Johnson Kinyua

Lateral flow Loop-Mediated Isothermal amplification test with stem primers: Detection of cryptosporidium species in Kenyan children presenting with diarrhea

Background. Cryptosporidium is a protozoan parasite and a major cause of diarrhea in children and immunocompromised patients. Current diagnostic methods for cryptosporidiosis such as microscopy have low sensitivity while techniques such as PCR indicate higher sensitivity levels but are seldom used in developing countries due to their associated cost. 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 test has a detection limit of 10 pg/µl (~100 oocysts/ml) indicating a need for more sensitive diagnostic tools. This study developed 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). Results. The stem LFD LAMP test showed analytical sensitivity of 10 oocysts/ml compared to 100 oocysts/ml (10 pg/ul) for each of the SAM-1 LAMP test and nested PCR. The stem LFD LAMP and nested PCR detected 29/39 and 25/39 positive samples of previously identified C. parvum and C. hominis DNA, respectively. The SAM-1 LAMP detected 27/39. On detection of Cryptosporidium DNA in 67 clinical samples, the stem LFD LAMP detected 16 samples and SAM-2 LAMP 14 and nested PCR identified 11. Preheating 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, and no cross-amplification was recorded with nontarget DNA. Conclusion. The developed stem LFD LAMP test is an appropriate method for the 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.

In Silico Characterization and Structural Modeling of Dermacentor andersoni p36 Immunosuppressive Protein

Ticks cause approximately

Diversity and distribution of soil nematodes in Ngere tea catchment area of Murang'a county, Kenya

17–19 billion economic losses to the livestock industry globally. Development of recombinant antitick vaccine is greatly hindered by insufficient knowledge and understanding of proteins expressed by ticks. Ticks secrete immunosuppressant proteins that modulate the hosts immune system during blood feeding; these molecules could be a target for antivector vaccine development. Recombinant p36, a 36 kDa immunosuppressor from the saliva of female Dermacentor andersoni, suppresses T-lymphocytes proliferation in vitro. To identify potential unique structural and dynamic properties responsible for the immunosuppressive function of p36 proteins, this study utilized bioinformatic tool to characterize and model structure of D. andersoni p36 protein. Evaluation of p36 protein family as suitable vaccine antigens predicted a p36 homolog in Rhipicephalus appendiculatus, the tick vector of East Coast fever, with an antigenicity score of 0.7701 that compares well with that of Bm86 (0.7681), the protein antigen that constitute commercial tick vaccine Tickgard™. Ab initio modeling of the D. andersoni p36 protein yielded a 3D structure that predicted conserved antigenic region, which has potential of binding immunomodulating ligands including glycerol and lactose, found located within exposed loop, suggesting a likely role in immunosuppressive function of tick p36 proteins. Laboratory confirmation of these preliminary results is necessary in future studies.

Development and evaluation of a Loop Mediated Isothermal Amplification (LAMP) technique for the detection of hookworm (Necator americanus) infection in fecal samples.

A survey was conducted to determine the diversity and distribution of soil nematodes associated with tea in Ngere tea catchment area in Kenya. Soil samples were collected from six electoral zones of Ngere factory in Gatanga division Thika district, Murang’a County, Kenya. Nematodes were extracted and recovered from soil samples using a modified Baermann funnel method and identified under a light microscope based on their morphological characters. They were also classified according to their feeding habits. Ten genera belonging to, bacteriovores, fungivores, and omnivores were identified. Fungal feeding and parasitic nematodes were the most widely distributed trophic groups across the tea catchment area. Plant-parasitic nematodes recovered included Pratylenchus spp., Helicotylenchus spp., Rotylenchus spp., Aphelenchus spp., Rotylenchulus spp., and Xiphinema spp. Tylenchus spp was the most frequently occurring species in the soil (60.47%) where the population was 429 followed by Pratylenchus spp with 55.81% frequency rating and a population of 404 while Aphelenchus spp had frequency rating of 48.84% and a population of 530. Ditylenchus and Rotylenchus spp had the lowest frequency rating of 6.98 and 4.65%, respectively, while Rotylenchus spp had the lowest population of 33. Six genera of plant parasitic nematodes were encountered in the collected soil samples. These plant parasitic nematodes were identified as Pratylenchus spp., Aphelenchus spp., Helicotylenchus spp., Tylenchus spp., Xiphinema spp and Duotylenchus spp. Pratylenchus spp. and Tylenchus spp. were the most frequently occurring species in the soil (50%) with a population of 25 and 21, respectively, which was followed by Helicotylenchus spp., Aphelenchus spp., Xiphinema spp., and Duotylenchus spp. with 16.67% frequency rating and a population of 10, 10, 3 and 20, respectively. Pratylenchus spp had the highest nematode population of 28.09% and the lowest nematode population was Xiphinema spp., having a population of 3.37%. The correlation between nematode population counts and total organic carbon content was not significant at P≥0.05.