Development of a novel sensitive single-tube nested PCR assay for the detection of African swine fever virus.

African swine fever (ASF) is a highly fatal and contagious viral disease caused by African swine fever virus (ASFV). It has caused significant economic losses to the swine industry and poses a serious threat to food security worldwide. Diagnostic tests with high sensitivity are essential for the effective management of ASF. Here, we describe a single-tube nested PCR (STN-PCR) assay for the detection of ASFV in which two consecutive amplification steps are carried out within a single tube. Two pairs of primers (outer and inner) were designed to target the p72 gene of ASFV. The primer concentrations, annealing temperatures, and number of amplification cycles were optimized to ensure the consecutive utilization of outer and inner primer pairs during amplification while minimizing the likelihood of amplicon contamination. In comparison with two conventional endpoint PCR assays (one of which is recommended by the World Organization for Animal Health), the newly developed STN-PCR assay demonstrated a 100-fold improvement in the limit of detection (LOD), detecting 100 copies of ASFV genomic DNA, whereas the endpoint PCR assays could detect no fewer than 10,000 copies. The clinical performance of the STN-PCR assay was validated using 95 tissue samples suspected of being positive for ASFV, and the assay showed 100% specificity. A Cohen's kappa value of 0.91 indicated perfect agreement between the assays. This new STN-PCR assay is a potentially valuable tool that will facilitate the control of ASF.

Serpent strikes, sapien sways: A rare case of bilateral cerebellar infarct following viper bite.

Ischemic stroke following snake bite is rare. We report a posterior circulation infarct involving bilateral cerebellum and occipital lobe following Russell's viper bite in a previously healthy individual. A 50 years old healthy individual, soon after being bitten by the Russel viper on the left foot he developed pain and swelling followed by drooping of eyelids, slurring of speech and giddiness with multiple episodes of vomiting. The patient was administered ASV as well as neostigmine and atropine injections. Following this, the neurological manifestations resolved except dysarthria. CT brain study done was normal. On day 2 of hospitalization, he developed left cerebellar signs (positive finger nose finger test, rebound phenomenon, dysdiadochokinesia, a positive heel shin test). Subsequently, an MRI with MR angiogram was done which showed acute infarcts in bilateral cerebellar hemispheres, with the left superior cerebellar peduncle showing restricted diffusion and low ADC values. Ischemic infarction following viper envenomation has been described by only few authors. In majority of the cases reported, ischemic infarction involved the anterior circulation. The possible mechanisms of infarction in this scenario are discussed. Patient was treated with anti-snake venom and showed a good recovery. Early imaging and early treatment with anti-snake venom is important for a favourable outcome.

Résumé L'AVC ischémique consécutif à une morsure de serpent est rare. Nous rapportons un infarctus de la circulation postérieure impliquant le cervelet et le lobe occipital bilatéraux suite à une morsure de vipère de Russell chez un individu auparavant en bonne santé. Un individu en bonne santé de 50 ans, peu de temps après avoir été mordu par la vipère Russel au pied gauche, il a développé une douleur et un gonflement suivis d'un affaissement des paupières, d'un trouble de l'élocution et de vertiges avec de multiples épisodes de vomissements. Le patient a reçu de l'ASV ainsi que des injections de néostigmine et d'atropine. Suite à cela, les manifestations neurologiques ont disparu sauf la dysarthrie. L'étude cérébrale réalisée par TDM était normale. Au 2ème jour d'hospitalisation, il a développé des signes cérébelleux gauches (test doigt nez doigt positif, phénomène de rebond, dysdiadochokinésie, test talon tibia positif). Par la suite, une IRM avec angiographie IRM a été réalisée qui a montré des infarctus aigus dans les hémisphères cérébelleux bilatéraux, le pédoncule cérébelleux supérieur gauche montrant une diffusion restreinte et de faibles valeurs d'ADC. L'infarctus ischémique consécutif à une envenimation par vipère n'a été décrit que par quelques auteurs. Dans la majorité des cas rapportés, l'infarctus ischémique impliquait la circulation antérieure. Les mécanismes possibles de l'infarctus dans ce scénario sont discutés. Le patient a été traité avec du venin anti-serpent et a montré une bonne récupération. Une imagerie précoce et un traitement précoce avec du venin anti-serpent sont importants pour un résultat favorable. Mots-clés: Infarctus cérébelleux, étude cérébrale par imagerie par résonance magnétique, morsure de vipère.

Seroepidemological investigation of Toxoplasma gondii and Trichinella spp. in pigs reared by tribal communities and small-holder livestock farmers in Northeastern India.

Toxoplasma gondii and Trichinella spp. are critical tissue-dwelling foodborne zoonotic parasites associated with pork consumption and pig rearing. Despite being a major pig-rearing region in the country, Northeastern India has not undergone any investigation regarding the presence of T. gondii and Trichinella spp. in pigs. Therefore, this study aims to determine the seroprevalence of T. gondii and Trichinella spp. and identify associated risk factors in pigs reared by tribal communities and small-holder livestock farmers in the northeastern region of India. In a cross-sectional serological survey, 400 pigs from 400 households across five northeastern states of India underwent testing for the seroprevalence of porcine toxoplasmosis and trichinellosis. Serum samples (80 from each state) were analyzed using commercially available ELISA assays. Data on backyard farm characteristics and various management aspects were collected, and risk factors linked with prevalence were analyzed through univariate and multivariate logistic regression analysis. The findings revealed that the apparent and true prevalence of anti-T. gondii antibodies were 45% (40.12-49.88, 95% CI) and 45.7% (40.7-50.69, 95% CI), respectively. As for anti- Trichinella antibodies, both the apparent and true prevalence were 0.75% (-0.1-1.6, 95% CI). The univariate and multivariate analyses indicated that age above 24 months (OR 7.20, 95% CI 2.45-23.71), exposure to cats (OR = 5.87, 95% CI 2.55-14.05), and farms operating for breeding purposes (OR = 5.60, 95% CI 3.01-11.04) were significant risk factors associated with the seroprevalence of T. gondii. This study marks the initial documentation of the seroprevalence of T. gondii and Trichinella spp. in pigs reared by tribal communities in Northeastern India. The results emphasize the significance of these parasites as foodborne zoonotic threats in the region, potentially posing substantial public health risks, especially within tribal and rural communities. The insights derived from this research could be valuable in formulating targeted preventive and control strategies against T. gondii and Trichinella spp. in pigs, not only in this region but also in areas with similar rearing practices.

Novel helix loop-mediated isothermal amplification (HAMP) assay for colorimetric detection of Staphylococcus aureus in milk.

Staphylococcus aureus is an important and leading cause of foodborne diseases worldwide. Prompt detection and recall of contaminated foods are crucial to prevent untoward health consequences caused by S. aureus. Helix loop-mediated isothermal amplification (HAMP) is an exciting recent addition to the array of available isothermal-based nucleic acid amplification techniques. This study aimed to develop and evaluate a HAMP assay for detecting S. aureus in milk and milk products. The assay is completed in 75 minutes of isothermal temperature incubation (64 ˚C) and dye-based visual interpretation of results based on colour change. The specificity of the developed assay was ascertained using 27 S. aureus and 17 non S. aureus bacterial strains. The analytical sensitivity of the developed HAMP assay was 9.7 fg/µL of pure S. aureus DNA. The detection limit of the HAMP assay in milk (86 CFU/mL) was 1000x greater than the routinely used endpoint PCR (86 × 103 CFU/mL). The practicality of applying the HAMP assay was also assessed by analysing milk and milk product samples (n = 95) obtained from different dairy farms and retail outlets. The developed test is a more rapid, sensitive, and user-friendly method for the high-throughput screening of S. aureus in food samples and may therefore be suitable for field laboratories. To our knowledge, this is the first study to develop and evaluate the HAMP platform for detecting S. aureus.

First Seroepidemiological Investigation of Hepatitis E Virus Infection in Backyard Pigs from Northeastern India: Prevalence and Associated Risk Factors.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis globally, with zoonotic potential, and pigs are considered the major reservoir. To determine the seroprevalence of HEV infection in pigs reared in backyard conditions in the northeastern region of India, blood samples were collected from 400 pigs from five northeastern states (80 samples from each state) and tested for IgG antibodies against HEV using an ELISA assay. Questionnaires on farm characteristics and management practices were completed, and risk factors associated with HEV were studied using univariate and multivariate analysis. The apparent seroprevalence of HEV infection was 51% (46.1-55.9, 95% CI), with a true prevalence of 52.98% (47.22-58.75, 95% CI). The risk factors significantly associated with higher HEV seropositivity were as follows: lack of disinfection (OR 4.65), feeding swill (restaurant and bakery waste) (OR 2.55), failure to follow the all-in-all-out production system (OR 3.47), and medium holding size (OR 9.83), which refers to mixed rearing of younger and older age groups. This study demonstrates that HEV is widespread among pigs reared in northeastern India. The risk factor analysis conducted in this study provides valuable insights into the prevalence of HEV in the region.

Quality improvement project to assess patient satisfaction towards outpatient services of a tertiary care teaching hospital, South India - A cross-sectional study.

Background: Patient satisfaction survey is a valuable tool to measure the outcomes of care rendered by hospitals. Outpatient department (OPD) is a very crucial area of any hospital services as huge number of patient's visits this area for clinical services. Information about quality of services in OPD will serve two purposes, i.e., identifying areas of improvement in the services offered and highlighting the need for corrective actions. Patient satisfaction is the key determinant for assessing the quality of patient care and is the desired outcome of every hospital. The present study was conducted to measure the levels of patient satisfaction and to ascertain any service gaps in OPD of a tertiary care teaching hospital, Mysuru, India. Materials and Methods: This cross-sectional, descriptive study was conducted among 600 patients using a structured questionnaire for assessing the levels of patient satisfaction. Results: The overall satisfaction rate in this study was 97.2%, with major satisfiers being clinical care provided by doctors and nursing staffs and few patients were not satisfied with cleanliness of toilets and medications' availability. Conclusion: Patient satisfaction survey may be a good audit tool and it should be used to improve the standard of services. According to the patient's opinion, the study showed good satisfaction with respect to reception and registration services, physical facilities, doctor's services, nursing services, laboratory services, and pharmacy services. Few deficient areas are identified in terms of service delivery and the same will be bridged as early as possible to achieve universal patient satisfaction.

RésuméContexte: L'enquête sur la satisfaction des patients est un outil précieux pour mesurer les résultats des soins rendus par les hôpitaux. Le service ambulatoire (OPD) est un domaine très crucial de tous les services hospitaliers car un grand nombre de visites de patients dans ce domaine pour des services cliniques. Les informations sur la qualité des services dans OPD serviront à deux fins, à savoir l'identification des domaines d'amélioration des services offerts et la mise en évidence de la nécessité de mesures correctives. La satisfaction des patients est le principal déterminant pour évaluer la qualité des soins aux patients et est le résultat souhaité pour chaque hôpital. La présente étude a été menée pour mesurer les niveaux de satisfaction des patients et pour déterminer les éventuelles lacunes de service dans le DPO d'un hôpital d'enseignement de soins tertiaires, Mysuru, Inde. Matériel et méthodes: Cette étude descriptive transversale a été menée auprès de 600 patients à l'aide d'un questionnaire structuré pour évaluer les niveaux de satisfaction des patients. Résultats: Le taux de satisfaction global de cette étude était de 97,2%, les principaux éléments de satisfaction étant les soins cliniques fournis par les médecins et le personnel infirmier et peu de patients n'étaient pas satisfaits de la propreté des toilettes et de la disponibilité des médicaments. Conclusion: L'enquête sur la satisfaction des patients peut être un bon outil d'audit et elle devrait être utilisée pour améliorer la qualité des services. De l'avis du patient, l'étude a montré une bonne satisfaction en ce qui concerne les services d'accueil et d'inscription, les installations physiques, les services médicaux, les services infirmiers, les services de laboratoire et les services de pharmacie. Peu de domaines déficients sont identifiés en termes de prestation de services et les mêmes seront comblés le plus tôt possible pour atteindre la satisfaction universelle des patients.

Pro-inflammatory cytokine expression studies of subclinical and clinical endometritis in endometrial tissues of buffaloes.

The objective of the study was to investigate the role of pro-inflammatory cytokines in the pathogenesis of endometritis in buffaloes, which can lead to early diagnosis of endometritis. The expression of CD14, TLR-4, and IL-1ß encoding gene in endometrium of buffaloes with subclinical endometritis were 1.87, 2.71, and 3.48-fold, while samples with clinical endometritis showed only 2.90, 4.76, and 8.02-fold for CD14, TLR-4, and IL-1ß, respectively, though numerical value of expression was higher in subclinical and clinical endometritis but it was at par with control. The expression profile for IL-6, IL-8, and TNF-α mRNA for subclinical endometritis were 11.95, 14.87, and 12.95-fold while for clinical endometritis, values were 18.17, 37.97, and 28.83-fold for IL-6, IL-8, and TNF-α mRNA, respectively, which were significantly higher in subclinical and clinical endometritis as compared to apparently normal samples at follicular phase. In the luteal group, the subclinical endometritis sample showed 2.13, 6.43, 14.52, 22.38, 18.64, and 2.82-fold respectively, for CD14, IL-1ß IL-6, IL-8, TNF-α, and TLR-4 mRNA. Except TLR-4, the values of expression were significantly higher when compared with apparently normal. Whereas, the expression values of clinical endometritic uteri were 4.01, 7.15, 17.20, 45.55, 52.58, and 32.95-fold respectively, for CD14, TLR-4, IL-1ß, IL-6, IL-8, and TNF-α mRNA which were significantly higher as compared to apparently normal uteri. An increase in the mRNA expression of CD14, TLR-4, and pro-inflammatory cytokines having higher diagnostic importance in uterine inflammation.

Experimental atherosclerosis in goats.

Varying degrees of aortic sudanophilia (5 to 75 per cent) comparable to human atherosclerotic lesions (fatty streaks/spots) developed in all nine experimental goats receiving 2 g of protected cholesterol daily for two, four and nine months. From the fifth month onward, the serum cholesterol level stabilised at 450 to 525 mg 100 ml-1. Aortic sudanophilia developed after two months but without any appreciable intimal thickening. Pronounced intimal thickening with foam cells was seen after four to nine months. The development of fatty streaks/spots was directly proportional to the degree of hypercholesterolaemia and duration of protected cholesterol feeding. It was concluded that goats could be useful in comparative atherosclerosis research.