Field-Evaluation of a New Lateral Flow Assay for Detection of Cellular and Humoral Immunity against Mycobacterium leprae

Abstract

BackgroundField-applicable tests detecting asymptomatic Mycobacterium leprae (M. leprae) infection or predicting progression to leprosy, are urgently required. Since the outcome of M. leprae infection is determined by cellular- and humoral immunity, we aim to develop diagnostic tests detecting pro-/anti-inflammatory and regulatory cytokines as well as antibodies against M. leprae. Previously, we developed lateral flow assays (LFA) for detection of cytokines and anti-PGL-I antibodies. Here we evaluate progress of newly developed LFAs for applications in resource-poor settings.MethodsThe combined diagnostic value of IP-10, IL-10 and anti-PGL-I antibodies was tested using M. leprae-stimulated blood of leprosy patients and endemic controls (EC). For reduction of the overall test-to-result time the minimal whole blood assay time required to detect distinctive responses was investigated. To accommodate LFAs for field settings, dry-format LFAs for IP-10 and anti-PGL-I antibodies were developed allowing storage and shipment at ambient temperatures. Additionally, a multiplex LFA-format was applied for simultaneous detection of anti-PGL-I antibodies and IP-10. For improved sensitivity and quantitation upconverting phosphor (UCP) reporter technology was applied in all LFAs.ResultsSingle and multiplex UCP-LFAs correlated well with ELISAs. The performance of dry reagent assays and portable, lightweight UCP-LF strip readers indicated excellent field-robustness. Notably, detection of IP-10 levels in stimulated samples allowed a reduction of the whole blood assay time from 24 h to 6 h. Moreover, IP-10/IL-10 ratios in unstimulated plasma differed significantly between patients and EC, indicating the feasibility to identify M. leprae infection in endemic areas.ConclusionsDry-format UCP-LFAs are low-tech, robust assays allowing detection of relevant cytokines and antibodies in response to M. leprae in the field. The high levels of IP-10 and the required shorter whole blood assay time, render this cytokine useful to discriminate between leprosy patients and EC.

Longitudinal IP-10 Serum Levels Are Associated with the Course of Disease Activity and Remission in Patients with Rheumatoid Arthritis

“…The level of IP-10 at a single time point is therefore not informative whatsoever with respect to disease activity, as it may fluctuate significantly between different individuals, regardless of their clinical status. Similar results were obtained for sera derived from leprosy patients with or without leprosy reactions (8,26). Longitudinal monitoring is therefore vital for management of inflammatory diseases and can provide…”

“…Quantitative lateral flow (LF)-based assays were applied to detect IP-10 as described previously (26,37,38). Briefly, a mixture of 100 ng of a cytokine-specific UCP reporter conjugate and a diluted (1:30) serum sample was incubated for 60 min on a thermal shaker at 37°C and 900 rpm.…”

Description of Leprosy Classification at Baseline Among Patients Enrolled at the Uniform Multidrug Therapy Clinical Trial for Leprosy Patients in Brazil

“…6,7 The detection of anti-PGL-I antibodies can be a useful adjunct method to identify patients with higher bacterial load and point-of-care serological tests such as ML Flow test, performed at diagnosis can contribute *Address correspondence to Samira Bü hrer-Sé kula, IPTSP-Sala 335, Rua 235, s/n, Setor Leste Universitá rio, Goiania, Goias, Brazil 74605-050. E-mail: samira@buhrer.net to the prompt identification of MB leprosy patients, 6,8,9 which are the ones with higher risk to develop reactions. [10][11][12] This study used the U-MDT/CT-BR database to describe and report the performance of available tools to classify PB and MB leprosy patients at baseline.…”

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

“…Additionally, besides having high sensitivity due to the complete absence of background fluorescence, UCP-LF test strips can be stored as permanent records, allowing reanalysis in a reference laboratory. To develop diagnostic assays for application in resource-limited areas, we previously developed UCP-LFAs for single detection of cytokines (IFN-␥, IP-10, interleukin 10 [IL-10], CCL4) as well as antibodies against M. leprae PGL-I for the diagnosis of nonreactional leprosy and tuberculosis (18)(19)(20). Generally, the performance of one biomarker can be significantly enhanced by using a custom-made grouping of independent biomarkers called a biomarker profile or signature.…”

“…Concentrations of antibodies against PGL-I and IP-10 were measured in all sera using a single UCP-LFA for either IP-10 or anti-PGL-I IgM and a multiplex UCP-LFA for both markers. Simultaneous detection of IP-10 and anti-PGL-I IgM was performed following a two-phase protocol described for single analyte detection (18,24,25). The protocol included a preflow incubation (60 min, 37°C, 900 rpm) of 10 l 100-fold-diluted sample with 90 l LF assay buffer containing 100 ng of the UCP ␣IP-10 conjugate and 100 ng of the UCP ␣IgM conjugate (18).…”