Abstract
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.
Highlights
The application of nucleic acid amplification technologies (NAAT) has had a significant and beneficial impact on the diagnosis of infectious diseases
For assays incubated for 20 minutes at #30uC, performance of the HIV-1 Recombinase polymerase amplification (RPA) assay on 10 copies of HIV-1 proviral DNA was not optimal, with only 3 temperatures producing any positive results (1/3 at 15uC and 29uC, or 2/3 at 30uC), and all 3 replicates failing at temperatures between 20uC and 27uC (Table 1)
At ambient temperatures below 31uC, an exothermic chemical heater could be used to provide supplementary heat for optimal RPA performance to as low an ambient temperature of 10uC. These results demonstrate that, even in the absence of electricity or a battery dependent heater, RPA is significantly faster than other isothermal assays that are being developed as tools for NAAT based diagnostics in low resource settings (LRS)
Summary
The application of nucleic acid amplification technologies (NAAT) has had a significant and beneficial impact on the diagnosis of infectious diseases. There remains a critical need for high quality diagnostics that can be performed closer to the point of care in low resource settings (LRS) to aid in the clinical diagnosis and subsequent treatment of endemic diseases such as HIV, tuberculosis and malaria. The infrastructure and equipment requirements to perform PCR-based HIV diagnostics results in the need to ship specimens from rural clinics to centralized facilities for testing. Rapid treatment can potentially lead to a reduced viral reservoir and better outcomes, further emphasizing the need for rapid point of care diagnostics [5]
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