In situ Tissue Regeneration in the Cornea from Bench to Bedside.

Corneal blindness accounts for 5.1% of visual deficiency and is the fourth leading cause of blindness globally. An additional 1.5-2 million people develop corneal blindness each year, including many children born with or who later develop corneal infections. Over 90% of corneal blind people globally live in low- and middle-income regions (LMIRs), where corneal ulcers are approximately 10-fold higher compared to high-income countries. While corneal transplantation is an effective option for patients in high-income countries, there is a considerable global shortage of corneal graft tissue and limited corneal transplant programs in many LMIRs. In situ tissue regeneration aims to restore diseases or damaged tissues by inducing organ regeneration. This can be achieved in the cornea using biomaterials based on extracellular matrix (ECM) components like collagen, hyaluronic acid, and silk. Solid corneal implants based on recombinant human collagen type III were successfully implanted into patients resulting in regeneration of the corneal epithelium, stroma, and sub-basal nerve plexus. As ECM crosslinking and manufacturing methods improve, the focus of biomaterial development has shifted to injectable, in situ gelling formulations. Collagen, collagen-mimetic, and gelatin-based in situ gelling formulas have shown the ability to repair corneal wounds, surgical incisions, and perforations in in-vivo models. Biomaterial approaches may not be sufficient to treat inflammatory conditions, so other cell-free therapies such as treatment with tolerogenic exosomes and extracellular vesicles may improve treatment outcomes. Overall, many of the technologies described here show promise as future medical devices or combination products with cell or drug-based therapies. In situ tissue regeneration, particularly with liquid formulas, offers the ability to triage and treat corneal injuries and disease with a single regenerative solution, providing alternatives to organ transplantation and improving patient outcomes.

Usability and acceptability of a multimodal respiratory rate and pulse oximeter device in case management of children with symptoms of pneumonia: A cross-sectional study in Ethiopia.

AIM: Pneumonia is the leading infectious cause of death among children under five globally. Many pneumonia deaths result from inappropriate treatment due to misdiagnosis of signs and symptoms. This study aims to identify whether health extension workers (HEWs) in Ethiopia, using an automated multimodal device (Masimo Rad-G), adhere to required guidelines while assessing and classifying under five children with cough or difficulty breathing and to understand device acceptability. METHODS: A cross-sectional study was conducted in three districts of Southern Nations, Nationalities, and Peoples' Region, Ethiopia. Between September and December 2018, 133 HEWs were directly observed using Rad-G while conducting 599 sick child consultations. Usability was measured as adherence to the World Health Organization requirements to assess fast breathing and device manufacturer instructions for use. Acceptability was assessed using semi-structured interviews with HEWs, first-level health facility workers and caregivers. RESULTS: Adherence using the Rad-G routinely for 2 months was 85.3% (95% CI 80.2, 89.3). Health workers and caregivers stated a preference for Rad-G. Users highlighted a number of device design issues. CONCLUSION: While demonstrating high levels of acceptability and usability, the device modifications to consider include better probe fit, improved user interface with exclusive age categories and simplified classification outcomes.

Automated Respiratory Rate Counter to Assess Children for Symptoms of Pneumonia: Protocol for Cross-Sectional Usability and Acceptability Studies in Ethiopia and Nepal.

BACKGROUND: Manually counting a child's respiratory rate (RR) for 60 seconds using an acute respiratory infection timer is the World Health Organization (WHO) recommended method for detecting fast breathing as a sign of pneumonia. However, counting the RR is challenging and misclassification of an observed rate is common, often leading to inappropriate treatment. To address this gap, the acute respiratory infection diagnostic aid (ARIDA) project was initiated in response to a call for better pneumonia diagnostic aids and aimed to identify and assess automated RR counters for classifying fast breathing pneumonia when used by front-line health workers in resource-limited community settings and health facilities. The Children's Automated Respiration Monitor (ChARM), an automated RR diagnostic aid using accelerometer technology developed by Koninklijke Philips NV, and the Rad-G, a multimodal RR diagnostic and pulse oximeter developed by Masimo, were the two devices tested in these studies conducted in the Southern Nations, Nationalities, and Peoples' Region in Ethiopia and in the Karnali region in Nepal. OBJECTIVE: In these studies, we aimed to understand the usability of two new automated RR diagnostic aids for community health workers (CHWs; health extension workers [Ethiopia] and female community health volunteers [Nepal]) and their acceptability to CHWs in Ethiopia and Nepal, first-level health facility workers (FLHFWs) in Ethiopia only, and caregivers in both Ethiopia and Nepal. METHODS: This was a prospective, cross-sectional study with a mixed methods design. CHWs and FLHFWs were trained to use both devices and provided with refresher training on all WHO requirements to assess fast breathing. Immediately after training, CHWs were observed using ARIDA on two children. Routine pneumonia case management consultations for children aged 5 years and younger and the device used for these consultations between the first and second consultations were recorded by CHWs in their patient log books. CHWs were observed a second time after 2 months. Semistructured interviews were also conducted with CHWs, FLHFWs, and caregivers. The proportion of consultations with children aged 5 years and younger where CHWs using an ARIDA and adhered to all WHO requirements to assess fast breathing and device manufacturer instructions for use after 2 months will be calculated. Qualitative data from semistructured interviews will be analyzed using a thematic framework approach. RESULTS: The ARIDA project was funded in November 2015, and data collection was conducted between April and December 2018. Data analysis is currently under way and the first results are expected to be submitted for publication in 2020. CONCLUSIONS: This is the first time the usability and acceptability of automated RR counters in low-resource settings have been evaluated. Outcomes will be relevant for policy makers and are important for future research of this new class of diagnostic aids for the management of children with suspected pneumonia. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/14405.

Determining the Agreement Between an Automated Respiratory Rate Counter and a Reference Standard for Detecting Symptoms of Pneumonia in Children: Protocol for a Cross-Sectional Study in Ethiopia.

BACKGROUND: Acute respiratory infections (ARIs), primarily pneumonia, are the leading infectious cause of under-5 mortality worldwide. Manually counting respiratory rate (RR) for 60 seconds using an ARI timer is commonly practiced by community health workers to detect fast breathing, an important sign of pneumonia. However, correctly counting breaths manually and classifying the RR is challenging, often leading to inappropriate treatment. A potential solution is to introduce RR counters, which count and classify RR automatically. OBJECTIVE: This study aims to determine how the RR count of an Automated Respiratory Infection Diagnostic Aid (ARIDA) agrees with the count of an expert panel of pediatricians counting RR by reviewing a video of the child's chest for 60 seconds (reference standard), for children aged younger than 5 years with cough and/or difficult breathing. METHODS: A cross-sectional study aiming to enroll 290 children aged 0 to 59 months presenting to pediatric in- and outpatient departments at a teaching hospital in Addis Ababa, Ethiopia, was conducted. Enrollment occurred between April and May 2017. Once enrolled, children participated in at least one of three types of RR evaluations: (1) agreement-measure the RR count of an ARIDA in comparison with the reference standard, (2) consistency-measure the agreement between two ARIDA devices strapped to one child, and (3) RR fluctuation-measure RR count variability over time after ARIDA attachment as measured by a manual count. The agreement and consistency of expert clinicians (ECs) counting RR for the same child with the Mark 2 ARI timer for 60 seconds was also measured in comparison with the reference standard. RESULTS: Primary outcomes were (1) mean difference between the ARIDA and reference standard RR count (agreement) and (2) mean difference between RR counts obtained by two ARIDA devices started simultaneously (consistency). CONCLUSIONS: Study strengths included the design allowing for comparison between both ARIDA and the EC with the reference standard RR count. A limitation is that exactly the same set of breaths were not compared between ARIDA and the reference standard since ARIDA can take longer than 60 seconds to count RR. Also, manual RR counting, even when aided by a video of the child's chest movements, is subject to human error and can result in low interrater reliability. Further work is needed to reach global consensus on the most appropriate reference standard and an acceptable level of agreement to provide ministries of health with evidence to make an informed decision on whether to scale up new automated RR counters. TRIAL REGISTRATION: ClinicalTrials.gov NCT03067558; https://clinicaltrials.gov/ct2/show/NCT03067558. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): RR1-10.2196/16531.

Usability and acceptability of an automated respiratory rate counter to assess children for symptoms of pneumonia: A cross-sectional study in Ethiopia.

AIM: Manually counting respiratory rate (RR) is commonly practiced by community health workers to detect fast breathing, an important sign of childhood pneumonia. Correctly counting and classifying breaths manually is challenging, often leading to inappropriate treatment. This study aimed to determine the usability of a new automated RR counter (ChARM) by health extension workers (HEWs), and its acceptability to HEWs, first-level health facility workers (FLHFWs) and caregivers in Ethiopia. METHODS: A cross-sectional study was conducted in one region of Ethiopia between May and August 2018. A total of 131 HEWs were directly observed conducting 262 sick child consultations after training and 337 after 2 months. Usability was measured as adherence to the WHO requirements to assess fast breathing and device manufacturer instructions for use (IFU). Acceptability was measured through semi-structured interviews. RESULTS: After 2 months, HEWs were shown to adhere to the requirements in 74.6% consultations; an increase of 18.6% after training (P < .001). ChARM is acceptable to users and caregivers, with HEWs suggesting that ChARM increased client flow and stating a willingness to use ChARM in future. CONCLUSION: Further research on the performance, cost-effectiveness and implementation of this device is warranted to inform policy decisions in countries with a high childhood pneumonia burden.

Usability and acceptability of an automated respiratory rate counter to assess childhood pneumonia in Nepal.

AIM: Pneumonia is the leading cause of child death after the neonatal period, resulting from late care seeking and inappropriate treatment. Diagnosis involves counting respiratory rate (RR); however, RR counting remains challenging for health workers and miscounting, and misclassification of RR is common. We evaluated the usability of a new automated RR counter, the Philips Children's Respiratory Monitor (ChARM), to Female Community Health Volunteers (FCHVs), and its acceptability to FCHVs and caregivers in Nepal. METHODS: A cross-sectional study was conducted in Jumla district, Nepal. About 133 FCHVs were observed between September and December 2018 when using ChARM during 517 sick child consultations, 264 after training and 253 after 2 months of routine use of ChARM. Acceptability of the ChARM was explored using semi-structured interviews. RESULTS: FCHV adherence to guidelines after 2 months of using ChARM routinely was 52.8% (95% CI 46.6-58.9). The qualitative findings suggest that ChARM is acceptable to FCHVs and caregivers; however, capacity constraints such as older age and low literacy and impacted device usability were mentioned. CONCLUSION: Further research on the performance, cost-effectiveness and implementation feasibility of this device is recommended, especially among low-literate CHWs.

Performance of a High-Sensitivity Rapid Diagnostic Test for Plasmodium falciparum Malaria in Asymptomatic Individuals from Uganda and Myanmar and Naive Human Challenge Infections.

Sensitive field-deployable diagnostic tests can assist malaria programs in achieving elimination. The performance of a new Alere™ Malaria Ag P.f Ultra Sensitive rapid diagnostic test (uRDT) was compared with the currently available SD Bioline Malaria Ag P.f RDT in blood specimens from asymptomatic individuals in Nagongera, Uganda, and in a Karen Village, Myanmar, representative of high- and low-transmission areas, respectively, as well as in pretreatment specimens from study participants from four Plasmodium falciparum-induced blood-stage malaria (IBSM) studies. A quantitative reverse transcription PCR (qRT-PCR) and a highly sensitive enzyme-linked immunosorbent assay (ELISA) test for histidine-rich protein II (HRP2) were used as reference assays. The uRDT showed a greater than 10-fold lower limit of detection for HRP2 compared with the RDT. The sensitivity of the uRDT was 84% and 44% against qRT-PCR in Uganda and Myanmar, respectively, and that of the RDT was 62% and 0% for the same two sites. The specificities of the uRDT were 92% and 99.8% against qRT-PCR for Uganda and Myanmar, respectively, and 99% and 99.8% against the HRP2 reference ELISA. The RDT had specificities of 95% and 100% against qRT-PCR for Uganda and Myanmar, respectively, and 96% and 100% against the HRP2 reference ELISA. The uRDT detected new infections in IBSM study participants 1.5 days sooner than the RDT. The uRDT has the same workflow as currently available RDTs, but improved performance characteristics to identify asymptomatic malaria infections. The uRDT may be a useful tool for malaria elimination strategies.

Colorimetric tests for diagnosis of filarial infection and vector surveillance using non-instrumented nucleic acid loop-mediated isothermal amplification (NINA-LAMP).

Accurate detection of filarial parasites in humans is essential for the implementation and evaluation of mass drug administration programs to control onchocerciasis and lymphatic filariasis. Determining the infection levels in vector populations is also important for assessing transmission, deciding when drug treatments may be terminated and for monitoring recrudescence. Immunological methods to detect infection in humans are available, however, cross-reactivity issues have been reported. Nucleic acid-based molecular assays offer high levels of specificity and sensitivity, and can be used to detect infection in both humans and vectors. In this study we developed loop-mediated isothermal amplification (LAMP) tests to detect three different filarial DNAs in human and insect samples using pH sensitive dyes for enhanced visual detection of amplification. Furthermore, reactions were performed in a portable, non-instrumented nucleic acid amplification (NINA) device that provides a stable heat source for LAMP. The efficacy of several strand displacing DNA polymerases were evaluated in combination with neutral red or phenol red dyes. Colorimetric NINA-LAMP assays targeting Brugia Hha I repeat, Onchocerca volvulus GST1a and Wuchereria bancrofti LDR each exhibit species-specificity and are also highly sensitive, detecting DNA equivalent to 1/10-1/5000th of one microfilaria. Reaction times varied depending on whether a single copy gene (70 minutes, O. volvulus) or repetitive DNA (40 min, B. malayi and W. bancrofti) was employed as a biomarker. The NINA heater can be used to detect multiple infections simultaneously. The accuracy, simplicity and versatility of the technology suggests that colorimetric NINA-LAMP assays are ideally suited for monitoring the success of filariasis control programs.

A Field-Tailored Reverse Transcription Loop-Mediated Isothermal Assay for High Sensitivity Detection of Plasmodium falciparum Infections.

Highly sensitive and field deployable molecular diagnostic tools are critically needed for detecting submicroscopic, yet transmissible levels of malaria parasites prevalent in malaria endemic countries worldwide. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed and evaluated in comparison with thick blood smear microscopy, an antigen-based rapid diagnostic test (RDT), and an in-house RT-PCR targeting the same RT-LAMP transcript. The optimized assay detected Plasmodium falciparum infections in as little as 0.25ng of total parasite RNA, and exhibited a detection limit of 0.08 parasites/ µL when tested directly on infected whole blood lysates, or ~0.0008 parasites/ µL when using RNA extracts. Assay positivity was observed as early as eight minutes from initiation of the RT-LAMP and in most cases the reaction was complete before twenty minutes. Clinical evaluation of the assay on 132 suspected malaria cases resulted in a positivity rate of 90% for RT-LAMP using extracted RNA, and 85% when using whole blood lysates. The positivity rates were 70% for P. falciparum-specific RDT, 83% for RT-PCR, and 74% for thick blood smear microscopy (Mean parasite density = 36,986 parasites/ µL). Concordance rates between the developed RT-LAMP and comparator tests were greater than 75%, the lowest being with light microscopy (78%, McNemar's test: P = 0.0002), and the highest was with RT-PCR (87%, McNemar's test: P = 0.0523). Compared to reference RT-PCR, assay sensitivity was 90% for RT-LAMP on whole blood, and 96% for RT-LAMP using corresponding RNA extracts. Electricity-free heaters were further developed and evaluated in comparison with a battery-operated isothermal amplification machine for use with the developed test in resource-limited settings. Taken together, the data highlight the benefits of targeting high abundant RNA transcripts in molecular diagnosis, as well as the potential usefulness of the developed RT-LAMP-assay in malaria diagnosis in low to high parasite density settings.

Single-use, electricity-free amplification device for detection of HIV-1.

Early and accurate diagnosis of HIV is key for the reduction of transmission and initiation of patient care. The availability of a rapid nucleic acid test (NAT) for use at the point-of-care (POC) will fill a gap in HIV diagnostics, improving the diagnosis of acute infection and HIV in infants born to infected mothers. In this study, we evaluated the performance of non-instrumented nucleic acid amplification, single-use disposable (NINA-SUD) devices for the detection of HIV-1 in whole blood using reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) with lyophilized reagents. The NINA-SUD heating device harnesses the heat from an exothermic chemical reaction initiated by the addition of saline to magnesium iron powder. Reproducibility was demonstrated between NINA-SUD units and comparable, if not superior, performance for detecting clinical specimens was observed as compared to the thermal cycler. The stability of the lyophilized HIV-1 RT-LAMP reagents was also demonstrated following storage at -20, 4, 25, and 30°C for up to one month. The single-use, disposable NAT minimizes hands-on time and has the potential to facilitate HIV-1 testing in resource-limited settings or at the POC.

NINA-LAMP compared to microscopy, RDT, and nested PCR for the detection of imported malaria.

Microscopy and field adaptable rapid diagnostic tests (RDTs) are not sensitive and specific in certain conditions such as poor training of microscopists, lack of electricity, or lower sensitivity in the detection of non-falciparum malaria. More sensitive point-of-care testing (POCT) would reduce delays in diagnosis and initiation of therapy. In the current study, we have evaluated the efficacy of noninstrumented nucleic acid amplification (NINA) coupled with loop-mediated isothermal amplification (LAMP) for detection of traveler's malaria (n=140) in comparison with microscopy, nested PCR, and the only Food and Drug Administration-approved rapid diagnostic test. NINA-LAMP was 100% sensitive and 98.6% specific when compared to nested PCR. For non-falciparum detection, NINA-LAMP sensitivity was 100% sensitive compared to nested PCR, whereas RDT sensitivity was 71%. LAMP is highly sensitive and specific for symptomatic malaria diagnosis regardless of species.

Design of a New Type of Compact Chemical Heater for Isothermal Nucleic Acid Amplification.

Previous chemical heater designs for isothermal nucleic acid amplification have been based on solid-liquid phase transition, but using this approach, developers have identified design challenges en route to developing a low-cost, disposable device. Here, we demonstrate the feasibility of a new heater configuration suitable for isothermal amplification in which one reactant of an exothermic reaction is a liquid-gas phase-change material, thereby eliminating the need for a separate phase-change compartment. This design offers potentially enhanced performance and energy density compared to other chemical and electric heaters.

Assessment of the availability of technology for trauma care in Nepal.

BACKGROUND: We sought to assess the availability of technology-related equipment for trauma care in Nepal and to identify factors leading to optimal availability as well as deficiencies. We also sought to identify potential solutions addressing the deficits in terms of health systems management and product development. METHODS: Thirty-two items for large hospitals and sixteen items for small hospitals related to the technological aspect of trauma care were selected from the World Health Organization's Guidelines for Essential Trauma Care for the current study. Fifty-six small and 29 large hospitals were assessed for availability of these items in the study area. Site visits included direct inspection and interviews with administrative, clinical, and bioengineering staff. RESULTS: Deficiencies of many specific items were noted, including many that were inexpensive and which could have been easily supplied. Shortage of electricity was identified as a major infrastructural deficiency present in all parts of the country. Deficiencies of pulse oximetry and ventilators were observed in most hospitals, attributed in most part to frequent breakdowns and long downtimes because of lack of vendor-based service contracts or in-house maintenance staff. Sub-optimal oxygen supply was identified as a major and frequent deficiency contributing to disruption of services. All equipment was imported except for a small percent of suction machines and haemoglobinometers. CONCLUSIONS: The study identified a range of items which were deficient and whose availability could be improved cost-effectively and sustainably by better planning and organisation. The electricity deficit has been dealt with successfully in a few hospitals via direct feeder lines and installation of solar panels; wider implementation of these methods would help solve a large portion of the technological deficiencies. From a health systems management view-point, strengthening procurement and stocking of low cost items especially in remote parts of the country is needed. From a product development view-point, there is a need for robust pulse-oximeters and ventilators that are lower cost and which have longer durability and less need for repairs. Increasing capabilities for local manufacture is another potential method to increase availability of a range of equipment and spare parts.

Evaluation of non-instrumented nucleic acid amplification by loop-mediated isothermal amplification (NINA-LAMP) for the diagnosis of malaria in Northwest Ethiopia.

BACKGROUND: Malaria is a major public health problem in sub-Saharan African countries including Ethiopia. Early and accurate diagnosis followed by prompt and effective treatment is among the various tools available for prevention, control and elimination of malaria. This study aimed to evaluate the performance of non-instrumented nucleic acid amplification loop-mediated isothermal amplification (NINA-LAMP) compared to standard thick and thin film microscopy and nested PCR as gold standard for the sensitive diagnosis of malaria in Northwest Ethiopia. METHODS: A cross-sectional study was conducted in North Gondar, Ethiopia from March to July 2014. Eighty-two blood samples were collected from malaria suspected patients visiting Kola Diba Health Centre and analysed for Plasmodium parasites by microscopy, NINA-LAMP and nested PCR. The NINA-LAMP method was performed using the Loopamp Malaria Pan/Pf detection kits for detecting DNA of the genus Plasmodium and more specifically Plasmodium falciparum using an electricity-free heater. Diagnostic accuracy outcome measures (analytical sensitivity, specificity, predictive values, and Kappa scores) of NINA-LAMP and microscopy were compared to nested PCR. RESULTS: A total of 82 samples were tested in the primary analysis. Using nested PCR as reference, the sensitivity and specificity of the primary NINA-LAMP assay were 96.8% (95% confidence interval (CI), 83.2% - 99.5%) and 84.3% (95% CI, 71.4% - 92.9%), respectively for detection of Plasmodium genus, and 100% (95% CI, 75.1% - 100%) and 81.2% (95% CI, 69.9% - 89.6%), respectively for detection of P. falciparum parasite. Microscopy demonstrated sensitivity and specificity of 93.6% (95% CI, 78.5% - 99.0%) and 98.0% (95% CI, 89.5% - 99.7%), respectively for the detection of Plasmodium parasites. Post-hoc repeat NINA-LAMP analysis showed improvement in diagnostic accuracy, which was comparable to nested PCR performance and superior to microscopy for detection at both the Plasmodium genus level and P. falciparum parasites. CONCLUSION: NINA-LAMP is highly sensitive for the diagnosis of malaria and detection of Plasmodium parasite infection at both the genus and species level when compared to nested PCR. NINA-LAMP is more sensitive than microscopy for the detection of P. falciparum and differentiation from non-falciparum species and may be a critical diagnostic modality in efforts to eradicate malaria from areas of low endemicity.

Assessment of the availability of technology for trauma care in India.

BACKGROUND: We sought to assess the status of availability of technology for trauma care in a state in India and to identify factors contributing to both adequate levels of availability and to deficiencies. We also sought to identify potential solutions to deficiencies in terms of health system management and product development. METHODS: Thirty-two technology-related items were selected from the World Health Organization's Guidelines for Essential Trauma Care. The status of these items was assessed at 43 small and large hospitals in Gujarat State. Site visits utilized direct inspection and interviews with administrative, clinical, and bioengineering staff. RESULTS: Many specific individual items could be better supplied, including many that were very low cost (e.g., chest tubes). Many deficiencies arose because of mismatch of resources, such as availability of equipment in the absence of personnel trained to use it. Several locally manufactured items were fairly well supplied: pulse oximetry, image intensification, and X-ray machines. Ventilators were often deficient because of inadequate numbers of units and frequent breakdowns. CONCLUSIONS: Availability of a range of lower-cost items could be improved by better organization and planning, such as: better procurement and stock management; eliminating mismatch of resources, including optimizing training for use of existing resources; and by strengthening service contracts and in-house repair capabilities. From a product development viewpoint, there is a need for lower cost, more durable, and easier to repair ventilators. Promoting increased capacity for local manufacturing should also be considered as a potential method to decrease cost and increase availability of a range of equipment.

Electricity-free amplification and detection for molecular point-of-care diagnosis of HIV-1.

In resource-limited settings, the lack of decentralized molecular diagnostic testing and sparse access to centralized medical facilities can present a critical barrier to timely diagnosis, treatment, and subsequent control and elimination of infectious diseases. Isothermal nucleic acid amplification methods, including reverse transcription loop-mediated isothermal amplification (RT-LAMP), are well-suited for decentralized point-of-care molecular testing in minimal infrastructure laboratories since they significantly reduce the complexity of equipment and power requirements. Despite reduced complexity, however, there is still a need for a constant heat source to enable isothermal nucleic acid amplification. This requirement poses significant challenges for laboratories in developing countries where electricity is often unreliable or unavailable. To address this need, we previously developed a low-cost, electricity-free heater using an exothermic reaction thermally coupled with a phase change material. This heater achieved acceptable performance, but exhibited considerable variability. Furthermore, as an enabling technology, the heater was an incomplete diagnostic solution. Here we describe a more precise, affordable, and robust heater design with thermal standard deviation <0.5°C at operating temperature, a cost of approximately US$.06 per test for heater reaction materials, and an ambient temperature operating range from 16°C to 30°C. We also pair the heater with nucleic acid lateral flow (NALF)-detection for a visual readout. To further illustrate the utility of the electricity-free heater and NALF-detection platform, we demonstrate sensitive and repeatable detection of HIV-1 with a ß-actin positive internal amplification control from processed sample to result in less than 80 minutes. Together, these elements are building blocks for an electricity-free platform capable of isothermal amplification and detection of a variety of pathogens.

Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA.

Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25-43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in LRS.

The essential role of infection-detection technologies for malaria elimination and eradication.

Recent emphasis on malaria elimination and eradication (E&E) goals is changing the way that experts evaluate malaria diagnostic tools and tactics. As prevalence declines, the focus of malaria management is pivoting toward low-density, subclinical infections and geographically and demographically concentrated reservoirs. These and other changes present challenges and opportunities for innovations in malaria diagnostics aimed at meeting the needs of malaria elimination programs. Developing such technologies requires a review of the operational approaches to detecting malaria infections in areas of declining prevalence. Here we review recent research on epidemiology and biology related to malaria elimination and operational factors that influence E&E strategies. We further propose use-scenarios and a target product profile framework to define and prioritize the required attributes of infection-detection technologies.

Prioritizing investments in innovations to protect women from the leading causes of maternal death.

PATH, an international nonprofit organization, assessed nearly 40 technologies for their potential to reduce maternal mortality from postpartum hemorrhage and preeclampsia and eclampsia in low-resource settings. The evaluation used a new Excel-based prioritization tool covering 22 criteria developed by PATH, the Maternal and Neonatal Directed Assessment of Technology (MANDATE) model, and consultations with experts. It identified five innovations with especially high potential: technologies to improve use of oxytocin, a uterine balloon tamponade, simplified dosing of magnesium sulfate, an improved proteinuria test, and better blood pressure measurement devices. Investments are needed to realize the potential of these technologies to reduce mortality.

Molecular diagnostics in a teacup: Non-Instrumented Nucleic Acid Amplification (NINA) for rapid, low cost detection of Salmonella enterica.

We report on the use of a novel non-instrumented platform to enable a Loop Mediated isothermal Amplification (LAMP) based assay for Salmonella enterica. Heat energy is provided by addition of a small amount (<150 g) of boiling water, and the reaction temperature is regulated by storing latent energy at the melting temperature of a lipid-based engineered phase change material. Endpoint classification of the reaction is achieved without opening the reaction tube by observing the fluorescence of sequence-specific FRET-based assimilating probes with a simple handheld fluorometer. At or above 22°C ambient temperature the non-instrumented devices could maintain reactions above a threshold temperature of 61°C for over 90 min-significantly longer than the 60 min reaction time. Using the simple format, detection limits were less than 20 genome copies for reactions run at ambient temperatures ranging from 8 to 36°C. When used with a pre-enrichment step and non-instrumented DNA extraction device, trace contaminations of Salmonella in milk close to 1 CFU/mL could be reliably detected. These findings illustrate that the non- instrumented amplification approach is a simple, viable, low-cost alternative for field-based food and agricultural diagnostics or clinical applications in developing countries.