The Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis (Johne's disease), a systemic and chronic inflammation of intestine that affects bovine, small ruminants like goat and sheep. The disease has a greater economic importance in cattle and in small ruminants. But its effective control is impeded due to lack of rapid and accurate diagnostics. The present study is aimed at developing a LAMP-coupled lateral flow device (LFD) for rapid detection of paratuberculosis in livestock animal species such as cattle and in small ruminants at resource-limited areas. LAMP primers with biotin and FITC end tags were designed for IS900 gene specific for MAP. To determine sensitivity of LAMP assay, 10-fold serial dilutions were made from 10 ng/μl MAP stock DNA and were compared with PCR. The detection limits of LAMP-coupled LFD were defined and reactions were repeated for reproducibility. The specificity was evaluated using other infectious bacteria such as M. bovis, M. tuberculosis, Brucella abortus, Leptospira interrogan, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogens, and Staphylococcus aureus. A total of 95 samples turned positive for LAMP-coupled LFD out of 389 fecal samples. All the cultural-positive and PCR-positive samples showed positive in LAMP-coupled LFD. Nine samples with negative cultures turned positive in LAMP assay. The overall sensitivity and specificity of the LAMP-coupled LFD assays were 100% and 97.02% respectively in comparison with the culture as the gold standard method. The sensitivity detection limit of developed assay was 10 fg/μl and specificity was 100%. This assay successfully detected MAP not only by using bacterial DNA but also in clinical fecal samples. The clear band formation at control and test positions was observed on LAMP-coupled LFD. The developed assay is a simple, rapid, easy to perform, and is very useful in early diagnosis of Mycobacterium avium subsp. paratuberculosis at point of care resource-limited areas. Keywords Loop-mediated isothermal amplification (LAMP). Lateral flow device (LFD). Insertion sequence 900 (IS900) gene. Mycobacterium avium subsp. paratuberculosis (MAP). Fluorescein isothiocyanate (FITC). Polymerase chain reaction (PCR) .

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Malaria is a fatal life-threatening parasitic infection and a leading cause of morbidity and mortality. The present study was aimed to evaluate simple, inexpensive, accurate, reliable, easily available better diagnostic for rapid detection of malaria at point of care (POC). The study includes 1403 samples collected from the patients, of which 1227 were clinically suspected cases and 176 from consecutive feverish patients. Among the suspected cases only 338 samples were confirmed positive and 889 samples were negative for Plasmodium species by PCR. All the 889 samples showed negative result for plasmodium species by microscopy, Malarial Ag rapid kits but only 867 samples were confirmed negative with malarial Ab rapid kits. Of the 338 PCR positive samples, 337 samples were confirmed positive by microscopy and Malarial Ag rapid kits, but only 284 samples were confirmed positive using malarial Ab rapid kits. Overall the microscopy and the malaria antigen-based lateral flow assay exhibited similar sensitivity, specificity, PPV, NPV and efficiency, respectively, whereas the PCR assay had 100% sensitivity, specificity, PPV, NPV and efficiency. But the evolutionary data for malaria antibody lateral flow assay has 92.81% sensitivity, 94.13% specificity, 84.02% PPV, 97.52% NPV and 93.80% efficiency. The developed Malaria pf/pv antigen and antibody field-deployable kits are simple, rapid, accurate, reliable, inexpensive, user friendly, POC. In addition the kits are highly sensitive and species-specific. The pf/pv antigen kit is found to be more accurate with 99.7% sensitivity and 100% specificity than to Malaria pf/pv antibody rapid kits. Of the two rapid kits developed, Malaria pf/pv antigen kit is found be more accurate with 99.7% sensitivity and 100% specificity than to Malaria pf/pv antibody rapid kits. KeywordsPoint of care (POC) • Immuno chromatographic test (ICT) • Plasmodium Lactate Dehydrogenase (pLDH) • Histidine Rich Protein II (HRPII) • Plasmodium falciparum (pf) • Plasmodium vivax (pv) • Antigen (Ag) • Antibody (Ab) • Rapid diagnostic test (RDT) • Polymerase chain reaction (PCR)