Diagnostic Tests to Support Late-Stage Control Programs for Schistosomiasis and Soil-Transmitted Helminthiases

<div><p>Global efforts to address schistosomiasis and soil-transmitted helminthiases (STH) include deworming programs for school-aged children that are made possible by large-scale drug donations. Decisions on these mass drug administration (MDA) programs currently rely on microscopic examination of clinical specimens to determine the presence of parasite eggs. However, microscopy-based methods are not sensitive to the low-intensity infections that characterize populations that have undergone MDA. Thus, there has been increasing recognition within the schistosomiasis and STH communities of the need for improved diagnostic tools to support late-stage control program decisions, such as when to stop or reduce MDA. Failure to adequately address the need for new diagnostics could jeopardize achievement of the 2020 London Declaration goals. In this report, we assess diagnostic needs and landscape potential solutions and determine appropriate strategies to improve diagnostic testing to support control and elimination programs. Based upon literature reviews and previous input from experts in the schistosomiasis and STH communities, we prioritized two diagnostic use cases for further exploration: to inform MDA-stopping decisions and post-MDA surveillance. To this end, PATH has refined target product profiles (TPPs) for schistosomiasis and STH diagnostics that are applicable to these use cases. We evaluated the limitations of current diagnostic methods with regards to these use cases and identified candidate biomarkers and diagnostics with potential application as new tools. Based on this analysis, there is a need to develop antigen-detecting rapid diagnostic tests (RDTs) with simplified, field-deployable sample preparation for schistosomiasis. Additionally, there is a need for diagnostic tests that are more sensitive than the current methods for STH, which may include either a field-deployable molecular test or a simple, low-cost, rapid antigen-detecting test.</p></div

The steps required for gold standard microscopy in deworming programs.

<p>In the typical surveillance testing performed to assess the prevalence of helminth infection and the impact of deworming programs, stool samples (or sometimes urine for schistosomiasis) are collected and transported to a nearby laboratory space for microscopic analysis and follow-on reporting. There are numerous factors affecting each step of the process that contribute to making this analysis less than optimal.</p

Overview of STH and Schistosomaiasis (SCH) diagnostic landscapes.

<p>Overview of STH and Schistosomaiasis (SCH) diagnostic landscapes.</p

Workflow for the use of the lyophilized control with a glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test.

<p>Workflow for the use of the lyophilized control with a glucose-6-phosphate dehydrogenase (G6PD) rapid diagnostic test.</p

Stability of the reconstituted lyophilized recombinant glucose-6-phosphate dehydrogenase (r-G6PD).

<p>Lyophilized controls were reconstituted under four conditions to determine stability after rehydration. Three concentrations were assessed: normal (blue), high intermediate (purple), and intermediate (green). The controls were rehydrated in protein storage buffer (PSB) or deionized (DI) water and kept on ice or at room temperature after rehydration: PSB on ice (filled squares), PSB at room temperature (filled circles), DI water on ice (filled triangles), and DI water at room temperature (filled diamonds). Activity was measured on the Trinity Biotech quantitative assay at time zero, 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, and overnight 24 hours later. Thresholds are shown to indicate whether each measurement is still within an acceptable range of G6PD enzyme activity based on the ranges of Trinity controls: lower acceptable normal (blue dotted line), upper intermediate (black dotted line), lower intermediate (green dotted line), and upper deficient (red dotted line).</p

Target product profile for quality control reagents to support point-of-care diagnostic tests for glucose-6-phosphate dehydrogenase (G6PD) deficiency.

<p>Minimal and optimal requirements are described for different product specifications. The unitized recombinant G6PD tubes are expected to meet most specifications with the exception of the shelf-life of the reconstituted reagents (not achieved).</p

Titration of lyophilized human recombinant glucose-6-phosphate dehydrogenase (r-G6PD).

<p>Lyophilized human r-G6PD (blue line) was titrated and tested in the Trinity Biotech quantitative assay alongside the Trinity controls (grey line). From these data, three lyophilized concentrations were chosen to represent normal, intermediate, and deficient enzyme activity.</p

Performance of the positive controls on a qualitative glucose-6-phosphate dehydrogenase (G6PD) test.

<p><b>A.</b> Evaluation of human recombinant glucose-6-phosphate dehydrogenase (r-G6PD) standards. The lyophilized r-G6PD controls were tested in the newly developed rapid diagnostic test for G6PD. Line intensity versus G6PD activity was generated using over 20 replicates for each enzyme control. The data show that there are significant differences when comparing intermediate and deficient results to normal. B. Representative images of visual results with r-G6PD standards. Visual results presented here show that the control lines gave similar output across the standards tested. Differences were seen between all groups however, the intermediate high and low test line intensities were similar to the trained eye.</p

Descriptive statistics for data illustrated in Fig 6.

<p>Testing of the rapid diagnostic test with recombinant human glucose-6-phosphate dehydrogenase (G6PD) standards, with test-line intensities being measured using a low-cost hand-held reader. Mean difference in readings and lower to upper bounds for a 95% confidence interval (95% CI) as determined by the Dunnetts multiple comparisons test are shown for each control compared to the Normal control readings.</p

Performance of human recombinant glucose-6-phosphate dehydrogenase (r-G6PD) controls on qualitative tests for G6PD.

<p>Two lots of three concentrations (normal, intermediate, and deficient) of human r-G6PD were tested on two qualitative assays for G6PD: the fluorescent spot test (panels A and B) and a novel prototype rapid diagnostic test for G6PD (panels C and D). In panels A and B, the top row represents the Trinity normal controls for reference; the second, third, and fourth rows show the signals for the normal, intermediate, and deficient human r-G6PD controls, respectively. In panels C and D, the line intensity of test output correlates with expected enzyme activity, referring to normal, intermediate, and deficient activity. The signal for one lot of freshly lyophilized human r-G6PD (A and C) is compared to a second lot that had been stored at 4°C for more than one year (B and D).</p