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.

Comparison of depth of necrosis using cryotherapy by gas and number of freeze cycles.

OBJECTIVE: This study aimed to establish the noninferiority of a single-freeze application with CO2 or N2O compared with the standard double freeze with N2O for cryotherapy treatment. MATERIALS AND METHODS: Sixty women undergoing hysterectomy for reasons other than cervical cancer or precancer were randomized to 1 of 3 techniques as follows: (1) double freeze with N2O, (2) single freeze with N2O, or (3) single freeze with CO2. The cervix was separated and cut into anterior and posterior segments, and the deepest area of necrosis was recorded. Comparisons were made using regression analysis. The margin of noninferiority was defined as 0.8 mm. RESULTS: On the anterior lip, a single freeze with N2O was noninferior to a double freeze of the same gas, but on the posterior lip, the single freeze was not. The single freeze of CO2 did not provide sufficient depth of necrosis in either lip to infer noninferiority versus the double freeze with N2O. CONCLUSIONS: A single freeze with N2O is noninferior to a double-freeze technique in the anterior but not the posterior lip. However, the result for posterior lips was close to reaching statistical significance. In addition, CO2 had approximately 1 mm shallower depth of necrosis compared with N2O techniques; however, the clinical implications are unknown. Given the extensive use of CO2 globally, further clinical evaluation is needed.

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.

Instrument-free exothermic heating with phase change temperature control for paper microfluidic devices.

Many infectious diseases, as well as some cancers, that affect global health are most accurately diagnosed through nucleic acid amplification and detection. There is a great need to simplify nucleic acid-based assay systems for use in global health in low-resource settings as well as in settings that do not have convenient access to laboratory staff and equipment such as doctors' offices and home care settings. In developing countries, unreliable electric power, inadequate supply chains, and lack of maintenance for complex diagnostic instruments are all common infrastructure shortfalls. Many elements of instrument-free, disposable, nucleic acid amplification assays have been demonstrated in recent years. However, the problem of instrument-free, low-cost, temperature-controlled chemical heating remains unsolved. In this paper we present the current status and results of work towards developing disposable, low-cost, temperature-controlled heaters designed to support isothermal nucleic acid amplification assays that are integrated with a two-dimensional paper network. Our approach utilizes the heat generated through exothermic chemical reactions and controls the heat through use of engineered phase change materials to enable sustained temperatures required for nucleic acid amplification. By selecting appropriate exothermic and phase change materials, temperatures can be controlled over a wide range, suitable for various isothermal amplification methods, and maintained for over an hour at an accuracy of +/- 1°C.

Isothermal amplification using a chemical heating device for point-of-care detection of HIV-1.

BACKGROUND: To date, the use of traditional nucleic acid amplification tests (NAAT) for detection of HIV-1 DNA or RNA has been restricted to laboratory settings due to time, equipment, and technical expertise requirements. The availability of a rapid NAAT with applicability for resource-limited or point-of-care (POC) settings would fill a great need in HIV diagnostics, allowing for timely diagnosis or confirmation of infection status, as well as facilitating the diagnosis of acute infection, screening and evaluation of infants born to HIV-infected mothers. Isothermal amplification methods, such as reverse-transcription, loop-mediated isothermal amplification (RT-LAMP), exhibit characteristics that are ideal for POC settings, since they are typically quicker, easier to perform, and allow for integration into low-tech, portable heating devices. METHODOLOGY/SIGNIFICANT FINDINGS: In this study, we evaluated the HIV-1 RT-LAMP assay using portable, non-instrumented nucleic acid amplification (NINA) heating devices that generate heat from the exothermic reaction of calcium oxide and water. The NINA heating devices exhibited stable temperatures throughout the amplification reaction and consistent amplification results between three separate devices and a thermalcycler. The performance of the NINA heaters was validated using whole blood specimens from HIV-1 infected patients. CONCLUSION: The RT-LAMP isothermal amplification method used in conjunction with a chemical heating device provides a portable, rapid and robust NAAT platform that has the potential to facilitate HIV-1 testing in resource-limited settings and POC.

A simple, inexpensive device for nucleic acid amplification without electricity-toward instrument-free molecular diagnostics in low-resource settings.

BACKGROUND: Molecular assays targeted to nucleic acid (NA) markers are becoming increasingly important to medical diagnostics. However, these are typically confined to wealthy, developed countries; or, to the national reference laboratories of developing-world countries. There are many infectious diseases that are endemic in low-resource settings (LRS) where the lack of simple, instrument-free, NA diagnostic tests is a critical barrier to timely treatment. One of the primary barriers to the practicality and availability of NA assays in LRS has been the complexity and power requirements of polymerase chain reaction (PCR) instrumentation (another is sample preparation). METHODOLOGY/PRINCIPAL FINDINGS: In this article, we investigate the hypothesis that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays. We assess the heater's equivalence to commercially available PCR instruments through the characterization of the temperature profiles produced, and a minimal method comparison. Versions of the prototype for several different isothermal techniques are presented. CONCLUSIONS/SIGNIFICANCE: We demonstrate that an electricity-free heater based on exothermic chemical reactions and engineered phase change materials can successfully incubate isothermal NA amplification assays, and that the results of those assays are not significantly different from ones incubated in parallel in commercially available PCR instruments. These results clearly suggest the potential of the non-instrumented nucleic acid amplification (NINA) heater for molecular diagnostics in LRS. When combined with other innovations in development that eliminate power requirements for sample preparation, cold reagent storage, and readout, the NINA heater will comprise part of a kit that should enable electricity-free NA testing for many important analytes.

Non-Instrumented Nucleic Acid Amplification (NINA) for Rapid Detection of Ralstonia solanacearum Race 3 Biovar 2.

We report on the use of a non-instrumented device for the implementation of a loop-mediated amplification (LAMP) based assay for the select-agent bacterial-wilt pathogen Ralstonia solanacearum race 3 biovar 2. Heat energy is generated within the device by the exothermic hydration of calcium oxide, and the reaction temperature is regulated by storing latent energy at the melting temperature of a renewable lipid-based engineered phase-change material. Endpoint detection of the LAMP reaction is achieved without opening the reaction tube by observing the fluorescence of an innovative FRET-based hybridization probe with a simple custom fluorometer. Non-instrumented devices could maintain reactions near the design temperature of 63°C for at least an hour. Using this approach DNA extracted from the pathogen could be detected at fewer than ten copies within a 25 µL reaction mix, illustrating the potential of these technologies for simple, powerful agricultural diagnostics in the field. Furthermore, the assay was just as reliable when implemented in a tropical environment at 31°C as it was when implemented in an air-conditioned lab maintained at 22°C, illustrating the potential value of the technology for field conditions in the tropics and subtropics.

Non-instrumented nucleic acid amplification (NINA): instrument-free molecular malaria diagnostics for low-resource settings.

We have achieved the first complete, non-instrumented nucleic acid amplification test (NAAT) using a calcium oxide heat source thermally linked to an engineered phase change material. These two components alone maintain a thermal profile suitable for the loop-mediated isothermal amplification assay. Starting with computational fluid dynamics analysis, we identified nominal geometry for the exothermic reaction chamber, phase change material chamber, thermal insulation, and packaging. Using this model, we designed and fabricated an alpha prototype assay platform. We have verified the function of this multi-pathogen-capable platform with both fluorescent and visual turbidity indications using samples spiked with malaria DNA. Both the exothermically heated platform samples and samples heated on a Perkin-Elmer GeneAmp9600 thermocycler were first incubated at 62°C for 45 minutes, then heated to 95°C to terminate enzyme activity, then analyzed. Results from the exothermically heated, non-instrumented platform were comparable to those from the thermocycler. These developments will enable point-of-care diagnostics using accurate NAATs which until now have required a well-equipped laboratory. The aim of this research is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where assays such as immunochromatographic strip tests are successfully used but where there is no access to the infrastructure and logistics required to operate and maintain instrument-based diagnostics.

Performance of cryotherapy devices using nitrous oxide and carbon dioxide.

OBJECTIVE: To compare temperatures reached by 4 different cryotherapy devices commonly used to treat precancerous cervical lesions in low-resource countries using nitrous oxide (N(2)O) and carbon dioxide (CO(2)) as well as with and without a gas conditioner. METHODS: Bench testing was conducted using 4 different cryotherapy devices and locally obtained N(2)O and medical- and industrial-grade CO(2) refrigerant with and without a gas conditioner. A thermocouple was used to continuously measure the temperature of the cryotherapy tip. Comparison across the cryotherapy devices was based on the mean and lowest temperatures. RESULTS: Without the gas conditioner, all of the devices tested reached mean temperatures colder than -50°C with N(2)O, although 2 devices reached warmer temperatures in a proportion of N(2)O tests. Only 2 of the devices reached mean temperatures colder than -50°C with CO(2). One-way analysis of variance identified the device as the dominant factor for the temperature differences, while the gas was not a determinant of temperature variation. The gas conditioner hindered the performance of 2 of the devices, and only improved the performance of 1 device. CONCLUSION: Both N(2)O and CO(2) reach appropriate freezing temperatures with some cryotherapy devices. Performance of some cryotherapy devices is suboptimal.

Effect of the "cough technique" on cryotherapy freezing temperature.

OBJECTIVE: To assess the impact of the cough technique on the tip temperature reached in a cryotherapy device commonly used to treat precancerous cervical lesions. METHODS: During February to March 2009, bench testing was conducted in Peru on 3 Wallach LL100 cryotherapy units using locally obtained carbon dioxide as the refrigerant. The temperature at the cryo tip was measured continuously with a thermocouple. Mean temperatures recorded with and without the cough technique were compared across the 3 cryotherapy devices. RESULTS: Higher mean temperatures were observed with the cough technique (-21.7 degrees C [95% CI, -23.0 to -20.4]; -22.0 degrees C [95% CI, -24.2 to -19.7]; -29.4 degrees C [95% CI, -30.3 to -28.6]) compared with mean temperatures observed with the standard procedure (-38.6 degrees C [95% CI, -44.8 to -32.4]; -36.0 degrees C [95% CI, -43.5 to -28.5]; -41.4 degrees C [95% CI, -49.8 to -33.0]). No test performed with the cough technique reached a temperature below -33 degrees C. CONCLUSION: Higher temperatures were observed when the cough technique was used. Caution should be exercised by practitioners who use this procedure when performing cryotherapy because the temperatures obtained may not be sufficient to destroy precancerous tissue.