Quantitative lateral flow strip assays as user-friendly tools to detect biomarker profiles for leprosy

Leprosy is a debilitating, infectious disease caused by Mycobacterium leprae. Despite the availability of multidrug therapy, transmission is unremitting. Thus, early identification of M. leprae infection is essential to reduce transmission. The immune response to M. leprae is determined by host genetics, resulting in paucibacillary (PB) and multibacillary (MB) leprosy associated with dominant cellular or humoral immunity, respectively. This spectral pathology of leprosy compels detection of immunity to M. leprae to be based on multiple, diverse biomarkers. In this study we have applied quantitative user friendly lateral flow assays (LFAs) for four immune markers (anti-PGL-I antibodies, IL-10, CCL4 and IP-10) for whole blood samples from a longitudinal BCG vaccination field-trial in Bangladesh. Different biomarker profiles, in contrast to single markers, distinguished M. leprae infected from non-infected test groups, patients from household contacts (HHC) and endemic controls (EC), or MB from PB patients. The test protocol presented in this study merging detection of innate, adaptive cellular as well as humoral immunity, thus provides a convenient tool to measure specific biomarker profiles for M. leprae infection and leprosy utilizing a field-friendly technology

Development of lateral flow assays to detect host proteins in cattle for improved diagnosis of bovine tuberculosis

Bovine tuberculosis (bTB), caused by Mycobacterium bovis (M. bovis) infection in cattle, is an economically devastating chronic disease for livestock worldwide. Efficient disease control measures rely on early and accurate diagnosis using the tuberculin skin test (TST) and interferon-gamma release assays (IGRAs), followed by culling of positive animals. Compromised performance of TST and IGRA, due to BCG vaccination or co-infections with non-tuberculous mycobacteria (NTM), urges improved diagnostics. Lateral flow assays (LFAs) utilizing luminescent upconverting reporter particles (UCP) for quantitative measurement of host biomarkers present an accurate but less equipment- and labor-demanding diagnostic test platform. UCP-LFAs have proven applications for human infectious diseases. Here, we report the development of UCP-LFAs for the detection of six bovine proteins (IFN-γ, IL-2, IL-6, CCL4, CXCL9, and CXCL10), which have been described by ELISA as potential biomarkers to discriminate M. bovis infected from naïve and BCG-vaccinated cattle. We show that, in line with the ELISA data, the combined PPDb-induced levels of IFN-γ, IL-2, IL-6, CCL4, and CXCL9 determined by UCP-LFAs can discriminate M. bovis challenged animals from naïve (AUC range: 0.87–1.00) and BCG-vaccinated animals (AUC range: 0.97–1.00) in this cohort. These initial findings can be used to develop a robust and user-friendly multi-biomarker test (MBT) for bTB diagnosis

Prototype multi-biomarker test for point-of-care leprosy diagnostics

To end the decade-long, obstinately stagnant number of new leprosy cases, there is an urgent need for field-applicable diagnostic tools that detect infection with Mycobacterium leprae, leprosy's etiologic agent. Since immunity against M. leprae is characterized by humoral and cellular markers, we developed a lateral flow test measuring multiple host proteins based on six previously identified biomarkers for various leprosy phenotypes. This multi-biomarker test (MBT) demonstrated feasibility of quantitative detection of six host serum proteins simultaneously, jointly allowing discrimination of patients with multibacillary and paucibacillary leprosy from control individuals in high and low leprosy endemic areas. Pilot testing of fingerstick blood showed similar MBT performance in point-of-care (POC) settings as observed for plasma and serum. Thus, this newly developed prototype MBT measures six biomarkers covering immunity against M. leprae across the leprosy spectrum. The MBT thereby provides the basis for immunodiagnostic POC tests for leprosy with potential for other (infectious) diseases as well.Diagnostic Technique in Health Technology; Applied Microbiology; Biotechnolog

Application of new host biomarker profiles in quantitative point-of-care tests facilitates leprosy diagnosis in the field

Background: Transmission of Mycobacterium leprae, the pathogen causing leprosy, is still persistent. To facilitate timely (prophylactic) treatment and reduce transmission it is vital to both early diagnose leprosy, and identify infected individuals lacking clinical symptoms. However, leprosy-specific biomarkers are limited, particularly for paucibacillary disease. Therefore, our objective was to identify new biomarkers for leprosy and assess their applicability in point-of-care (POC) tests. Methods: Using multiplex-bead-arrays, 60 host-proteins were measured in a cross-sectional approach in 24-h whole blood assays (WBAs) collected in Bangladesh (79 patients; 54 contacts; 51 endemic controls (EC)). Next, 17 promising biomarkers were validated in WBAs of a separate cohort (55 patients; 27 EC). Finally, in a third cohort (36 patients; 20 EC), five candidate markers detectable in plasma were assessed for application in POC tests. Findings: This study identified three new biomarkers for leprosy (ApoA1, IL-1Ra, S100A12), and confirmed five previously described biomarkers (CCL4, CRP, IL-10, IP-10, αPGL-I IgM). Overnight stimulation in WBAs provided increased specificity for leprosy and was required for IL-10, IL-1Ra and CCL4. The remaining five biomarkers were directly detectable in plasma, hence suitable for rapid POC tests. Indeed, lateral flow assays (LFAs) utilizing this five-marker profile detected both multi- and paucibacillary leprosy patients with variable immune responses. Interpretation: Application of novel host-biomarker profiles to rapid, quantitative LFAs improves leprosy diagnosis and allows POC testing in low-resource settings. This platform can thus aid diagnosis and classification of leprosy and also provides a tool to detect M.leprae infection in large-scale contact screening in the field

Detection and localization of early- and late-stage cancers using platelet RNA

Cancer patients benefit from early tumor detection since treatment outcomes are more favorable for less advanced cancers. Platelets are involved in cancer progression and are considered a promising biosource for cancer detection, as they alter their RNA content upon local and systemic cues. We show that tumor-educated platelet (TEP) RNA-based blood tests enable the detection of 18 cancer types. With 99% specificity in asymptomatic controls, thromboSeq correctly detected the presence of cancer in two-thirds of 1,096 blood samples from stage I–IV cancer patients and in half of 352 stage I–III tumors. Symptomatic controls, including inflammatory and cardiovascular diseases, and benign tumors had increased false-positive test results with an average specificity of 78%. Moreover, thromboSeq determined the tumor site of origin in five different tumor types correctly in over 80% of the cancer patients. These results highlight the potential properties of TEP-derived RNA panels to supplement current approaches for blood-based cancer screening

Field-Friendly Test for Monitoring Multiple Immune Response Markers during Onset and Treatment of Exacerbated Immunity in Leprosy

Acute inflammatory reactions represent the major cause of irreversible neuropathy in leprosy. These tissue-destroying episodes have considerable overlap with acute immunological complications (flares) in several chronic (autoimmune) diseases that similarly warrant early detection. However, the lack of diagnostic tests impedes early diagnosis of these reactions. Here, we evaluated a user-friendly multiplex lateral flow assay for the simultaneous detection of IP-10 and anti-phenolic glycolipid I antibodies for longitudinally monitoring early onset and treatment of leprosy reactions

Household Contacts of Leprosy Patients in Endemic Areas Display a Specific Innate Immunity Profile

Leprosy is a chronic infectious disease, caused by Mycobacterium leprae, that can lead to severe life-long disabilities. The transmission of M. leprae is continuously ongoing as witnessed by the stable new case detection rate. The majority of exposed individuals does, however, not develop leprosy and is protected from infection by innate immune mechanisms. In this study the relation between innate immune markers and M. leprae infection as well as the occurrence of leprosy was studied in household contacts (HCs) of leprosy patients with high bacillary loads. Serum proteins associated with innate immunity (ApoA1, CCL4, CRP, IL-1Ra, IL-6, IP-10, and S100A12) were determined by lateral flow assays (LFAs) in conjunction with the presence of M. leprae DNA in nasal swabs (NS) and/or slit-skin smears (SSS). The HCs displayed ApoA1 and S100A12 levels similar to paucibacillary patients and could be differentiated from endemic controls based on the levels of these markers. In the 31 households included the number (percentage) of HCs that were concomitantly diagnosed with leprosy, or tested positive for M. leprae DNA in NS and SSS, was not equally divided. Specifically, households where M. leprae infection and leprosy disease was not observed amongst members of the household were characterized by higher S100A12 and lower CCL4 levels in whole blood assays of HCs in response to M. leprae. Lateral flow assays provide a convenient diagnostic tool to quantitatively measure markers of the innate immune response and thereby detect individuals which are likely infected with M. leprae and at risk of developing disease or transmitting bacteria. Low complexity diagnostic tests measuring innate immunity markers can therefore be applied to help identify who should be targeted for prophylactic treatment

Multi-center evaluation of a user-friendly lateral flow assay to determine IP-10 and CCL4 levels in blood of TB and non-TB cases in Africa

Objective: Multi-center evaluation of a user-friendly lateral flow test for detection of IP-10 and CCL4 levels in Mycobacterium tuberculosis (Mtb) antigen-stimulated whole blood samples from tuberculosis (TB) suspects. Design and methods: A quantitative lateral flow (LF)-based assay platform was applied to detect chemokines IP-10 and CCL4. Chemokine quantitation was achieved using interference-free, fluorescent up-converting phosphor (UCP) labels. The new assays allowed worldwide shipping and storage without requiring a cold chain and were tested at seven institutes (including Ethiopia, Malawi, The Gambia, South Africa, Uganda and Namibia) employing portable lightweight readers for detection of the UCP label. At each site, clinical samples, confirmed TB and non-TB (i.e. other respiratory diseases (ORD)) cases, were collected and analyzed simultaneously with quality control (QC) human IP-10 or CCL4 standards. Results: Performance of the UCP-LF assay in Africa using QC standards indicated high robustness allowing quantitative detection between 100 and 100,000 pg/mL. The optimized assays allowed successful determination of chemokine levels using 1 μL whole blood sample from the locally recruited subjects with TB or ORD. Conclusion: This African multi-center trial further demonstrated the applicability of the low-tech and robust UCP-LF platform as a convenient quantitative assay for chemokine detection in whole blood. Ambient shipping and storage of all assay reagents and the availability of lightweight standalone readers were acknowledged as essential requirement for test implementation in particular in remote and resource-limited settings