Nucleic Acid Test to Diagnose Cryptosporidiosis: Lab Assessment in Animal and Patient Specimens

Abstract

Diarrheal diseases cause more morbidity and mortality around the world than human immunodeficiency virus (HIV), malaria, or tuberculosis. Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance. The protozoan parasites of the genus Cryptosporidium are increasingly recognized to be responsible for a significant portion of diarrhea morbidity. We present a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the isothermal amplification technique recombinase polymerase amplification (RPA) to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 min; products are then detected visually on simple lateral flow strips. The RPA-based Cryptosporidium assay (RPAC assay) was developed and optimized using DNA from human stool samples spiked with pathogen. It was then tested using DNA extracted from the stool of infected mice where it correctly identified the presence or absence of 27 out of 28 stool samples. It was finally tested using DNA extracted from the stool of infected patients where it correctly identified the presence or absence of 21 out of 21 stool samples. The assay was integrated into a foldable, paper and plastic device that enables DNA amplification with only the use of pipets, pipet tips, and a heater. The performance of the integrated assay is comparable to or better than polymerase chain reaction (PCR), without requiring the use of thermal cycling equipment. This platform can easily be adapted to detect DNA from multiple pathogens.

Quantification of HIV-1 DNA Using Real-Time Recombinase Polymerase Amplification

“…4,5 For these reasons, a number of recent reports have proposed RPA-based strategies for the detection of pathogens. [6][7][8][9][10] Although some papers demonstrate a relationship between nucleic acid concentration and onset of amplification, 10,11 to the best of our knowledge, RPA has not yet been implemented to quantify sample concentration using a standard curve. Moreover, the accuracy with which samples can be quantified with real-time RPA has not yet been characterized.…”

Detection of Entamoeba histolytica by Recombinase Polymerase Amplification

“…Studies are in progress by our group to combine this assay with the previously described RPA assays for Cryptosporidium and Giardia in a multiplex assay. 13,14 In addition, simpler methods of DNA extraction will be optimal for widespread use. Future studies are needed to evaluate the feasibility of implementing RPA assays in endemic areas.…”

“…Recently, we have demonstrated the feasibility to detect protozoan parasites in stool samples. 13,14 In this work, we have developed a specific and sensitive assay to detect E. histolytica that can be used at POC.…”

Recombinase Polymerase Amplification-Based Assay to Diagnose Giardia in Stool Samples

“…Although positive and negative samples could easily be identified using the naked eye, we also used a previously described method to objectively differentiate positive from negative samples. 20 Briefly, for objective bench top characterization of positive and negative RPAG assay results, lateral flow strips were immediately scanned with a flatbed scanner after 5 minutes of lateral flow time. Using the scanned image, the signal/background ratio of the test region was calculated using a custom MATLAB (The MathWorks, Natick, MA) script.…”