Development, confirmation, and application of a seeded Escherichia coli process control organism to validate Salmonella enterica serovar Typhi environmental surveillance methods.

Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of Typhoid fever. Blood culture is the gold standard for clinical diagnosis, but this is often difficult to employ in resource limited settings. Environmental surveillance of waste-impacted waters is a promising supplement to clinical surveillance, however validating methods is challenging in regions where S. Typhi concentrations are low. To evaluate existing S. Typhi environmental surveillance methods, a novel process control organism (PCO) was created as a biosafe surrogate. Using a previous described qPCR assay, a modified PCR amplicon for the staG gene was cloned into E. coli. We developed a target region that was recognized by the Typhoid primers in addition to a non-coding internal probe sequence. A multiplex qPCR reaction was developed that differentiates between the typhoid and control targets, with no cross-reactivity or inhibition of the two probes. The PCO was shown to mimic S. Typhi in lab-based experiments with concentration methods using primary wastewater: filter cartridge, recirculating Moore swabs, membrane filtration, and differential centrifugation. Across all methods, the PCO seeded at 10 CFU/mL and 100 CFU/mL was detected in 100% of replicates. The PCO is detected at similar quantification cycle (Cq) values across all methods at 10 CFU/mL (Average = 32.4, STDEV = 1.62). The PCO was also seeded into wastewater at collection sites in Vellore (India) and Blantyre (Malawi) where S. Typhi is endemic. All methods tested in both countries were positive for the seeded PCO. The PCO is an effective way to validate performance of environmental surveillance methods targeting S. Typhi in surface water.

SARS-CoV-2 recombinase polymerase amplification assay with lateral flow readout and duplexed full process internal control.

Nucleic acid amplification tests for the detection of SARS-CoV-2 have been an important testing mechanism for the COVID-19 pandemic. While these traditional nucleic acid diagnostic methods are highly sensitive and selective, they are not suited to home or clinic-based uses. Comparatively, rapid antigen tests are cost-effective and user friendly but lack in sensitivity and specificity. Here we report on the development of a one-pot, duplexed reverse transcriptase recombinase polymerase amplification SARS-CoV-2 assay with MS2 bacteriophage as a full process control. Detection is carried out with either real-time fluorescence or lateral flow readout with an analytical sensitivity of 50 copies per reaction. Unlike previously published assays, the RNA-based MS2 bacteriophage control reports on successful operation of lysis, reverse transcription, and amplification. This SARS-CoV-2 assay features highly sensitive detection, visual readout through an LFA strip, results in less than 25 minutes, minimal instrumentation, and a useful process internal control to rule out false negative test results.

Correction: Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

[This corrects the article DOI: 10.1371/journal.pone.0274415.].

A Reagent and Virus Benchmarking Panel for a Uniform Analytical Performance Assessment of N Antigen-Based Diagnostic Tests for COVID-19.

Rapid diagnostic tests (RDTs) that detect antigen indicative of severe acute respiratory syndrome coronavirus-2 (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 reverse transcription (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 antigen (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 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 comparisons between diagnostic tests. IMPORTANCE This study reports the results for severe acute respiratory syndrome coronavirus-2 (SARS-COV-2) nucleocapsid (N) and spike (S) antigen quantification assays and their performance against clinical reverse transcription (RT)-PCR results, thus describing an open-access quantification method for two important SARS-CoV-2 protein analytes. Characterized N antigen panels were used to evaluate the limits of detection of four different rapid tests for SARS-CoV-2 against multiple sources of nucleocapsid antigen, demonstrating proof-of-concept materials and methodology to evaluate SARS-CoV-2 rapid antigen detection tests. Quantification of N antigen was used to characterize the relationship between viral count and antigen concentration among clinical samples and panel members of both clinical sample and viral culture origin. This contributes to a deeper understanding of protein antigen and molecular analytes and presents analytical methods complementary to clinical evaluation for characterizing the performance of both laboratory-based and point-of-care rapid diagnostics for SARS-CoV-2.

Multiplexed micronutrient, inflammation, and malarial antigenemia assessment using a plasma fractionation device.

Processing and storing blood samples for future analysis of biomarkers can be challenging in resource limited environments. The preparation of dried blood spots (DBS) from finger-stick collection of whole blood is a widely used and established method as DBS are biosafe, and allow simpler field processing, storage, and transport protocols than serum or plasma. Therefore, DBS are commonly used in population surveys to assess infectious disease and/or micronutrient status. Recently, we reported that DBS can be used with the Q-plex™ Human Micronutrient 7-plex Array (MN 7-plex), a multiplexed immunoassay. This tool can simultaneously quantify seven protein biomarkers related to micronutrient deficiencies (iodine, iron and vitamin A), inflammation, and malarial antigenemia using plasma or serum. Serum ferritin, an iron biomarker, cannot be measured from DBS due to red blood cell (RBC) ferritin content confounding the results. In this study, we assess a simple blood fractionation tool that passively separates plasma from other blood components via diffusion through a membrane into a plasma collection disc (PCD). We evaluated the concordance of MN 7-plex analyte concentrations from matched panels of eighty-eight samples of PCD, DBS, and wet plasma prepared from anticoagulated venous whole blood. The results showed good correlations of >0.93 between the eluates from PCD and DBS for each analyte except ferritin; while correlations seen for plasma/PCD were weaker. However, the recovery rate of the analytes from the PCD were better than those from DBS. The serum ferritin measures from the PCD were highly correlated to wet plasma samples (0.85). This suggests that surveillance for iron status in low resource settings can be improved over the current methods restricted to only measuring sTfR in DBS. When used in combination with the MN 7-plex, all seven biomarkers can be simultaneously measured using eluates from the PCDs.

Rapid detection of hepatitis C virus using recombinase polymerase amplification.

Over 71 million people are infected with hepatitis C virus (HCV) worldwide, and approximately 400,000 global deaths result from complications of untreated chronic HCV. Pan-genomic direct-acting antivirals (DAAs) have recently become widely available and feature high cure rates in less than 12 weeks of treatment. The rollout of DAAs is reliant on diagnostic tests for HCV RNA to identify eligible patients with viremic HCV infections. Current PCR-based HCV RNA assays are restricted to well-resourced central laboratories, and there remains a prevailing clinical need for expanded access to decentralized HCV RNA testing to provide rapid chronic HCV diagnosis and linkage to DAAs in outpatient clinics. This paper reports a rapid, highly accurate, and minimally instrumented assay for HCV RNA detection using reverse transcription recombinase polymerase amplification (RT-RPA). The assay detects all HCV genotypes with a limit of detection of 25 copies per reaction for genotype 1, the most prevalent in the United States and worldwide. The clinical sensitivity and specificity of the RT-RPA assay were both 100% when evaluated using 78 diverse clinical serum specimens. The accuracy, short runtime, and low heating demands of RT-RPA may enable implementation in a point-of-care HCV test to expand global access to effective treatment via rapid chronic HCV diagnosis.

Performance of novel antibodies for lipoarabinomannan to develop diagnostic tests for Mycobacterium tuberculosis.

Lipoarabinomannan (LAM), a component of the Mycobacterium tuberculosis (MTB) cell wall, is detectable in the urine of MTB infected patients with active tuberculosis (TB). LAM-specific antibodies (Igs) have been developed by a variety of traditional and recombinant methods for potential use in a rapid diagnostic test (RDT). We evaluated the analytical performance of the TB LAM Igs to identify pairs that offer superior performance over existing urine LAM tests. We assessed 25 new and 4 existing Igs in a matrixed format using a multiplex electrochemiluminescence-based liquid immunoassay. A total of 841 paired Ig combinations were challenged with in vitro cultured LAM (cLAM) derived from MTB strains representing diverse phylogenetic lineages, alongside urinary LAM (uLAM) from the urine of adults with active pulmonary TB. Analytical sensitivity of down-selected Ig pairs was determined using MTB Aoyama-B cLAM, while diagnostic accuracy was determined using clinical samples. When testing cLAM, the reactivity of Ig pairs was similar across MTB lineages 1-4 but lineage 5:6 had significantly more reactivity among Ig pairs. Overall, 41 Ig pairs had a strong binding affinity to cLAM, as compared to the reference pair of S4-20/A194-01, and 28 Ig pairs therein exhibited a strong affinity for both cLAM and uLAM. Retrospective testing on clinical urine specimens demonstrated varying sensitivities (12-80%) and specificities (14-100%). The five top pairs had a similar analytical limit of detection to the reference pair but in four instances, the sensitivity and specificity with clinical uLAM samples was poor. Overall, epitopes presented by uLAM are different from cLAM, which may affect antibody performance when testing uLAM in patient samples. Several new Ig pairs had similar ranges of high sensitivity to cLAM but overall, there were no new candidate Ig pairs identified in this round of screening with increased performance with uLAM as compared to an existing optimal pair.

HIV pre-exposure prophylaxis adherence test using reverse transcription isothermal amplification inhibition assay.

Current HIV antiretroviral therapy (ART) or pre-exposure prophylaxis (PrEP) therapy adherence monitoring relies on either patient self-reported adherence or monitored drug dispensing, which are not reliable. We report a proof-of-concept adherence monitoring assay which directly measures nucleotide reverse transcriptase inhibitor (NRTI) concentration using a reverse transcription isothermal amplification inhibition assay. We measure the concentration of Tenofovir diphosphate (TFV-DP) - an NRTI that functions as a deoxyadenosine triphosphate (dATP) analog and long-term adherence marker for PrEP - by measuring the inhibition of the reverse transcription of an RNA template. The completion or inhibition of reverse transcription is evaluated by recombinase polymerase amplification (RPA), an isothermal nucleic acid amplification assay commonly used for point-of-care diagnostics. We present and validate a model that predicts the amplification probability as a function of dATP and TFV-DP concentrations, nucleotide insertion sites on the RNA template, and RNA template concentration. The model can be used to rationally design and optimize the assay to operate at clinically relevant TFV-DP concentrations. We provide statistical analysis that demonstrates how the assay may be used as a qualitative or semi-quantitative tool for measuring adherence to NRTI drugs and used to support patient compliance. Due to its simple instrumentation and short runtime (<1 hour), this assay has the potential for implementation in low-complexity laboratories or point-of-care settings, which may improve access to ART and PrEP adherence monitoring.

A multicenter analytical performance evaluation of a multiplexed immunoarray for the simultaneous measurement of biomarkers of micronutrient deficiency, inflammation and malarial antigenemia.

A lack of comparative data across laboratories is often a barrier to the uptake and adoption of new technologies. Furthermore, data generated by different immunoassay methods may be incomparable due to a lack of harmonization. In this multicenter study, we describe validation experiments conducted in a single lab and cross-lab comparisons of assay results to assess the performance characteristics of the Q-plex™ 7-plex Human Micronutrient Array (7-plex), an immunoassay that simultaneously quantifies seven biomarkers associated with micronutrient (MN) deficiencies, inflammation and malarial antigenemia using plasma or serum; alpha-1-acid glycoprotein, C-reactive protein, ferritin, histidine-rich protein 2, retinol binding protein 4, soluble transferrin receptor, and thyroglobulin. Validations included repeated testing (n = 20 separately prepared experiments on 10 assay plates) in a single lab to assess precision and linearity. Seven independent laboratories tested 76 identical heparin plasma samples collected from a cohort of pregnant women in Niger using the same 7-plex assay to assess differences in results across laboratories. In the analytical validation experiments, intra- and inter-assay coefficients of variation were acceptable at <6% and <15% respectively and assay linearity was 96% to 99% with the exception of ferritin, which had marginal performance in some tests. Cross-laboratory comparisons showed generally good agreement between laboratories in all analyte results for the panel of 76 plasma specimens, with Lin's concordance correlation coefficient values averaging ≥0.8 for all analytes. Excluding plates that would fail routine quality control (QC) standards, the inter-assay variation was acceptable for all analytes except sTfR, which had an average inter-assay coefficient of variation of ≥20%. This initial cross-laboratory study demonstrates that the 7-plex test protocol can be implemented by users with some experience in immunoassay methods, but familiarity with the multiplexed protocol was not essential.

Screening Antibodies Raised against the Spike Glycoprotein of SARS-CoV-2 to Support the Development of Rapid Antigen Assays.

Severe acute respiratory coronavirus-2 (SARS-CoV-2) is a novel viral pathogen and therefore a challenge to accurately diagnose infection. Asymptomatic cases are common and so it is difficult to accurately identify infected cases to support surveillance and case detection. Diagnostic test developers are working to meet the global demand for accurate and rapid diagnostic tests to support disease management. However, the focus of many of these has been on molecular diagnostic tests, and more recently serologic tests, for use in primarily high-income countries. Low- and middle-income countries typically have very limited access to molecular diagnostic testing due to fewer resources. Serologic testing is an inappropriate surrogate as the early stages of infection are not detected and misdiagnosis will promote continued transmission. Detection of infection via direct antigen testing may allow for earlier diagnosis provided such a method is sensitive. Leading SARS-CoV-2 biomarkers include spike protein, nucleocapsid protein, envelope protein, and membrane protein. This research focuses on antibodies to SARS-CoV-2 spike protein due to the number of monoclonal antibodies that have been developed for therapeutic research but also have potential diagnostic value. In this study, we assessed the performance of antibodies to the spike glycoprotein, acquired from both commercial and private groups in multiplexed liquid immunoassays, with concurrent testing via a half-strip lateral flow assays (LFA) to indicate antibodies with potential in LFA development. These processes allow for the selection of pairs of high-affinity antispike antibodies that are suitable for liquid immunoassays and LFA, some of which with sensitivity into the low picogram range with the liquid immunoassay formats with no cross-reactivity to other coronavirus S antigens. Discrepancies in optimal ranking were observed with the top pairs used in the liquid and LFA formats. These findings can support the development of SARS-CoV-2 LFAs and diagnostic tools.

HIV detection from human serum with paper-based isotachophoretic RNA extraction and reverse transcription recombinase polymerase amplification.

The number of people living with HIV continues to increase with the current total near 38 million, of which about 26 million are receiving antiretroviral therapy (ART). These treatment regimens are highly effective when properly managed, requiring routine viral load monitoring to assess successful viral suppression. Efforts to expand access by decentralizing HIV nucleic acid testing in low- and middle-income countries (LMICs) has been hampered by the cost and complexity of current tests. Sample preparation of blood samples has traditionally relied on cumbersome RNA extraction methods, and it continues to be a key bottleneck for developing low-cost POC nucleic acid tests. We present a microfluidic paper-based analytical device (µPAD) for extracting RNA and detecting HIV in serum, leveraging low-cost materials, simple buffers, and an electric field. We detect HIV virions and MS2 bacteriophage internal control in human serum using a novel lysis and RNase inactivation method, paper-based isotachophoresis (ITP) for RNA extraction, and duplexed reverse transcription recombinase polymerase amplification (RT-RPA) for nucleic acid amplification. We design a specialized ITP system to extract and concentrate RNA, while excluding harsh reagents used for lysis and RNase inactivation. We found the ITP µPAD can extract and purify 5000 HIV RNA copies per mL of serum. We then demonstrate detection of HIV virions and MS2 bacteriophage in human serum within 45-minutes.

Review of Methods Suitable for Environmental Surveillance of Salmonella Typhi and Paratyphi.

Typhoid fever is an enteric disease caused by the pathogens Salmonella Typhi and Salmonella Paratyphi. Clinical surveillance networks are lacking in many affected areas, thus presenting a need to understand transmission and population prevalence. Environmental surveillance (ES) has been suggested as a potentially effective method in the absence of (or in supplement to) clinical surveillance. This review summarizes methods identified in the literature for sampling and detection of typhoidal Salmonella from environmental samples including drinking water, wastewater, irrigation water, and surface waters. Methods described use a trap or grab sampling approach combined with various selective culture and molecular methods. The level to which the performance of identified methods is characterized for ES in the literature is variable, thus arguing for the optimization and standardization of ES techniques.

Enzymatic and Chemical-Based Methods to Inactivate Endogenous Blood Ribonucleases for Nucleic Acid Diagnostics.

There are ongoing research efforts into simple and low-cost point-of-care nucleic acid amplification tests (NATs) addressing widespread diagnostic needs in resource-limited clinical settings. Nucleic acid testing for RNA targets in blood specimens typically requires sample preparation that inactivates robust blood ribonucleases (RNases) that can rapidly degrade exogenous RNA. Most NATs rely on decades-old methods that lyse pathogens and inactivate RNases with high concentrations of guanidinium salts. Herein, we investigate alternatives to standard guanidinium-based methods for RNase inactivation using an activity assay with an RNA substrate that fluoresces when cleaved. The effects of proteinase K, nonionic surfactants, SDS, dithiothreitol, and other additives on RNase activity in human serum are reported. Although proteinase K has been widely used in protocols for nuclease inactivation, it was found that high concentrations of proteinase K are unable to eliminate RNase activity in serum, unless used in concert with denaturing concentrations of SDS. It was observed that SDS must be combined with proteinase K, dithiothreitol, or both for irreversible and complete RNase inactivation in serum. This work provides an alternative chemistry for inactivating endogenous RNases for use in simple, low-cost point-of-care NATs for blood-borne pathogens.

Point-of-Care HIV Viral Load Testing: an Essential Tool for a Sustainable Global HIV/AIDS Response.

The global public health community has set ambitious treatment targets to end the HIV/AIDS pandemic. With the notable absence of a cure, the goal of HIV treatment is to achieve sustained suppression of an HIV viral load, which allows for immunological recovery and reduces the risk of onward HIV transmission. Monitoring HIV viral load in people living with HIV is therefore central to maintaining effective individual antiretroviral therapy as well as monitoring progress toward achieving population targets for viral suppression. The capacity for laboratory-based HIV viral load testing has increased rapidly in low- and middle-income countries, but implementation of universal viral load monitoring is still hindered by several barriers and delays. New devices for point-of-care HIV viral load testing may be used near patients to improve HIV management by reducing the turnaround time for clinical test results. The implementation of near-patient testing using these new and emerging technologies may be an essential tool for ensuring a sustainable response that will ultimately enable an end to the HIV/AIDS pandemic. In this report, we review the current and emerging technology, the evidence for decentralized viral load monitoring by non-laboratory health care workers, and the additional considerations for expanding point-of-care HIV viral load testing.

Performance and workflow assessment of six nucleic acid extraction technologies for use in resource limited settings.

Infectious disease nucleic acid amplification technologies (NAAT) have superior sensitivity, specificity, and rapid time to result compared to traditional microbiological methods. Recovery of concentrated, high quality pathogen nucleic acid (NA) from complex specimen matrices is required for optimal performance of several NA amplification/detection technologies such as polymerase chain reaction (PCR). Fully integrated NAAT platforms that enable rapid sample-to-result workflows with minimal user input are generally restricted to larger reference lab settings, and their complexity and cost are prohibitive to widespread implementation in resource limited settings (RLS). Identification of component technologies for incorporation of reliable and affordable sample preparation with pathogen NA amplification/detection into an integrated platform suitable for RLS, is a necessary first step toward achieving the overarching goal of reducing infectious disease-associated morbidity and mortality globally. In the current study, we evaluate the performance of six novel NA extraction technologies from different developers using blinded panels of stool, sputum and blood spiked with variable amounts of quality-controlled DNA- and/or RNA-based microbes. The extraction efficiencies were semi-quantitatively assessed using validated real-time reverse transcription (RT)-PCR assays specific for each microbe and comparing target-specific RT-PCR results to those obtained with reference NA extraction methods. The technologies were ranked based on overall diagnostic accuracy (analytical sensitivity and specificity). Sample input and output volumes, total processing time, user-required manual steps and cost estimates were also examined for suitability in RLS. Together with the performance analysis, these metrics were used to select the more suitable candidate technologies for further optimization of integrated NA amplification and detection technologies for RLS.

Assessment of eight nucleic acid amplification technologies for potential use to detect infectious agents in low-resource settings.

Nucleic acid amplification technologies (NAATs) are high-performance tools for rapidly and accurately detecting infectious agents. They are widely used in high-income countries to diagnose disease and improve patient care. The complexities associated with test methods, reagents, equipment, quality control and assurance require dedicated laboratories with trained staff, which can exclude their use in low-resource and decentralized healthcare settings. For certain diseases, fully integrated NAAT devices and assays are available for use in environmentally-controlled clinics or emergency rooms where relatively untrained staff can perform testing. However, decentralized settings in many low- and middle-income countries with large burdens of infectious disease are challenged by extreme environments, poor infrastructure, few trained staff and limited financial resources. Therefore, there is an urgent need for low-cost, integrated NAAT tools specifically designed for use in low-resource settings (LRS). Two essential components of integrated NAAT tools are: 1) efficient nucleic acid extraction technologies for diverse and complex sample types; and 2) robust and sensitive nucleic acid amplification and detection technologies. In prior work we reported the performance and workflow capacity for the nucleic acid extraction component. In the current study we evaluated performance of eight novel nucleic acid amplification and detection technologies from seven developers using blinded panels of RNA and/or DNA from three pathogens to assess both diagnostic accuracy and suitability as an essential component for low-cost NAAT in LRS. In this exercise, we noted significant differences in performance among these technologies and identified those most promising for potential further development.

Measurement of micronutrient deficiency associated biomarkers in dried blood spots using a multiplexed immunoarray.

Simplifying blood collection is often critical when collecting specimens in remote and/or austere settings. The use of dried blood spots (DBS) offers a practical collection method suitable for a wide variety of analytes. A small volume of whole blood can be obtained rapidly through a minimally invasive heel- or finger-stick using a disposable safety lancet. Once dried, the samples require no further processing, are stable for months or longer, pose minimal risk of disease transmission, and are easy to ship. DBS are often used in demographic health surveys to assess infectious disease status in vulnerable populations. These samples can be used to screen biomarkers of micronutrient deficiency (MND) and inflammation. We recently described a multiplexed immunoarray, the Q-plex human micronutrient array, which can simultaneously quantify seven biomarkers related to MND, inflammation and malarial antigenemia using plasma (alpha-1-acid glycoprotein, C-reactive protein, ferritin, histidine-rich protein 2, retinol binding protein, soluble transferrin receptor, and thyroglobulin). In this work, we present a protocol for preparing eluates from DBS samples and their measurement using a modified protocol for this new tool. We evaluated the concordance of analyte concentrations (excluding ferritin) from a panel ninety samples of DBS prepared from anticoagulated venous blood and paired K2-EDTA plasma. The results show high correlation between DBS eluates and wet plasma for five of the six analytes screened, suggesting the Q-plex human micronutrient array can be used with DBS samples, but also highlighting that anticoagulants can have a negative effects on some test components.

Semiquantitative Nucleic Acid Test with Simultaneous Isotachophoretic Extraction and Amplification.

Nucleic acid amplification tests (NAATs) provide high diagnostic accuracy for infectious diseases and quantitative results for monitoring viral infections. The majority of NAATs require complex equipment, cold chain dependent reagents, and skilled technicians to perform the tests. This largely confines NAATs to centralized laboratories and can significantly delay appropriate patient care. Low-cost, point-of-care (POC) NAATs are especially needed in low-resource settings to provide patients with diagnosis and treatment planning in a single visit to improve patient care. In this work, we present a rapid POC NAAT with integrated sample preparation and amplification using electrokinetics and paper substrates. We use simultaneous isotachophoresis (ITP) and recombinase polymerase amplification (RPA) to rapidly extract, amplify, and detect target nucleic acids from serum and whole blood in a paper-based format. We demonstrate simultaneous ITP and RPA can consistently detect 5 copies per reaction in buffer and 10 000 copies per milliliter of human serum with no intermediate user steps. We also show preliminary extraction and amplification of DNA from whole blood samples. Our test is rapid (results in less than 20 min) and made from low-cost materials, indicating its potential for detecting infectious diseases and monitoring viral infections at the POC in low resource settings.

Simultaneous assessment of iodine, iron, vitamin A, malarial antigenemia, and inflammation status biomarkers via a multiplex immunoassay method on a population of pregnant women from Niger.

Deficiencies of vitamin A, iron, and iodine are major public health concerns in many low- and middle-income countries, but information on their status in populations is often lacking due to high costs and logistical challenges associated with assessing micronutrient status. Accurate, user-friendly, and low-cost analytical tools are needed to allow large-scale population surveys on micronutrient status. We present the expansion of a 7-plex protein microarray tool for the simultaneous measurement of up to seven biomarkers with relevance to the assessment of the key micronutrients iron, iodine, and vitamin A, and inflammation and malaria biomarkers: α-1-acid glycoprotein, C-reactive protein, ferritin, retinol binding protein 4, soluble transferrin receptor, thyroglobulin, and histidine-rich protein II. Assay performance was assessed using international reference standards and then verified by comparing the multiplexed and conventional immunoassay results on a training panel of plasma samples collected from US adults. These data were used to assign nominal concentrations to the calibrators of the assay to further improve performance which was then assessed by interrogating plasma samples from a cohort of pregnant women from Niger. The correlation between assays for each biomarker measured from this cohort was typically good, with the exception of thyroglobulin, and the sensitivity ranged from 74% to 93%, and specificity from 81% to 98%. The 7-Plex micronutrient assay has the potential for use as an affordable tool for population surveillance of vitamin A, iron, and iodine deficiencies as well as falciparum malarial parasitemia infectivity and inflammation. The assay is easy-to-use, requires minimal sample volume, and is scalable, rapid, and accurate-needing only a low-cost reader and basic equipment present in most reference laboratory settings and so may be employed by low and middle income countries for micronutrient surveillance to inform on status in key populations. Micronutrient deficiencies including iron, iodine, and vitamin A affect a significant portion of the world's population. Efforts to assess the prevalence of these deficiencies in vulnerable populations are challenging, partly due to measurement tools that are inadequate for assessing multiple micronutrients in large-scale population surveys. We have developed a 7-plex immunoassay for the simultaneous measurement of seven biomarkers relevant to assessing iodine, iron, and vitamin A status, inflammation and Plasmodium falciparum parasitemia by measuring levels of thyroglobulin, ferritin, soluble transferrin receptor, retinol binding protein 4, α-1-acid glycoprotein, C-reactive protein, and histidine-rich protein II. This 7-plex immunoassay technique has potential as a rapid and effective tool for use in large-scale surveys and assessments of nutrition intervention programs in low- and middle-income countries.

Factors influencing Recombinase polymerase amplification (RPA) assay outcomes at point of care.

Recombinase Polymerase Amplification (RPA) can be used to detect pathogen-specific DNA or RNA in under 20 min without the need for complex instrumentation. These properties enable its potential use in resource limited settings. However, there are concerns that deviations from the manufacturer's protocol and/or storage conditions could influence its performance in low resource settings. RPA amplification relies upon viscous crowding agents for optimal nucleic acid amplification, and thus an interval mixing step after 3-6 min of incubation is recommended to distribute amplicons and improve performance. In this study we used a HIV-1 RPA assay to evaluate the effects of this mixing step on assay performance. A lack of mixing led to a longer time to amplification and inferior detection signal, compromising the sensitivity of the assay. However lowering the assay volume from 50 µL to 5 µL showed similar sensitivity with or without mixing. We present the first peer-reviewed study that assesses long term stability of RPA reagents without a cold chain. Reagents stored at -20 °C, and 25 °C for up to 12 weeks were able to detect 10 HIV-1 DNA copies. Reagents stored at 45 °C for up to 3 weeks were able to detect 10 HIV-1 DNA copies, with reduced sensitivity only after >3 weeks at 45 °C. Together our results show that reducing reaction volumes bypassed the need for the mixing step and that RPA reagents were stable even when stored for 3 weeks at very high temperatures.