Improved methods for point of care detection of blood-borne pathogens

Preventing the spread of blood-borne infectious diseases is vital to improving global health outcomes, particularly for low- and middle-income countries (LMICs). Sensitive and accurate diagnosis of infections is vital to this effort. Nucleic acid amplification tests (NAATs), which amplify pathogen nucleic acids, are gold-standard techniques for detection and quantification of pathogen levels. However, standard NAATs such as polymerase chain reaction (PCR) require expensive equipment for blood sample processing and DNA/RNA amplification, making them challenging to implement in resource-limited areas of LMICs. In this work, I developed two methods to simplify sample processing and amplification to make 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 and a paper-and-plastic sample capture device were developed to isolate, purify, and store pathogen DNA and RNA on a paper capture membrane. Extracted nucleic acids can be eluted and used in standard NAATs or in developmental amplification assays. I demonstrated successful isolation of HIV virion RNA and P. falciparum parasite DNA from whole blood samples over several concentrations with >60% recovery. Extracted RNA remains stable on the capture membrane for two weeks at room temperature and 37°C, alleviating the need for cold storage after sample collection. These results are a promising step toward using this method for simplified sample extraction and storage in low-resource settings in LMICs. The second method I developed is a novel isothermal amplification technique for P. falciparum DNA. Sensitive diagnosis of P. falciparum infection is vital to identify and treat low-density, asymptomatic infections and move closer to eliminating malaria. Highly sensitive PCR assays are difficult to deploy in resource-limited areas of LMICs and existing isothermal methods require complex assay design and are often not sensitive enough to diagnose asymptomatic infections. Here, I developed a novel isothermal technique which amplifies multiple regions of the P. falciparum genome, generating a large amount of DNA for better analytical sensitivity. The assay achieves a lower limit of detection of ~23.4 fg P. falciparum gDNA/µL (~1 parasite/µL) in 30 minutes, similar gold-standard PCR assay while using a fraction of the resources required for PCR. Lastly, I adapted the assay for implementation at the point of care. I showed that the assay directly amplifies P. falciparum parasite DNA captured on paper with the paper-and-plastic device previously developed. I also incorporated visual assay readout with lateral flow strips, eliminating the need for specialized equipment to detect amplified DNA. I explored methods to eliminate cold storage of reagents by stabilizing amplification enzymes at room temperature. The work described in this thesis represents two enhanced methods for point of care detection of blood borne pathogens. By simplifying sample extraction, amplification, and detection, the methods described here make NAATs more accessible to low-resource areas of LMICs. 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. The flexibility built in to these methods enables easier integration into existing workflows in LMICs.2021-05-18T00:00:00

A paperfluidic platform to detect Neisseria gonorrhoeae in clinical samples

Globally, the microbe Neisseria gonorrhoeae (NG) causes 106 million newly documented sexually transmitted infections each year. Once appropriately diagnosed, NG infections can be readily treated with antibiotics, but high-risk patients often do not return to the clinic for treatment if results are not provided at the point of care. A rapid, sensitive molecular diagnostic would help increase NG treatment and reduce the prevalence of this sexually transmitted disease. Here, we report on the design and development of a rapid, highly sensitive, paperfluidic device for point-of-care diagnosis of NG. The device integrates patient swab sample lysis, nucleic acid extraction, thermophilic helicase-dependent amplification (tHDA), an internal amplification control (NGIC), and visual lateral flow detection within an 80 min run time. Limits of NG detection for the NG/NGIC multiplex tHDA assay were determined within the device, and clinical performance was validated retroactively against qPCR-quantified patient samples in a proof-of-concept study. This paperfluidic diagnostic has a clinically relevant limit of detection of 500 NG cells per device with analytical sensitivity down to 10 NG cells per device. In triplicate testing of 40 total urethral and vaginal swab samples, the device had 95% overall sensitivity and 100% specificity, approaching current laboratory-based molecular NG diagnostics. This diagnostic platform could increase access to accurate NG diagnoses to those most in need.This work was funded by the National Institute of Health National Institute of Allergy and Infectious Diseases award number R01 AI113927 to Boston University and the NIH National Institute of Biomedical and Bioengineering award number U54 EB007958 to Johns Hopkins University. (R01 AI113927 - National Institute of Health National Institute of Allergy and Infectious Diseases; U54 EB007958 - NIH National Institute of Biomedical and Bioengineering)Accepted manuscrip

Development and clinical validation of Iso-IMRS: a novel diagnostic assay for P. falciparum malaria

In many countries targeting malaria elimination, persistent malaria infections can have parasite loads significantly below the lower limit of detection (LLOD) of standard diagnostic techniques, making them difficult to identify and treat. The most sensitive diagnostic methods involve amplification and detection of Plasmodium DNA by polymerase chain reaction (PCR), which requires expensive thermal cycling equipment and is difficult to deploy in resource-limited settings. Isothermal DNA amplification assays have been developed, but they require complex primer design, resulting in high nonspecific amplification, and show a decrease in sensitivity than PCR methods. Here, we have used a computational approach to design a novel isothermal amplification assay with a simple primer design to amplify P. falciparum DNA with analytical sensitivity comparable to PCR. We have identified short DNA sequences repeated throughout the parasite genome to be used as primers for DNA amplification and demonstrated that these primers can be used, without modification, to isothermally amplify P. falciparum parasite DNA via strand displacement amplification. Our novel assay shows a LLOD of ∼1 parasite/μL within a 30 min amplification time. The assay was demonstrated with clinical samples using patient blood and saliva. We further characterized the assay using direct amplicon next-generation sequencing and modified the assay to work with a visual readout. The technique developed here achieves similar analytical sensitivity to current gold standard PCR assays requiring a fraction of time and resources for PCR. This highly sensitive isothermal assay can be more easily adapted to field settings, making it a potentially useful tool for malaria elimination.Accepted manuscrip