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Title: “Improved Methods for Point of Care Detection of Blood-Borne Pathogens”
Committee:
Catherine Klapperich, PhD – BU BME (Co-Advisor)
Mario Cabodi, PhD – BU BME (Co-Advisor)
James Galagan, PhD – BU BME (Chair)
Muhammad Zaman, PhD – BU BME
Jesse Gitaka, PhD – Research Fellow, Mount Kenya University
Abstract:
Preventing the spread of blood-borne infectious diseases is vital to improving global health, particularly in low- and middle-income countries (LMICs). Sensitive and accurate diagnosis of infections is critical to this effort. While nucleic acid amplification tests (NAATs), are the gold-standard techniques for detection and quantification of pathogen levels, they are resource-intensive, making them challenging to implement in some areas of LMICs. This work outlines the development of two methods to make accurate, sensitive NAATs more accessible for use at the point of care in resource-limited areas of LMICs.
The first method enables instrument-free nucleic acid extraction from whole blood. A room temperature lysis chemistry, coupled with a paper-and-plastic device, was used to isolate, purify, and store pathogen DNA and RNA on a paper capture membrane. Successful isolation of HIV virion RNA and P. falciparum parasite DNA from whole blood was achieved at a recovery rate greater than 60% over a wide range of pathogen concentrations. The viability of RNA storage on paper was demonstrated at room temperature and at 37°C over two weeks, thereby eliminating the need for cold storage after sample collection. The second method is a novel isothermal amplification assay developed to identify asymptomatic patients infected with P. falciparum. Using a unique primer design, the assay uses Bst strand displacing polymerase to amplify multiple regions of the P. falciparum genome at 56°C, achieving an analytical sensitivity of ~23.4 fg/µL P. falciparum gDNA (~1 parasite/µL) in 30 minutes. The assay was adopted to a point of care format by amplifying captured parasite DNA directly on paper and using a visual readout, thereby eliminating the need for any specialized equipment. This point of care assay shows similar analytical sensitivity to a gold standard PCR assay while using a fraction of the resources required for PCR.
The whole blood nucleic acid extraction device and isothermal assay described in this work can be used together for sensitive diagnosis of P. falciparum malaria. The methods can also be used independently, or in combination with other techniques routinely used in the field, for detection of HIV, Plasmodium or other blood-borne pathogens. The flexibility built into these methods enables easier integration with existing testing workflows in LMICs.
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