Detection of Cryptosporidium Species in Kenya Using Lateral Flow Loop-Mediated Isothermal Amplification

Abstract

Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy depict inter-observer variability and do not allow for batch processing while techniques such as PCR indicate higher sensitivity levels, but are seldom used in developing countries due to the associated cost. This study aimed to develop a more sensitive lateral flow dipstick (LFD) LAMP test based on SAM-1 gene and with the addition of a second set of reaction accelerating primers (stem primers). A loop mediated isothermal amplification (LAMP) technique, a method with shorter time to result and with equal or higher sensitivity compared to PCR, has been developed and applied in the detection of Cryptosporidium species. The detection limit was determined using analytical sensitivity based on reference DNA, sensitivity was achieved using archived samples, while specificity was achieved using closely related DNA. This test has a detection limit of 10pg/ µl (~100 oocysts/ml) suggesting need for more sensitive diagnostic tools. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/µl) for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP, SAM-1 LAMP and nested PCR detected 29/39 (74.3%), 27/39 (69.2%), and 25/39 (64.1%) positive samples of previously identified C. parvum and C. hominis DNA, respectively. Using the 67 Cryptosporidium DNA clinical samples, the stem LFD LAMP detected 16/67 (23.8%) positive samples, SAM-2 LAMP detected 14/67 (20.8%) positive samples, while the nested PCR detected 11/67 (16.4%) positive samples. The 67 samples had not been sequenced and had not been tested to determine whether they were positive or negative. The positive samples may be a representation of the prevalence of the disease in the population. Pre-heating the templates increased detection by stem LFD LAMP to 19 samples. Time to results from master mix preparation step took ~80 minutes. The test was specific (100%), and no cross-amplification was recorded with non-target DNA. This stem LFD LAMP test is more appropriate for detection of C. hominis, C. parvum and C. meleagridis DNA in human stool samples. It can be used in algorithm with other diagnostic tests and may offer promise as an effective diagnostic tool in the control of cryptosporidiosis.