Objective: This study, conducted between March 2015 and July 2016, investigated the sensitivity and specificity of the OV-16 RDT and Dried Blood Spot (DBS) Enzyme-Linked Immunosorbent Assay (ELISA) for skin snip microfilaria (MF) detection in Ogun State. Methods: Five hundred and eighty-eight (588) members of 16 first-line communities in 8 endemic Local Government Areas (LGAs) provided fingerstick whole blood specimens examined for IgG4 antibodies against the O. volvulus antigen OV-16, using DBS OV-16 ELISA and OV-16 RDT. In consort with these specimens, 162 study participants consented to be evaluated by skin snip. Results: Results showed a seroprevalence of 106(18.0%) using ELISA and 102(17.3%) by RDT, with 92.2% agreement. The OV-16 RDT demonstrated a positive and negative agreement of 76.4 (95% CI 67.2 to 84.1%) and 95.6% (95% CI: 93.4 to 97.3), respectively, to the OV-16 ELISA results from the concordant dataset. Against the 64 positive skin snips microscopy results, sensitivity of the RDT was 79.7% (95% CI 73.43% to 85.27) and the ELISA was 70.3% (95% CI 64.72% to 75.37%). Conclusion: The results of this study provided information on the performance of the OV-16 RDT. This information will contribute data for comparison to studies in other areas where treatment is ongoing. In conclusion, the commercial launch of the OV-16 RDT has made available a point-of-care diagnostic tool that could serve to help local and country onchocerciasis elimination programs to measure changes and progress in onchocerciasis intervention.

A prototype of a self-contained, automated, disposable device for chemically amplified protein-based detection of influenza virus from nasal swab specimens was developed and evaluated in a clinical setting. The device required only simple specimen manipulation without any dedicated instrumentation or specialized training by the operator for interpretation. The device was based on a sandwich immunoassay for influenza virus nucleoprotein; it used an enzyme-labeled antibody and a chromogenic substrate to provide an amplified visible signal, in a two-dimensional paper network format. All reagents were stored within the device. Device performance was assessed at Seattle Children's Hospital; clinical staff collected nasal swab samples from 25 patients and then operated test devices on site to detect influenza A and B in those specimens. The total test time from device initiation to result was approximately 35 min. Device performance for influenza A detection was ∼70% accurate using in-house qRT-PCR influenza A as a gold-standard comparison. The ratio of valid to total completed device runs yielded a success rate of 92%, and the negative predictive value for both the influenza A and B assay was 81%. The ability to diagnose respiratory infections rapidly and close to the patient was well received by hospital staff, inspiring further optimization of device function.

Since 2005, Ethiopia has aggressively scaled up malaria prevention and case management. As a result, the number of malaria cases and deaths has significantly declined. In order to track progress towards the elimination of malaria in Amhara Region, coverage of malaria control tools and current malaria transmission need to be documented.

Background: This serological study conducted between March and July 2015 investigated the status of onchocerciasis in Ogun State, Nigeria after a decade of mass drug administration (MDA) with ivermectin. Baseline information from the rapid epidemiological mapping of onchocerciasis (REMO) prior to MDA had indicated that Ogun State was meso-endemic to onchocerciasis. Following years of treatment with ivermectin, it has become important to investigate the current status of the disease using more sensitive diagnostic methods. Methods: The study was conducted in 32 communities in eight onchocerciasis-endemic local government areas (LGAs). Using the Ov16 rapid diagnostic test (Ov16 RDT), finger-prick whole blood was obtained from 3 895 consented participants from age five and above. Skin snips were obtained from a subset of 481 corresponding participants as the majority of community members did not consent to skin snipping. Results: Study revealed a cumulative seroprevalence of 9.3% (CI 9.29–9.31%) by RDT and 17.3% (CI 16.73–18.34%) microfilaridermia prevalence by the skin snip subset. Seroprevalence among children between the ages of five and nine was 2.9% (CI 1.74–3.53%) across all LGAs. A community microfilaria load (CMFL) between 0 and 1.21 MF/skin snip was recorded in the different communities studied. Among the studied LGAs, children in Odeda LGA recorded the highest seroprevalence by RDT (14.9%) followed by Abeokuta North (5.1%), Abeokuta South (4.8%) and Imeko-Afon (0.6%), while Ewekoro, Ifo, Obafemi-Owode and Yewa North LGAs recorded zero prevalence. Conclusion: It appears that the elimination of onchocerciasis in some LGAs is possible considering the lack of new infection among children and the hypo-endemicity among the adult population. However, the microfilaria prevalence observed among adults is of concern as it may imply that mass treatment has not been effective. Increased therapeutic coverage is advised to fully maximise the potential of ivermectin treatment to achieve the disease elimination.

Background Serological assays for human IgG4 to the Onchocerca volvulus antigen Ov16 have been used to confirm elimination of onchocerciasis in much of the Americas and parts of Africa. A standardized source of positive control antibody (human anti-Ov16 IgG4) will ensure the quality of surveillance data using these tests. Methodology/Principal Findings A recombinant human IgG4 antibody to Ov16 was identified by screening against a synthetic human Fab phage display library and converted into human IgG4. This antibody was developed into different positive control formulations for enzyme-linked immunosorbent assay (ELISA) and rapid diagnostic test (RDT) platforms. Variation in ELISA results and utility as a positive control of the antibody were assessed from multiple laboratories. Temperature and humidity conditions were collected across seven surveillance activities from 2011–2014 to inform stability requirements for RDTs and positive controls. The feasibility of the dried positive control for RDT was evaluated during onchocerciasis surveillance activity in Togo, in 2014. When the anti-Ov16 IgG4 antibody was used as a standard dilution in horseradish peroxidase (HRP) and alkaline phosphatase (AP) ELISAs, the detection limits were approximately 1ng/mL by HRP ELISA and 10ng/mL by AP ELISA. Positive control dilutions and spiked dried blood spots (DBS) produced similar ELISA results. Used as a simple plate normalization control, the positive control antibody may improve ELISA data comparison in the context of inter-laboratory variation. The aggregate temperature and humidity monitor data informed temperature parameters under which the dried positive control was tested and are applicable inputs for testing of diagnostics tools intended for sub-Saharan Africa. As a packaged positive control for Ov16 RDTs, stability of the antibody was demonstrated for over six months at relevant temperatures in the laboratory and for over 15 weeks under field conditions. Conclusions The recombinant human anti-Ov16 IgG4 antibody-based positive control will benefit inter-laboratory validation of ELISA assays and serve as quality control (QC) reagents for Ov16 RDTs at different points of the supply chain from manufacturer to field use.

BACKGROUND:Diagnostics provide a means to measure progress toward disease elimination. Many countries in Africa are approaching elimination of onchocerciasis after successful implementation of mass drug administration programs as well as vector control. An understanding of how markers for infection such as skin snip microfilaria and Onchocerca volvulus-specific seroconversion perform in near-elimination settings informs how to best use these markers. METHODS:All-age participants from 35 villages in Togo were surveyed in 2013 and 2014 for skin snip Onchocerca volvulus microfilaria and IgG4 antibody response by enzyme-linked immunosorbent assay (ELISA) to the Onchocerca volvulus-specific antigen Ov16. A Gaussian mixture model applying the expectation-maximization (EM) algorithm was used to determine seropositivity from Ov16 ELISA data. For a subset of participants (n = 434), polymerase chain reaction (PCR) was performed on the skin snips taken during surveillance. RESULTS:Within the 2,005 participants for which there was Ov16 ELISA data, O. volvulus microfilaremia prevalence and Ov16 seroprevalence were, 2.5 and 19.7 %, respectively, in the total population, and 1.6 and 3.6 % in children under 11. In the subset of 434 specimens for which ELISA, PCR, and microscopy data were generated, it was found that in children under 11 years of age, the anti-Ov16 IgG4 antibody response demonstrate a sensitivity and specificity of 80 and 97 %, respectively, against active infections as determined by combined PCR and microscopy on skin snips. Further analysis was performed in 34 of the 35 villages surveyed. These villages were stratified by all-age seroprevalence into three clusters: < 15 %; 15-20 %; and > 20 %. Age-dependence of seroprevalence for each cluster was best reflected by a two-phase force-of-infection (FOI) catalytic model. In all clusters, the lower of the two phases of FOI was associated with a younger age group, as reflected by the seroconversion rates for each phase. The age at which transition from lower to higher seroconversion, between the two phases of FOI, was found to be highest (older) for the cluster of villages with < 15 % seroprevalence and lowest (younger) for the cluster with the highest all-age seroprevalence. CONCLUSIONS:The anti-Ov16 IgG4 antibody response is an accurate marker for active infection in children under 11 years of age in this population. Applying Ov16 surveillance to a broader age range provides additional valuable information for understanding progression toward elimination and can inform where targeted augmented interventions may be needed. Clustering of villages by all-age sero-surveillance allowed application of a biphasic FOI model to differentiate seroconversion rates for different age groups within the village cluster categories.

ABSTRACT Elimination programs for Wuchereria bancrofti and Onchocerca volvulus are in critical need of sensitive, specific, and point-of-contact (POC) tools that can be used for surveillance years beyond cessation of mass drug administration when infection intensities are low. Previously, Wb123 and Ov16 were identified individually as potential filarial antigens for an antibody-based POC test. The present study compares single-antigen Wb123- and Ov16-based POC tests with an integrated configuration to detect antibodies to Wb123 and Ov16 simultaneously. Wb123 and Ov16 isolates were striped onto lateral flow strips containing anti-IgG4. Sera from W. bancrofti-, O. volvulus-, and other helminth-infected or -uninfected individuals were added to the strips with buffer. Strips were read for the appearance of a positive or negative test line for both antigens at 20 min and following drying. Sensitivity, specificity, and predictive values were calculated for the single-antigen and biplex strips. Single and biplex lateral flow strips showed nearly identical results, with >90% sensitivity for Ov16 and >92% sensitivity for Wb123. Overall specificities for the single and biplex tests were 98% and 96% for Ov16 and Wb123, respectively. Biplex tests performed as well as the single-antigen tests regardless of the intensity of patient IgG4 response. The high sensitivity and specificity make these new biplex tests extremely useful for POC long-term surveillance following mass drug administration in Africa that should reduce time and cost in areas where bancroftian filariasis and onchocerciasis are coendemic.

Onchocerciasis is a neglected tropical disease caused by infection with the parasite Onchocerca volvulus (Ov). An estimated 180 million people are at risk for Ov infection, and 37 million people are infected, mostly in Africa. A lateral flow-based assay to detect human IgG4 antibodies to the Ov-specific antigen Ov-16 was developed as a rapid tool to detect exposure to Ov. The test, when performed on 449 sera specimens from patients with microfiladermia and Ov-negative patients, has a sensitivity of 89.1% (95% confidence interval: 86.2%-92.0%), and specificity of 97% (95% confidence interval: 95.4%-98.6%). Because the intended use of the test is for surveillance, it is highly desirable to have a stable, long-lasting result. An extended read window is thus desirable for a high-volume, busy workflow and facilitates post-surveillance quality assurance. The main restriction on achieving an extended read window for this assay was the erythrocyte lysis that can alter the signal-to-noise ratio, especially in those with low IgG4 levels (weak positives). We describe a test housing that incorporates a user-independent feature driven by assay fluid and an expanding wick that detaches the blood separation membrane from the nitrocellulose used in the assay, but before hemolysis occurs. We demonstrated material functionality at extreme operational conditions (37°C, 80% relative humidity) and a read window of a minimum of 70 days. The fluid-driven assay device performs equally as well with whole blood as with plasma, as demonstrated with 100 spiked clinical specimens (with a correlation coefficient of 0.96). We show a novel, inexpensive, and simple approach to actuating the detachment of the blood separation membrane from the nitrocellulose test with no impact on the performance characteristics of the test.

Criteria for testing development of rapid diagnostic test to support onchocerciasis control and elimination programs Allison L. Golden1, Dunia Faulx1, Lindsay Yokobe1, Eric Stevens1, Roger Peck1, Melissa Valdez1, Cathy Steel2, Thomas B. Nutman2, Michael Kalnoky3, Afework Hailemariam Tekle4, Gonzalo Domingo1, Tala de los Santos1 1PATH, Seattle, WA, United States, 2National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, United States, 3Tsuga Analytics, Seattle, WA, United States, 4African Programme for Onchocerciasis Control, Ouagadougou, Burkina Faso 263