80 to 1280. In addition, all immunised mice were completely protected, with no virus detection in tissues, weight loss, or mortality. The immunised splenocytes produced more cytokines that stimulate immune response (IFN-γ and TNF-α) than those that regulate it (IL-4 and IL-10). Taken together, the inactivated fragmented MERS-CoV vaccine is effective for the protection of mice against lethal MERS-CoV. Thus, the inactivated fragmented MERS-CoV vaccine warrants further testing in other hosts. Full article

Groundwater flow and contaminant migration tracing is a vital method of identifying and characterising pollutant source-pathway-receptor linkages in karst aquifers. Bacteriophages are an attractive alternative tracer to non-reactive fluorescent dye tracers, as high titres (>10¹² pfu mL⁻¹) can be safely released into the aquifer, offering improved tracer detectability. However, the interpretation of bacteriophage tracer breakthrough curves is complicated as their fate and transport are impacted by aquifer physicochemical conditions. A comparative tracer migration experiment was conducted in a peri-urban catchment in southeast England to characterise the behaviour of MS2 bacteriophage relative to sodium fluorescein dye in a karstic chalk aquifer. Tracers were released into a stream sink and detected at two abstraction boreholes located 3 km and 10 km away. At both sites, the loss of MS2 phage greatly exceeded that of the solute tracer. In contrast, the qualitative shape of the dye and phage breakthrough curves were visually very similar, suggesting that the bacteriophage arriving at each site was governed by comparable transport parameters to the non-reactive dye tracer. The colloid filtration theory was applied to explain the apparent contradiction of comparable tracer breakthrough patterns despite massive phage losses in the subsurface. One-dimensional transport models were also fitted to each breakthrough curve to facilitate a quantitative comparison of the transport parameter values. The model results suggest that the bacteriophage migrates through the conduit system slightly faster than the fluorescent dye, but that the former is significantly less dispersed. These results suggest that whilst the bacteriophage tracer cannot be used to predict receptor concentrations from transport via karstic flow paths, it can provide estimates for groundwater flow and solute contaminant transit times. This study also provides insight into the attenuation and transport of pathogenic viruses in karstic chalk aquifers. Full article

The aim of this study was to determine the thrombogenicity of lupus anticoagulant (LA) antibodies using a modified thrombin generation assay (TGA) with the addition of activated protein C (APC) in a group of 85 patients with LA-positive samples. Of these, 58 patients had clinical manifestations of antiphospholipid syndrome (APS) according to the Sydney criteria classification, i.e., each patient had thrombosis or foetal loss, and 27 patients did not show any clinical manifestations of APS. A comparison of the two groups’ TGA results revealed statistically significant differences (Fisher’s test p = 0.0016). The group of patients exhibiting clinical manifestations of APS showed higher thrombogenicity in 56.9% of patients, while the group of patients not yet exhibiting clinical manifestations of APS showed higher thrombogenicity in 25.9% of patients. There were no significant differences in the specificity of the TGA test between the groups of patients exhibiting similar clinical manifestations. Receiver operating characteristic curve analysis showed a more significant relationship (p = 0.0060) for TGA than for LA titre (p = 0.3387). These data suggest that the determination of LA thrombogenicity with the TGA assay leads to an increased prediction of the manifestation of a thromboembolic event. Our findings appear to be particularly relevant for the prediction of thrombotic events in patients with laboratory-expressed APS and no clinical manifestations. Full article

This study addresses the challenge of developing a cheap, patient-friendly alternative to antibiotics using bacteriophages for gastrointestinal applications. It explores the feasibility of manufacturing an enteric solid dosage form containing a salmonella-specific Myoviridae phage, Felix O1, encapsulated in spray-dried trehalose/Eudragit microparticles. The spray-dried powder was further formulated by combining the spray-dried microparticles with magnesium stearate to facilitate the fabrication of tablets using direct compression. The paper presents a comprehensive evaluation of the tablets with measurements of phage viability during tablet fabrication using a range of compression settings and, after tablet disintegration, dissolution and friability. Phage viability measurements were performed using storage stability testing of spray-dried powders and tablets in sealed vials at 4 °C, 20 °C and 30 °C and under different humidity conditions of 0%, 50% and 65% RH. The recommended compression force range was found to be 10–15 kN for a standard 10 mm diameter tablet. The storage of tablets at 4 °C/0% RH was found to be the most favourable condition resulting in a ~1 log loss in titre over a six-month storage period. Storage at higher temperatures and samples exposed to high levels of humidity resulted in a significant loss in phage viability. The paper highlights challenges in developing phage formulations suitable for direct-compression tableting, which afford the phages protection when exposed to temperatures and humidity levels that do not require a cold supply chain. Full article

Cancer patients have higher prevalences of antiphospholipid antibodies (aPLs), occasionally associated with thrombotic events. A cross-sectional study regarding the presence of criteria (IgG/IgM anti-cardiolipin-aCL, anti-β2 glycoprotein I-aβ2GPI) and non-criteria (IgG/IgM anti-phosphatidylserine-aPS, anti-phosphatidylethanolamine-aPE, anti-prothrombin-aPT) aPLs in 146 patients with involuntary weight loss was performed. None of the patients had thrombotic events during the study. Out of the 36 cancer patients, 33 had non-hematologic malignancies. In the cancer subgroup, 60% of the patients had at least one positive aPL, with significantly more patients being positive for aβ2GPI IgG compared with the non-cancer subgroup—p = 0.03, OR = 2.23 (1.02–4.88). When evaluating the titres, aCL IgG/IgM, aβ2GPI IgG, aPE IgG, and aPS IgG had significantly higher values in cancer patients, the best cancer predictor being aβ2GPI IgG—AUC 0.642 (0.542–0.742). Gastrointestinal cancer patients were studied separately, and aCL IgM positivity was significantly higher—p = 0.008, OR = 6.69 (1.35–33.02). Both the titres of aCL IgM (p = 0.006) and aPS IgM (p = 0.03) were higher in the gastrointestinal cancer subgroup, with aCL IgM being the best predictor for gastrointestinal cancer development—AUC 0.808 (0.685–0.932). Despite criteria and non-criteria aPLs being frequent in cancer, their connection with thrombosis in these patients is probably dependent on other important risk factors and needs further research. Full article

Clostridium botulinum serotypes C and D cause botulism in livestock, a neuroparalytic disease that results in substantial economic losses. Vaccination with aluminium-based toxoid vaccines is widely used to control the spread of botulism. Aluminium-based adjuvants are preferred owing to their apparent stimulation of the immune responses to toxoid vaccines when compared to other adjuvants. The aim of our study was to evaluate aluminium hydroxide nanoparticles as a potential substitute for alhydrogel in the botulism bivalent vaccine. Botulism vaccines were formulated with either alhydrogel or nanoalum and comparative efficacy between the two formulations was conducted by evaluating the immune response in vaccinated guinea pigs. A significant increase in immunological parameters was observed, with the antibody titres higher in the serum of guinea pigs (20 IU/mL of anti-BoNT C/D) injected with nanoalum-containing vaccine than guinea pigs inoculated with the standard alhydrogel-containing vaccine (8.7 IU/mL and 10 IU/mL of anti-BoNT C and anti-BoNT D, respectively). Additionally, the nanoalum-containing vaccine demonstrated potency in a multivalent vaccine (20 IU/mL of anti-BoNT C/D), while the standard alhydrogel-containing vaccine showed a decline in anti-BoNT C (5 IU/mL) antibody titres. Full article

Turnip yellows virus (TuYV), is one of the most important pathogens of oilseed rape, which has caused enormous yield losses in all growing regions of the world in recent years. Therefore, there is a need for resistant varieties for sustainable crop protection. We have investigated the resistance of known varieties and newly developed advanced-breeding lines of oilseed rape to TuYV in greenhouse and field trials. We have analysed the TuYV titre of individual genotypes inoculated with the virus using viruliferous aphids Myzus persicae. The genotypes ‘DK Temptation’ and ‘Rescator’ had the lowest and highest virus titres, respectively, and were used as resistant and susceptible models for comparative analyses with other genotypes. In the greenhouse, the best results were obtained with the genotypes ‘OP-8143 DH’ (2.94 × 10⁵ copies), OP-BN-72 (3.29 × 10⁵ copies), ‘Navajo’ (3.58 × 10⁵ copies) and ‘SG-C 21215’ (4.09 × 10⁵ copies), which reached virus titres about 2 times higher than the minimum virus concentration measured in ‘DK Temptation’ (1.80 × 10⁵ copies). In the field trials, the genotypes ‘Navajo’ (3.39 × 10⁵ copies), ‘OP-8148 DH’ (4.44 × 10⁵ copies), ‘SG-C 21215’ (6.80 × 10⁵ copies) and OP-8480 (7.19 × 10⁵ copies) had the lowest virus titres and reached about 3 times the virus titre of DK Temptation (2.54 × 10⁵ copies). Both trials showed that at least two commercial varieties (e.g., DK Temptation, Navajo) and three advanced breeding lines (e.g., OP-8143 DH, OP-BN-72, SG-C 21215) had low titres of the virus after TuYV infection. This indicates a high level of resistance to TuYV in ‘Navajo’ or the newly developed breeding lines and the basis of resistance is probably different from R54 (as in ‘DK Temptation’). Furthermore, the greenhouse trials together with RT -qPCR-based virus titre analysis could be a cost-effective and efficient method to assess the level of resistance of a given genotype to TuYV infection compared to the field trials. However, further research is needed to identify the underlying mechanisms causing this difference in susceptibility. Full article