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The relationship between N-antigen concentration and viral load within a specimen and across different specimens is essential for interpretation of rapid diagnostic tests (RDT) clinical performance in different use cases. A prospective study was conducted in Porto Velho, Brazil, to investigate RDT performance in different specimen types as a function of the correlation between antigen concentration and viral load. The study included 214 close contacts with recent exposures to confirmed cases, aged 12 years and older and with various levels of vaccination. Antigen concentration was measured in nasopharyngeal swab (NPS), anterior nares swab (ANS), and saliva specimens. Reverse transcriptase (RT)–PCR was conducted on the NPS and saliva specimens, and two RDTs were conducted on ANS and one on saliva. Antigen concentration correlated with viral load when measured in the same specimen type but not across specimen types. Antigen levels were higher in symptomatic cases compared to asymptomatic/oligosymptomatic cases and lower in saliva compared to NPS and ANS samples. Discordant results between the RDTs conducted on ANS and the RT-PCR on NPS were resolved by antigen concentration values. The analytical limit-of-detection of RDTs can be used to predict the performance of the tests in populations for which the antigen concentration is known. The antigen dynamics across different sample types observed in SARS-CoV-2 disease progression support use of RDTs in nasal samples. Given lower antigen concentrations in saliva, tests using saliva is expected to require improved analytical sensitivity to achieve clinical sensitivity similar to testing of nasal samples.

Rapid diagnostic tests (RDTs) that detect antigen indicative of SARS-CoV-2 infection can help in making quick health care decisions and regularly monitoring groups at risk of infection. With many RDT products entering the market, it is important to rapidly evaluate their relative performance. Comparison of clinical evaluation study results is challenged by protocol design variations and study populations. Laboratory assays were developed to quantify nucleocapsid (N) and spike (S) SARS-CoV-2 antigens. Quantification of the two antigens in nasal eluates confirmed higher abundance of N than S antigen. The median concentration of N antigen was 10 times greater than S per genome equivalent. The N antigen assay was used in combination with quantitative RT-PCR to qualify a panel composed of recombinant antigens, inactivated virus, and clinical specimen pools. This benchmarking panel was applied to evaluate the analytical performance of the SD Biosensor STANDARD Q COVID-19 Ag test, Abbott Panbio COVID-19 Ag Rapid Test, Abbott BinaxNOW COVID-19 Ag test, and the LumiraDx SARS-CoV-2 Ag Test. The four tests displayed different sensitivities toward the different panel members, but all performed best with the clinical specimen pool. The concentration for a 90% probability of detection across the four tests ranged from 21 pg/mL to 102 pg/mL of N antigen in the extracted sample. Benchmarking panels provide a quick way to verify the baseline performance of a diagnostic and enable direct comparison between diagnostic tests.

Background Point-of-care and decentralized testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical to inform public health responses. Performance evaluations in priority use cases such as contact tracing can highlight trade-offs in test selection and testing strategies. Methods A prospective diagnostic accuracy study was conducted among close contacts of coronavirus disease 2019 (COVID-19) cases in Brazil. Two anterior nares swabs (ANS), a nasopharyngeal swab (NPS), and saliva were collected at all visits. Vaccination history and symptoms were assessed. Household contacts were followed longitudinally. Three rapid antigen tests and 1 molecular method were evaluated for usability and performance against reference reverse-transcription polymerase chain reaction (RT-PCR) on nasopharyngeal swab specimens. Results Fifty index cases and 214 contacts (64 household) were enrolled. Sixty-five contacts were RT-PCR positive during ≥1 visit. Vaccination did not influence viral load. Gamma variants were most prevalent; Delta variants emerged increasingly during implementation. The overall sensitivity of evaluated tests ranged from 33% to 76%. Performance was higher among symptomatic cases and those with cycle threshold (Ct) values 70% and almost 90% after 4 days. Conclusions The near-immediate time to results for antigen tests significantly offsets lower analytical sensitivity in settings where RT-PCR results are delayed or unavailable.

BackgroundPoint-of-care and decentralized testing for SARS-CoV-2 is critical to inform public health responses. Performance evaluations in priority use cases such as contact tracing can highlight trade-offs in test selection and testing strategies.MethodsA prospective diagnostic accuracy study was conducted among close contacts of COVID-19 cases in Brazil. Two anterior nares swabs (ANS), a nasopharyngeal swab (NPS), and saliva were collected at all visits. Vaccination history and symptoms were assessed. Household contacts were followed longitudinally. Three rapid antigen tests and one molecular method were evaluated for usability and performance against reference RT-PCR on NPS.ResultsFifty index cases and 214 contacts (64 household) were enrolled. Sixty-five contacts were RT-PCR positive during at least one visit. Vaccination did not influence viral load. Gamma variants were most prevalent; Delta emerged increasingly during implementation. Overall sensitivity of evaluated tests ranged from 33%–76%. Performance was higher among symptomatic cases and cases with Ct70% and almost 90% after four days.ConclusionsThe near immediate time-to-result for antigen tests significantly offsets lower analytical sensitivity in settings where RT-PCR results are delayed or unavailable.

The SD Bioline® IgG4 rapid diagnostic test (RDT) detects IgG4 antibodies induced by the Onchocerca volvulus-specific antigen Ov16. We evaluated the stability of the RDT results over 1 month, at different time points after completion of each assay, using eluted dried blood spots collected in central Cameroon. Agreement coefficients regarding positivity between 30 minutes and 24 hours, 1, 2, 3, and 4 weeks were, 96.4%, 93.4%, 93.3%, 93.2%, and 93.2%, respectively. Between 30 minutes and 24 hours, 3.6% of the 15,444 tests showed inconsistent results with 81.2% of these tests changing from negative to positive, increasing O. volvulus antibody prevalence from 23.9% to 26.2% (P < 0.0001). This change from negative to positive outcome was confirmed at the subsequent timepoints. Depending on the desired accuracy of prevalence estimates, reading time may have to be redefined more strictly.

No rapid diagnostic test exists to screen individuals for primary antibody deficiencies (PAD) at or near the point of care. In settings at risk for polio where live oral polio vaccine is utilized, undiagnosed PAD patients and cases with delayed diagnosis constitute a potential reservoir for neurovirulent polioviruses, undermining polio eradication. This research aimed to develop a rapid screening test suited for use in resource-limited settings to identify individuals with low immunoglobulin G (IgG) levels, enabling early diagnosis and appropriate treatment.Three prototype tests distinguishing low and normal IgG levels were evaluated with a blinded panel of serum/plasma specimens from 32 healthy controls and 86 primary immunodeficiency-confirmed patients with agammaglobulinemia, common variable immunodeficiency, and hyper-IgM syndrome, including 57 not receiving IgG therapy. Prototype tests were compared to laboratory reference and clinical case definition.The leading prototype correctly identified 32 of 32 healthy controls. Among primary antibody deficiency patients not receiving IgG treatment, 17 of 19 agammaglobulinemia, 7 of 24 common variable immunodeficiency, and 5 of 14 hyper-IgM were correctly identified by the prototype, with 67% agreement with the reference assay.The Rapid IgG Screen (RIgGS) test can differentiate between low IgG levels associated with agammaglobulinemia and normal IgG antibody levels. Differentiating CVID and hyper IgM was challenging due to the wide range in IgG levels and influence of high IgM. This test can facilitate the identification of patients with primary antibody deficiencies and support polio surveillance initiatives.

Background The recent expansion of tools designed to accurately quantify malaria parasite-produced antigens has enabled us to evaluate the performance of rapid diagnostic tests (RDTs) as a function of the antigens they detect—typically histidine rich protein 2 (HRP2) or lactate dehydrogenase (LDH). Methods For this analysis, whole blood specimens from a longitudinal study in Bancoumana, Mali were used to evaluate the performance of the ultra-sensitive HRP2-based Alere™ Malaria Ag P.f RDT (uRDT). The samples were collected as part of a transmission-blocking vaccine trial in a high transmission region for Plasmodium falciparum malaria. Furthermore, antigen dynamics after successful anti-malarial drug treatment were evaluated in these samples using the Q-Plex Human Malaria Array (4-Plex) to quantify antigen concentrations. Results The uRDT had a 50% probability of a positive result at 207 pg/mL HRP2 [95% credible interval (CrI) 160–268]. Individuals with symptomatic infection remained positive by uRDT for a median of 33 days [95% confidence interval (CI) 28–47] post anti-malarial drug treatment. Biphasic exponential decay models accurately captured the population level post-treatment dynamics of both HRP2 and Plasmodium LDH (pLDH), with the latter decaying more rapidly. Motivated by these differences in rates of decay, a novel algorithm that used HRP2:pLDH ratios to predict if an individual had active versus recently cleared P. falciparum infection was developed. The algorithm had 77.5% accuracy in correctly classifying antigen-positive individuals as those with and without active infection. Conclusions These results characterize the performance of the ultra-sensitive RDT and demonstrate the potential for emerging antigen-quantifying technologies in the field of malaria diagnostics to be helpful tools in distinguishing between active versus recently cleared malaria infections.

Author(s): Jang, Ihn Kyung; Tyler, Abby; Lyman, Chris; Rek, John C; Arinaitwe, Emmanuel; Adrama, Harriet; Murphy, Maxwell; Imwong, Mallika; Proux, Stephane; Haohankhunnatham, Warat; Barney, Rebecca; Rashid, Andrew; Kalnoky, Michael; Kahn, Maria; Golden, Allison; Nosten, Francois; Greenhouse, Bryan; Gamboa, Dionicia; Domingo, Gonzalo J | Abstract: Malaria antigen detection through rapid diagnostic tests (RDTs) is widely used to diagnose malaria and estimate prevalence. To support more sensitive next-generation RDT development and screen asymptomatic malaria, we developed and evaluated the Q-Plex™ Human Malaria Array (Quansys Biosciences, Logan, UT), which quantifies the antigens commonly used in RDTs-Plasmodium falciparum-specific histidine-rich protein 2 (HRP2), P. falciparum-specific lactate dehydrogenase (Pf LDH), Plasmodium vivax -specific LDH (Pv LDH), and Pan malaria lactate dehydrogenase (Pan LDH), and human C-reactive protein (CRP), a biomarker of severity in malaria. At threshold levels yielding 99.5% or more diagnostic specificity, diagnostic sensitivities against polymerase chain reaction-confirmed malaria for HRP2, Pf LDH, Pv LDH, and Pan LDH were 92.7%, 71.5%, 46.1%, and 83.8%, respectively. P. falciparum culture strains and samples from Peru indicated that HRP2 and Pf LDH combined improves detection of P. falciparum parasites with hrp2 and hrp3 deletions. This array can be used for antigen-based malaria screening and detecting hrp2/3 deletion mutants of P. falciparum.

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.

The World Health Organization currently recommends assessing elimination of onchocerciasis by testing whether Ov16 antibody prevalence in children aged 0–9 years is below 0.1%. However, the certainty of evidence for this recommendation is considered to be low. We used the established ONCHOSIM model to investigate the predictive value of different Ov16-antibody prevalence thresholds in various age groups for elimination of onchocerciasis in a variety of endemic settings and for various mass drug administration scenarios. According to our simulations, the predictive value of Ov16 antibody prevalence for elimination depends highly on the precontrol epidemiologic situation, history of mass drug administration, the age group that is sampled, and the chosen Ov16-antibody prevalence threshold. The Ov16 antibody prevalence in children aged 5–14 years performs best in predicting elimination. Appropriate threshold values for this age group start at 2.0% for very highly endemic areas; for lower-endemic areas, even higher threshold values are safe to use. Guidelines can be improved by sampling school-aged children, which also is operationally more feasible than targeting children under age 10 years. The use of higher threshold values allows sampling of substantially fewer children. Further improvement can be achieved by taking a differentiated sampling approach based on precontrol endemicity.

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.

In the past few years, efforts to eliminate onchocerciasis from Africa have intensified. These efforts are primarily based on the mass distribution of the anti-helminthic drug Mectizan™ (ivermectin). This program has led to the development of new guidelines by the World Health Organization for the verification that transmission has been suppressed and eventually eliminated. The requirements of diagnostic tools for this purpose differ in many ways from tests used to diagnose infection in individuals. In this review, we summarize the progress that has been made to identify diagnostics that meet the specialized requirements needed to verify onchocerciasis elimination, discuss why these tests were selected and summarize the needs that still exist to complete the arsenal of diagnostic tools that will be useful as the goal of elimination is achieved.

Background Onchocerciasis is targeted for elimination in Africa through annual or biannual ivermectin mass drug administration (MDA). An immunodiagnostic test, based on the detection of human IgG4 antibodies in the blood to the Onchocerca volvulus-specific antigen Ov16, is one of the recommended tools for determining whether transmission is interrupted and mass treatment can stop. For different transmission settings, the relationship between post-MDA Ov16 antibody prevalence in children (measured 1 year after the last round of MDA) and the duration and coverage of MDA, the mf prevalence in the population, and the probability that onchocerciasis is eventually eliminated is explored through mathematical modelling. Methodology The ONCHOSIM model was extended with new output on the Ov16 antibody serostatus of individuals. Seroconversion was assumed to be triggered by the first worm establishing in the host, with seroconversion occurring either before maturation, after maturation or only after the start of mf production. We are mainly interested in seroconversion rates in children, and for now ignore the possibility of seroreversion to simplify the model. Principal findings Yearly repeated MDA leads to a strong reduction in the parasite acquisition rate in humans. This reduces the seroconversion rate in newborns and young children, while those who seroconverted before the start of control remain antibody positive. Both the microfiladermia prevalence in the population aged 5 years and above and the Ov16 antibody prevalence in children under 10 declined with increasing duration of MDA. The association between either of these indicators and the model-predicted probability of elimination was not influenced much by the assumed treatment coverage levels, but was found to depend on baseline endemicity levels, assumptions regarding the trigger of seroconversion, and diagnostic test characteristics (sensitivity and specificity). Conclusions Better understanding of the dynamics of Ov16 antibody responses is required for accurate interpretation of seroprevalence data and more precise estimation of endpoint for MDA. Our study demonstrates that this endpoint will be dependent on baseline endemicity levels, which should be taken into account in guidelines for defining when to stop MDA., Author Summary Onchocerciasis is targeted for elimination in Africa through annual or biannual ivermectin mass drug administration. The elimination target places high demands on monitoring and evaluation systems, for timely detection of ongoing transmission and possible recrudescence. Guidelines recommend using serological evaluation by Ov16, to determine the presence of IgG4 antibodies to the antigen Ov16 in children of less than 10 years in order to detect exposure to the O. volvulus parasite. In this paper, we use the mathematical model ONCHOSIM to explore how the post-MDA Ov16 antibody prevalence in children (measured 1 year after the last round of MDA) is related to local transmission conditions, observed trends in infection during mass drug administration the duration and coverage of mass treatment, test characteristics, mf prevalence and likelihood of interruption of transmission. The level of seroprevalence that is indicative of successful elimination was found to depend on local transmission conditions. This should be considered in survey methodology and criteria for defining whether mass drug administration can safely be interrupted. Remaining uncertainties about the dynamics of seroconversion and seroreversion further complicate the interpretation of seroprevalence data and the definition of thresholds.

Soil-transmitted helminths (STHs) affect more than 1.5 billion people. The global strategy to control STH infections requires periodic mass drug administration (MDA) based on prevalence among populations at risk determined by diagnostic testing. Widely used copromicroscopy methods to detect infection, however, have low sensitivity as the prevalence and intensity of STH infections decline with repeated MDA. More sensitive diagnostic tools are needed to inform program decision-making. Using an integrated product development process, PATH conducted qualitative and quantitative formative research to inform the design and development of a more sensitive test for STH infections. The research, grounded in a conceptual framework for ensuring access to health products, involved stakeholder analysis, key opinion leader interviews, observational site visits of ongoing STH surveillance programs, and market research including market sizing, costing and willingness-to-pay analyses. Stakeholder analysis identified key groups and proposed strategic engagement of stakeholders during product development. Interviews highlighted features, motivations and concerns that are important for guiding design and implementation of new STH diagnostics. Process mapping outlined current STH surveillance workflows in Kenya and the Philippines. Market sizing in 2016 was estimated around half a million tests for lower STH burden countries, and 1–2 million tests for higher STH burden countries. The cost of commodities per patient for a molecular STH diagnostic may be around $10, 3–4 times higher than copromicroscopy methods, though savings may be possible in time and staffing requirements. The market is highly price sensitive as even at $5 per test, only 27% of respondents thought the test would be used by surveillance programs. A largely subsidized STH control strategy and a semi-functional Kato-Katz test may have created few incentives for manufacturers to innovate in STH diagnostics. Diverse partnerships, as well as balancing needs and expectations for new STH diagnostics are necessary to ensure access to needed products., Author summary Soil-transmitted helminths affect more than 1.5 billion people, mostly in very poor regions without proper sanitation. To control infections, countries periodically give deworming drugs to populations at risk, such as school-age children, based on the results of diagnostic testing to determine the prevalence of infection. The current method to detect infection works when infections are prevalent, but is less useful after repeated deworming, when disease prevalence is lower. We conducted formative research—including user and market research—to inform development of a new diagnostic tool with improved sensitivity. Findings from this research highlight pain points with current methods and processes, which are also opportunities for innovation. Access to new STH diagnostics may improve program decision-making and eventually contribute to elimination of these infections.

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., Author Summary Serological markers such as antibody responses to pathogen-specific antigens are used to inform disease epidemiology in many elimination programs. A major challenge with program-scale serological testing, and with any diagnostic test validation, is access to consistent and unlimited control reagents with which to provide assay QC and facilitate data consolidation. In the context of disease elimination, clinical positive sera will be particularly difficult to source and use as routine, inter-laboratory reagents. This study reports on a recombinant antibody specific against a key serological marker for onchocerciasis: its selection, testing, and incorporation into protocols across relevant immunoassay platforms. We have demonstrated it is a viable reagent for integration into QC and QA protocols to support long-term serological testing for onchocerciasis to support disease elimination efforts. This approach should be generalizable to other diagnostic tools supporting programs to achieve the 2020 goals of the London Declaration on Neglected Tropical Diseases.

Novel typhoid diagnostics currently under development have the potential to improve clinical care, surveillance, and the disease burden estimates that support vaccine introduction. Blood culture is most often used as the reference method to evaluate the accuracy of new typhoid tests; however, it is recognized to be an imperfect gold standard. If no single gold standard test exists, use of a composite reference standard (CRS) can improve estimation of diagnostic accuracy. Numerous studies have used a CRS to evaluate new typhoid diagnostics; however, there is no consensus on an appropriate CRS. In order to evaluate existing tests for use as a reference test or inclusion in a CRS, we performed a systematic review of the typhoid literature to include all index/reference test combinations observed. We described the landscape of comparisons performed, showed results of a meta-analysis on the accuracy of the more common combinations, and evaluated sources of variability based on study quality. This wide-ranging meta-analysis suggests that no single test has sufficiently good performance but some existing diagnostics may be useful as part of a CRS. Additionally, based on findings from the meta-analysis and a constructed numerical example demonstrating the use of CRS, we proposed necessary criteria and potential components of a typhoid CRS to guide future recommendations. Agreement and adoption by all investigators of a standardized CRS is requisite, and would improve comparison of new diagnostics across independent studies, leading to the identification of a better reference test and improved confidence in prevalence estimates.

During the last decade, several large clinical studies have demonstrated that analysis of chromosomal abnormalities is an essential basis for therapeutic decisions in patients with acute myelogenous leukemia (AML), and cytogenetic studies should now be regarded as mandatory both for routine treatment and as a part of clinical investigations in AML. However, new techniques for detailed genetic characterization and analysis of gene expression as well as protein modulation will become important in the further classification of AML subsets and the development of risk-adapted therapeutic strategies. In this context, we emphasize the importance of population-based clinical studies as a basis for future therapeutic guidelines. Such studies will then require the inclusion of patients at small clinical centers without specialized hematological research laboratories. To document a high and uniform quality of the laboratory investigations, it will be necessary to collect material for later analysis in selected laboratories. In this article, we describe current methods for collection of biological samples that can be used for later preparation of DNA, RNA, and proteins. With the use of gradient-separated AML cells, it should be possible to establish the necessary techniques for collection and handling of biological samples even at smaller centers, and complete collections from all included patients should then be possible even in population-based clinical studies.

The Global Programme to Eliminate Lymphatic Filariasis has an urgent need for rapid assays to detect ongoing transmission of lymphatic filariasis (LF) following multiple rounds of mass drug administration (MDA). Current WHO guidelines support using the antigen card immunochromatographic test (ICT), which detects active filarial infection but does not detect early exposure to LF. Recent studies found that antibody-based assays better serve this function. In the present study, two tests, a rapid IgG4 enzyme-linked immunosorbent assay (ELISA) and a lateral-flow strip immunoassay, were developed based on the highly sensitive and specific Wuchereria bancrofti antigen Wb123. A comparison of W. bancrofti -infected and -uninfected patients (with or without other helminth infections) demonstrated that both tests had high sensitivities and specificities (93 and 97% [ELISA] and 92 and 96% [strips], respectively). When the W. bancrofti -uninfected group was separated into those with other filarial/helminth infections (i.e., onchocerciasis, loiasis, and strongyloidiasis) and those who were parasite uninfected, the specificities of the assays varied between 91 and 100%. In addition, the geometric mean response by ELISA of W. bancrofti -infected patients was significantly higher than the response of those without W. bancrofti infection ( P < 0.0001). Furthermore, the Wb123 ELISA and the lateral-flow strips had high positive and negative predictive values, giving valuable information on the size of survey population needed to be reasonably certain whether or not transmission is ongoing. These highly sensitive and specific IgG4 tests to the W. bancrofti Wb123 protein give every indication that they will serve as useful tools for post-MDA monitoring.

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.

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