Comparative Study of a Novel Lateral Flow Rapid Test with Conventional Serological Test Systems for the Diagnosis of Canine Leishmaniosis in Croatia and Brazil.

Control of canine infections with Leishmania infantum (L. infantum), a major zoonotic disease in Brazil and southern Europe, is becoming increasingly important due to its close proximity to humans, the increasing import of dogs from endemic regions and the impact of climate change on vector spreading. Simple, rapid and reliable diagnostic tests are therefore needed to detect infected dogs. Here, we re-evaluated different serological methods for the diagnosis of canine leishmaniosis (CanL) in Croatia and Brazil. The diagnostic performance of the indirect fluorescent antibody test (IFAT) and the VetLine® Leishmania ELISA (GSD Frankfurt, Germany) was compared with three rKLi8.3-based diagnostic test systems, the rKLi8.3 ELISA (GSD Frankfurt, Germany), the INgezim® Leishma CROM (GSD Madrid, Spain) lateral flow test (LFT) and the VetBlot®Leishmania LineBlot (GSD Frankfurt, Germany). CanL symptomatic dogs were efficiently diagnosed by all tests, except the VetLine® Leishmania ELISA, which is based on whole Leishmania antigens. The advantage of rKLi8.3 was also observed in oligo- and asymptomatic dogs from Brazil and Croatia, although with reduced diagnostic efficiency compared to symptomatic dogs. Similar to IFAT and rKLi8.3 ELISA, the LFT did not cross-react with other common canine pathogens; it showed very high specificity for healthy dogs from endemic regions in both countries and did not react with healthy, vaccinated dogs in Brazil. In conclusion, serodiagnostic tests based on the rKLi8.3 antigens are superior to whole parasite antigens, and the LFT has the advantage of providing a laboratory-independent, rapid and specific diagnosis of CanL.

Rapid Detection of SARS-CoV-2 Variants of Concern by Genomic Surveillance Techniques.

This chapter describes the application of genomic, transcriptomic, proteomic, and metabolomic methods in the study of SARS-CoV-2 variants of concern. We also describe the important role of machine learning tools to identify the most significant biomarker signatures and discuss the latest point-of-care devices that can be used to translate these findings to the physician's office or to bedside care. The main emphasis is placed on increasing our diagnostic capacity and predictability of disease outcomes to guide the most appropriate treatment strategies.

Hilab System Device in an Oncological Hospital: A New Clinical Approach for Point of Care CBC Test, Supported by the Internet of Things and Machine Learning.

The complete blood count (CBC) is a highly requested test that is generally restricted to centralized laboratories, which are limited by high cost, being maintenance-demanding, and requiring costly equipment. The Hilab System (HS) is a small, handheld hematological platform that uses microscopy and chromatography techniques, combined with machine learning (ML) and artificial intelligence (AI), to perform a CBC test. This platform uses ML and AI techniques to add higher accuracy and reliability to the results besides allowing for faster reporting. For clinical and flagging capability evaluation of the handheld device, the study analyzed 550 blood samples of patients from a reference institution for oncological diseases. The clinical analysis encompassed the data comparison between the Hilab System and a conventional hematological analyzer (Sysmex XE-2100) for all CBC analytes. The flagging capability study compared the microscopic findings from the Hilab System and the standard blood smear evaluation method. The study also assessed the sample collection source (venous or capillary) influences. The Pearson correlation, Student t-test, Bland-Altman, and Passing-Bablok plot of analytes were calculated and are shown. Data from both methodologies were similar (p > 0.05; r ≥ 0.9 for most parameters) for all CBC analytes and flagging parameters. Venous and capillary samples did not differ statistically (p > 0.05). The study indicates that the Hilab System provides humanized blood collection associated with fast and accurate data, essential features for patient wellbeing and quick physician decision making.

Novel and highly stable strategy for the development of microfluidic enzymatic assays based on the immobilization of horseradish peroxidase (HRP) into cotton threads.

The use of biological components in the development of new methods of analysis and point-of-care (POC) devices is an ever-expanding theme in analytical chemistry research, due to the immense potential for early diagnosis of diseases and monitoring of biomarkers. In the present work, the evaluation of an electrochemical microfluidic device based on the immobilization of horseradish peroxidase (HRP) enzyme into chemically treated cotton threads is described. This bioreactor was used as a channel for the build of the microfluidic device, which has allowed to use of a non-modified screen-printed electrode (SPE) as an amperometric detector. Cotton threads were treated using citric acid, and the immobilization of HRP has been performed by EDC/NHS crosslinking, connecting amine groups of the enzymes to carboxylic acids in the cellulosic structure. For the analytical evaluation, an amperometric assay for hydrogen peroxide detection was performed after the injection of H2O2 and hydroquinone (HQN) concomitantly. The enzymatic reaction consumes H2O2 leading to the formation of O-quinone, which is readily reducible at non-modified SPE. Several experimental parameters related to enzyme immobilization have been investigated and under the best set of conditions, a good analytical performance was obtained. In addition, the threads were freezer-stored and, after 12 weeks, 84% of hydrogen peroxide sensitivity was maintained, which is very reasonable for enzyme-based systems and still offers good analytical precision. Therefore, a simple and inexpensive microfluidic system was reported by crosslinking carboxylic groups to amine-containing macromolecules, suggesting a new platform for many other protein-based assays.

Clinical-genetic findings in a group of subjects with macular dystrophies due to mutations in rare inherited retinopathy genes.

PURPOSE: To describe the results of clinical and molecular analyses in a group of patients suffering from inherited macular dystrophies, in which next-generation sequencing (NGS) efficiently detected rare causative mutations. METHODS: A total of eight unrelated Mexican subjects with a clinical and multimodal imaging diagnosis of macular dystrophy were included. Visual assessment methods included best corrected visual acuity, color fundus photography, Goldmann visual field tests, kinetic perimetry, dark/light adapted chromatic perimetry, full-field electroretinography, autofluorescence imaging, and spectral domain-optical coherence tomography imaging. Genetic screening was performed by means of whole exome sequencing with subsequent Sanger sequencing validation of causal variants. RESULTS: All patients exhibited a predominantly macular or cone-dominant disease. Patients' ages ranged from 12 to 60 years. Three cases had mutations in genes associated with autosomal dominant inheritance (UNC119 and PRPH2) while the remaining five cases had mutations in genes associated with autosomal recessive inheritance (CNGA3, POC1B, BEST1, CYP2U1, and PROM1). Of the total of 11 different pathogenic alleles identified, three were previously unreported disease-causing variants. CONCLUSIONS: Macular dystrophies can be caused by defects in genes that are not routinely analyzed or not included in NGS gene panels. In this group of patients, whole exome sequencing efficiently detected rare genetic causes of hereditary maculopathies, and our findings contribute to expanding the current knowledge of the clinical and mutational spectrum associated with these disorders.

Accuracy of the urine point-of-care circulating cathodic antigen assay for diagnosing Schistosomiasis mansoni infection in Brazil: A multicenter study

ABSTRACT Background: The World Health Organization recommends a market-ready, urine-based point-of-care diagnostic test for circulating cathodic antigens (CCA) to determine the prevalence of S. mansoni. This study evaluated the performance of the URINE CCA (SCHISTO) ECO TESTE® (POC-ECO), which is currently available in Brazil. Methods: Residents from eight sites with different prevalence estimates provided one urine sample for POC-ECO and one stool sample for Kato-Katz (KK) and Helmintex® (HTX) testing as an egg-detecting reference for infection status. Results: None of the study sites had significantly higher POC-ECO accuracy than KK. Conclusions: POC-ECO is not currently recommended in Brazilian schistosomiasis elimination programs.

Comparison of the SARS-CoV-2 BD Veritor Nasal Antigen Test with Nasopharyngeal Reverse Transcription-PCR in Symptomatic Patients.

This study evaluated the BD Veritor system for rapid detection of SARS-CoV-2, an immunochromatographic point-of-care test, by comparing it with a standard reverse transcription-PCR (RT-PCR) methodology using samples from symptomatic patients. Samples from 146 symptomatic and 2 asymptomatic patients between the 1st and the 40th day of infection were evaluated. The nasopharyngeal and/or oropharyngeal swabs were inserted in a tube containing 0.9% saline solution and stored at refrigerator temperature until the moment of use. The samples were first tested with the Xpert Xpress SARS-CoV-2 (GeneXpert) kit (RT-PCR method), and the cycle thresholds (CTs) for the E and N2 genes encoding the SARS-CoV-2 envelope and nucleoprotein, respectively, were established. Subsequently, the same samples were tested using the Veritor rapid test. We analyzed the CTs of the N2 gene, which is detected in both methodologies, and observed sensitivities of 100%, 98.8%, 89.6%, and 82.7% for the CTs of <25, <27, and <30 and all the CTs, respectively. The greatest sensitivity was observed when we performed the test on patients within 5 days of symptom onset. The BD Veritor system's workflow is simple and fast, taking approximately 16 min from sample preparation to obtaining the test result. In addition to its satisfactory sensitivity, with results that correlate with those of the RT-PCR, the BD Veritor analyzer instrument reduces the subjectivity of unaided visual readings and consequent potential variation in result interpretation. Therefore, our results showed that the BD Veritor diagnostic test can provide a rapid and accurate diagnosis for SARS-CoV-2. IMPORTANCE This study provides important and useful information, especially for diagnostic laboratories, since the results show that the BD Veritor system can provide a fast and safe point-of-care antigen diagnostic test for rapid detection of COVID-19 that has high sensitivity, reproducibility, and accuracy.

Smartphone-Based Biosensor Devices for Healthcare: Technologies, Trends, and Adoption by End-Users.

Smart biosensors are becoming an important support for modern healthcare, even more so in the current context. Numerous smartphone-based biosensor developments were published in recent years, some highly effective and sensitive. However, when patents and patent applications related to smart biosensors for healthcare applications are analyzed, it is surprising to note that, after significant growth in the first half of the decade, the number of applications filed has decreased considerably in recent years. There can be many causes of this effect. In this review, we present the state of the art of different types of smartphone-based biosensors, considering their stages of development. In the second part, a critical analysis of the possible reasons why many technologies do not reach the market is presented. Both technical and end-user adoption limitations were addressed. It was observed that smart biosensors on the commercial stage are still scarce despite the great evolution that these technologies have experienced, which shows the need to strengthen the stages of transfer, application, and adoption of technologies by end-users.

Reliability of point-of-care circulating cathodic antigen assay for diagnosing schistosomiasis mansoni in urine samples from an endemic area of Brazil after one year of storage at -20 degrees Celsius

ABSTRACT Background The World Health Organization recommends reliable point-of-care (POC) diagnostic testing to eliminate schistosomiasis. Lateral flow immunoassay that detects schistosome circulating cathodic antigen (CCA) in urine to establish prevalence thresholds for intervention in endemic areas is recommended. Stored urine may be useful if surveying at-risk populations is delayed or interrupted by unforeseen circumstances, such as the current COVID-19 pandemic. This study evaluated the manufacturer's claim that Schistosoma mansoni infection can be reliably diagnosed in urine samples stored at -20°C for one year. Methods Two-hundred-forty-two subjects from an endemic site in Brazil provided one urine sample each for testing with URINE CCA (SCHISTO) ECO TESTE® (POC-ECO) and one stool sample each for testing with Kato-Katz (KK) and Helmintex® (HTX) as a robust reference standard for infection status. At least 2 ml of urine from each participant was stored at -20°C; after one year, 76 samples were randomly selected for POC-ECO retesting. Results: The POC-ECO agreement between freshly collected and stored urine was inadequate considering trace results as positive (Cohen's kappa coefficient κ = 0.08) and negative (κ = 0.36). POC-ECO accuracy was not significantly greater than that of routine KK (54%; 95% confidence interval: 42.1%-65.5%). Conclusions The precision and accuracy of POC-ECO have to be optimized in both freshly collected and stored urine before it can be recommended for use in control programs in Brazil.

Development of a Diagnostic Biosensor Method of Hypersensitivity Pneumonitis towards a Point-of-Care Biosensor.

In spite of a current increasing trend in the development of miniaturized, standalone point-of-care (PoC) biosensing platforms in the literature, the actual implementation of such systems in the field is far from being a reality although deeply needed. In the particular case of the population screenings for local or regional diseases related to specific pathogens, the diagnosis of the presence of specific antibodies could drastically modify therapies and even the organization of public policies. The aim of this work was to develop a fast, cost-effective detection method based on the manipulation of functionalized magnetic beads for an efficient diagnosis of hypersensitivity pneumonitis (HP), looking for the presence of anti-pigeon antigen antibodies (APAA) in a patient's serum. We presented a Diagnostic Biosensor Method (DBM) in detail, with validation by comparison with a traditional high-throughput platform (ELISA assay). We also demonstrated that it was compatible with a microfluidic chip that could be eventually incorporated into a PoC for easy and broad deployment using portable optical detectors. After standardization of the different reaction steps, we constructed and validated a plastic chip that could easily be scaled to high-volume manufacturing in the future. The solution proved comparable to conventional ELISA assays traditionally performed by the clinicians in their laboratory and should be compatible with other antibody detection directly from patient samples.

Diagnostic Methods for Non-Falciparum Malaria.

Malaria is a serious public health problem that affects mostly the poorest countries in the world, killing more than 400,000 people per year, mainly children under 5 years old. Among the control and prevention strategies, the differential diagnosis of the Plasmodium-infecting species is an important factor for selecting a treatment and, consequently, for preventing the spread of the disease. One of the main difficulties for the detection of a specific Plasmodium sp is that most of the existing methods for malaria diagnosis focus on detecting P. falciparum. Thus, in many cases, the diagnostic methods neglect the other non-falciparum species and underestimate their prevalence and severity. Traditional methods for diagnosing malaria may present low specificity or sensitivity to non-falciparum spp. Therefore, there is high demand for new alternative methods able to differentiate Plasmodium species in a faster, cheaper and easier manner to execute. This review details the classical procedures and new perspectives of diagnostic methods for malaria non-falciparum differential detection and the possibilities of their application in different circumstances.

Case Report: Diagnosis and Assessment of Cure Approaches for Acute Schistosomiasis in Pre-School Children.

Acute schistosomiasis (AS) manifests with a broad spectrum of clinical features in pediatric populations. Diagnosis may be difficult in the absence of detectable numbers of eggs. As a result, new approaches may be required to achieve an accurate diagnosis. Optimal praziquantel (PZQ) treatment regimen for young children is debatable. Also, the post-treatment response is still poorly evaluated due to the lack of reliable markers. A group of 6 children (a toddler and 5 pre-school children) and one pre-adolescent were investigated for AS clinical manifestations and followed-up for two years after treatment. Ova detection was performed by Kato-Katz (KK) and presence of Schistosoma mansoni DNA was assessed by real-time PCR (rt-PCR) in stool samples. IgG and IgE anti-Schistosoma levels and urinary antigen were detected by ELISA and point-of-care circulating cathodic antigen (POC-CCA) testing in serum and urine, respectively. AS clinical symptoms were present in 5/7 (71.4%) of the infected children, and hypereosinophilia was detected in all of them. Ova detection and serology were positive in only 3/7 (44.9%) and 4/7 (57.1%), respectively. However, real-time PCR (rt-PCR) showed the presence of Schistosoma DNA in 6/7 (85.7%) of the cases, and urinary antigen was detected in all infected children. The long-term follow-up after treatment with three doses of PZQ (80mg/kg/dose), showed high cure rates (CR) as demonstrated by the DNA-based assay as well as reduced levels of side effects. CR based on urinary antigen detection ranged from 28.6 to 100%, being the highest CR due to double testing the 2-year post-treatment samples. The results suggest that high dose and repeated treatment with PZQ might be effective for AS in young children. Also, new laboratory markers should be considered to diagnosis and monitor the drug response.

Low specificity of point-of-care circulating cathodic antigen (POCCCA) diagnostic test in a non-endemic area for schistosomiasis mansoni in Brazil.

A point-of-care test for detecting schistosome circulating cathodic antigen in urine (POCCCA) has been proposed for mapping infection and defining prevalence thresholds for mass drug administration (MDA). However, there is increasing evidence that POCCCA may yield false-positive results, which requires rigorous specificity evaluation in non-endemic areas. POCCCA was applied in an area known to be free from infection and devoid of any condition for schistosomiasis transmission as part of a multicentre study to evaluate the performance of POCCCA in Brazil's low or potentially endemic settings. Besides POCCCA detection in urine, a search for eggs in stool was performed by Kato-Katz (KK) and Helmintex (HTX) methods. One-hundred-and-seventy-four participants returned urine samples, 140 of which delivered stool samples. All these were HTX-negative for Schistosoma mansoni, and all 118 tested with KK were negative for both S. mansoni and soil-transmitted helminths. POCCCA results from freshly collected urine yielded a specificity of 62.1% (95% CI: 53.6% - 70.2%), taking trace outcomes as positive according to the manufacturer's instructions. Retesting urine from the 140 HTX-negatives after one-year storage at -20 °C with two new POCCCA batches simultaneously yielded significantly different specificities (34.3%; 95%CI: 26.5% - 42.8% and 75.0%; 95% CI: 67.0% - 81.9%). These two batches had a weak agreement (Cohen's kappa: 0.56; 95%CI: 0.44-0.68) among the 174 urine samples retested. At present, POCCCA cannot be recommended either as a cut-off point for MDA or a reliable diagnostic tool for treatment of the infection carriers (selective chemotherapy) in low endemic areas and at final stages of transmission interruption. Manufacturers should be required to optimize production standardization and to assure quality and reproducibility of the test. Extended rigorous performance evaluations by different users from different regions are needed before POCCCA is widely recommended.

Diagnostic comparison of stool exam and point-of-care circulating cathodic antigen (POC-CCA) test for schistosomiasis mansoni diagnosis in a high endemicity area in northeastern Brazil.

This study aimed to evaluate the performance of the point-of-care circulating cathodic antigen (POC-CCA) test in a highly endemic area in Brazil, comparing it to the Kato-Katz (KK) technique for sensitivity, specificity and the intensity of the reaction of the test in relation to the parasitic load. The community in Sergipe, Brazil, participated in the study, providing three stool samples, one of urine (POC-CCA) and fingers tick blood sample was tested by enzyme-linked immunosorbent assay (ELISA). Sensitivity, specificity, positive predictive value, negative predictive value, accuracy, kappa coefficient and Spearman's correlation were calculated for the POC-CCA test using the KK as the reference. The prevalence of schistosomiasis by KK testing was 48.82%; POC-CCA (t+) 66.14%; POC-CCA (t-) 45.24%. ELISA results showed 100% agreement in individuals with high and moderate eggs per gram (EPG). POC-CCA presented good diagnostic performance in individuals with medium and high EPG, but there were a high number of false negatives in individuals with low intensity infections. As observed, POC-CCA-filter test improves accuracy and sensitivity compared to a conventional test.

Development and validation of a simple and rapid way to generate low volume of plasma to be used in point-of-care HIV virus load technologies

ABSTRACT A new point-of-care HIV viral load, mPIMA HIV-1/2 VL, Abbott, USA, has been recently developed. This point-of-care viral load requires no skilled person to run and uses a small plasma volume (50 µL). However, obtaining 50 µL of plasma can be a challenge in limited resource settings. We validated a simple and easy method to obtain enough amount of plasma to run a point-of-care viral load. The study utilized 149 specimens from patients failing antiretroviral therapy. At least 250 µL of whole blood was collected in a microtube/EDTA from fingerstick (fs-plasma) and immediately centrifuged. Parallel collection of venous blood to obtain plasma (vp-plasma) was used to compare performance in a point-of-care viral load assay and in methodology used in centralized laboratories Abbott M2000, Abbott, USA. The procedure for plasma collection takes less than 10 min and in 94% of the cases only one fingerstick was sufficient to collect at least 250 µL of blood. The Pearson correlation coefficient value for vp-plasma versus fs-plasma ran on mPIMA was 0.990. The Bland-Altman mean difference (md) for this comparison were virtually zero (md = −0.001) with limits of agreement between −0.225 and 0.223. In addition, the Pearson correlation coefficient value for fs-plasma in mPIMA versus vp-plasma in Abbott M2000 was 0.948 for values above the mPIMA limit of quantification (LoQ; from 800 to 1,000,000 copies/mL). These results validate this simple plasma isolation method capable to be implemented in low resource countries where point-of-care decentralization is deeply needed.

Development and validation of a simple and rapid way to generate low volume of plasma to be used in point-of-care HIV virus load technologies.

A new point-of-care HIV viral load, mPIMA HIV-1/2 VL, Abbott, USA, has been recently developed. This point-of-care viral load requires no skilled person to run and uses a small plasma volume (50µL). However, obtaining 50µL of plasma can be a challenge in limited resource settings. We validated a simple and easy method to obtain enough amount of plasma to run a point-of-care viral load. The study utilized 149 specimens from patients failing antiretroviral therapy. At least 250µL of whole blood was collected in a microtube/EDTA from fingerstick (fs-plasma) and immediately centrifuged. Parallel collection of venous blood to obtain plasma (vp-plasma) was used to compare performance in a point-of-care viral load assay and in methodology used in centralized laboratories Abbott M2000, Abbott, USA. The procedure for plasma collection takes less than 10min and in 94% of the cases only one fingerstick was sufficient to collect at least 250µL of blood. The Pearson correlation coefficient value for vp-plasma versus fs-plasma ran on mPIMA was 0.990. The Bland-Altman mean difference (md) for this comparison were virtually zero (md=-0.001) with limits of agreement between -0.225 and 0.223. In addition, the Pearson correlation coefficient value for fs-plasma in mPIMA versus vp-plasma in Abbott M2000 was 0.948 for values above the mPIMA limit of quantification (LoQ; from 800 to 1,000,000copies/mL). These results validate this simple plasma isolation method capable to be implemented in low resource countries where point-of-care decentralization is deeply needed.

Schistosomiasis: an epidemiological update on Brazils southernmost low endemic area in Esteio

Abstract INTRODUCTION: Brazil's southernmost state, Rio Grande do Sul (RGS), was considered schistosomiasis-free until 1998 when a low endemic focus was identified in Esteio, a city located next to the capital of RGS. In the last two decades, the control interventions applied in the region have been apparently successful, and the absence of new cases indicated the possibility of interrupted schistosomiasis transmission. The objective of this study was to update the clinical and epidemiological data of schistosomiasis in Esteio. METHODS: We reviewed all 28 individuals diagnosed with the infection since 1997 and a survey was applied to a group of 29 school-aged children residing in Vila Pedreira, one of the most affected neighborhoods. RESULTS No eggs were detected in fecal samples using the Helmintex method, and all samples were negative for serum antibodies on examination by the western blot technique using the Schistosoma mansoni microsomal antigen (MAMA- WB). In contrast, 23 individuals (79%) tested positive for the cathodic circulating antigen with the point-of-care immunochromatographic test (POC-CCA) on urine samples. Of the 28 formerly infected individuals, only eight were located, of which four tested positive, and four tested negative for serum antibodies using the MAMA-WB technique. CONCLUSIONS: Current adverse conditions for S. mansoni transmission in Esteio and the absence of a confirmed diagnosis suggests that there is (i) a lack of specificity of the POC-CCA test in low endemic settings, and (ii) a high probability that interruption of schistosomiasis has been achieved in Esteio.

The use of the circulating cathodic antigen (CCA) urine cassette assay for the diagnosis and assessment of cure of Schistosoma mansoni infections in an endemic area of the Amazon region

Abstract INTRODUCTION Schistosomiasis is a poverty-related disease that affects people in 78 countries worldwide. This study aimed to evaluate the point-of-care circulating cathodic antigen (POC-CCA) test performance using sensitive parasitological methods as a reference standard (RS) in individuals before and after treatment. METHODS The RS was established by combining the results of 16 Kato-Katz slides and the Helmintex® method. Positivity rates of the POC-CCA test and Kato-Katz and Helmintex® methods were calculated before treatment and 30 days afterward. Furthermore, the sensitivity, specificity, accuracy, and kappa coefficient before treatment were determined by comparing the methods. The cure rate was defined 30 days after treatment. RESULTS Among the 217 participants, the RS detected a total of 63 (29.0%) positive individuals. The POC-CCA test identified 79 (36.4%) infections. The evaluation of POC-CCA test performance in relation to the RS revealed a sensitivity of 61.9%, specificity of 74.0%, accuracy of 70.5%, and kappa coefficient of 0.33. Out of the 53 remaining participants after treatment, a total of 45 (81.1%) showed egg negative results, and 8 (18.9%) were egg positive according to the RS. A total of 5 (9.4%) egg-positive and 37 (69.8%) egg-negative individuals were positive by the POC-CCA test. CONCLUSIONS Our data show that the POC-CCA test has potential as an auxiliary tool for the diagnosis of Schistosoma mansoni infection, yielding better results than 16 Kato-Katz slides from three different stool samples. However, the immunochromatographic test lacks sufficient specificity and sensitivity for verifying the cure rate after treatment.

Development and evaluation of LAMP-coupled lateral flow device for the detection of MAP in livestock at point of care resource-limited areas.

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.