A multi-country phase 2 study to evaluate the suitcase lab for rapid detection of SARS-CoV-2 in seven Sub-Saharan African countries: Lessons from the field.

BACKGROUND: The COVID-19 pandemic led to severe health systems collapse, as well as logistics and supply delivery shortages across sectors. Delivery of PCR related healthcare supplies continue to be hindered. There is the need for a rapid and accessible SARS-CoV-2 molecular detection method in low resource settings. OBJECTIVES: To validate a novel isothermal amplification method for rapid detection of SARS-CoV-2 across seven sub-Sharan African countries. STUDY DESIGN: In this multi-country phase 2 diagnostic study, 3,231 clinical samples in seven African sites were tested with two reverse transcription Recombinase-Aided Amplification (RT-RAA) assays (based on SARS-CoV-2 Nucleocapsid (N) gene and RNA-dependent RNA polymerase (RdRP) gene). The test was performed in a mobile suitcase laboratory within 15 min. All results were compared to a real-time RT-PCR assay. Extraction kits based on silica gel or magnetic beads were applied. RESULTS: Four sites demonstrated good to excellent agreement, while three sites showed fair to moderate results. The RdRP gene assay exhibited an overall PPV of 0.92 and a NPV of 0.88. The N gene assay exhibited an overall PPV of 0.93 and a NPV 0.88. The sensitivity of both RT-RAA assays varied depending on the sample Ct values. When comparing sensitivity between sites, values differed considerably. For high viral load samples, the RT-RAA assay sensitivity ranges were between 60.5 and 100% (RdRP assay) and 25 and 98.6 (N assay). CONCLUSION: Overall, the RdRP based RT-RAA test showed the best assay accuracy. This study highlights the challenges of implementing rapid molecular assays in field conditions. Factors that are important for successful deployment across countries include the implementation of standardized operation procedures, in-person continuous training for staff, and enhanced quality control measures.

Evaluation of a real-time recombinase polymerase amplification assay for rapid detection of Schistosoma haematobium infection in resource-limited setting.

Accurate diagnosis of urogenital schistosomiasis is vital for surveillance/control programs as well as achieving the WHO 2012-2020 road map for the total eradication of schistosomiasis. Recombinase polymerase amplification (RPA) has emerged as a rapid and simple molecular tool adaptable for fewer resources with diagnostic accuracy similar to polymerase chain reaction (PCR). This rapid molecular assay employs the use of enzymes for the amplification of nucleic acid taget at a constant temperature. The aim of this study was to validate a real-time RPA assay targeting the Dra 1 repittitive sequence of Schistosoma (S.) haematobium and evaluate its use in urogenital schistosomiasis diagnosis. S. haematobium Dra 1 molecular DNA standard was applied to determine the assay's analytical sensitivity. DNA extracts of S. haematobium, other Schistosoma species, protozoa and bacteria species were used to determine the specificity of the RPA assay. Clinical performance of the assay was validated with a panel of 135 urine samples from volunteers of schistosomiasis endemic communities. The developed assay was evaluated with urine samples extracted by just boiling and with SpeedXtract® DNA extraction kit. A specific fragment of S. haematobium Dra 1 repetitive sequence was amplified within 15 minutes at a constant 42˚C using the developed S. haematobium RPA assay. The detection limit was 15 copies of Dra1 molecular DNA standard per reaction. There was no cross-reaction with other protozoan and bacterial species except Schistosoma species, S. mansoni and S. japonicum. Using 135 urine samples, Schistosoma RPA assay had a clinical sensitivity and specificity of 98.4% (95% CI, 91.6-100) and 100% (95% CI, 94.9-99) respectively when compared to S. haematobium Dra 1 qPCR assay. The diagnostic performance of S. haematobium real-time RPA assay was not affected by the use of crude DNA extracted samples. The S. haematobium RPA assay can serve as an alternative to PCR, especially in low resource settings.