Trypanosoma Brucei Brucei Infection In Rat Model And Potential Of Urine As A Diagnostic Sample In Loop-Mediated Isothermal Amplification Assay

ABSTRACT

Human African Trypanosomiasis (HAT) is a protozoal disease that is caused by the extracellular parasite and also transmitted by vector known as tsetse fly that belong to genus Glossina. HAT commonly known as Sleeping sickness is a tropical disease that is neglected, historically endemic among 36 countries of Africa in sub-Saharan. In the last decade, WHO reports shows a tremendous decline in cases due to collaboration from both national and international efforts. Early diagnosis especially in cases of low parasitaemia is crucial in management of HAT. Loop-mediated isothermal amplification (LAMP) assay has been used to detect low number of trypanosomes in the host blood in an effort to improve management of HAT. There is need to improve on this diagnostic procedure by using non-invasive samples. The aim of current study was to assess urine use as the non-invasive diagnostic sample for trypanosomiasis in a rat model. Sixteen female Sprague-Dawley rats (Rattus norvegicus) weighing 181 - 281g were utilised in the current study. Rats were divided into three groups. Group I comprised seven rats which were inoculated with 1×105 trypanosomes, serial sacrificed at 14, 21 and 28 days post inoculation (dpi), or when paresis was observed. Group II had seven rats similarly inoculated and then treated with Diminazene Aceturate (40mg/kg of body weight (bwt) intraperitoneally (IP)) at 21 dpi, and then followed up till relapse. Group III was the control group and had two rats which were non-infected and served as negative control. Following infection, the parameters monitored included body weight, urine volume, haematology, parasitaemia and LAMP involving urine or blood. The infected rats developed parasitaemia and treatment with 40mg/kg bwt of DA cleared parasitaemia in rats within five days to non-detectable levels. However, relapse in parasitaemia occurred starting day 117 after infection. The decrease was significant (P=0.02728) in urine volume voided after treatment with DA compared to urine volume voided before treatment. Secondly, a decrease that was significant (P=0.03590) in weight at 21 days post infection compared to starting weight before infection was observed. Also, infected group recorded a significant reduction (P=0.00283) in PCV blood. The LAMP assay detected infection in both samples of urine and serum. However, even though the sensitivity of LAMP test when using serum samples for detection of the infected animals was higher compared to urine samples, the difference between the samples was not significant (P=0.48481) in terms of infection detection. Therefore urine provides a major advantage over serum considering that it is non-invasive. The LAMP test performed on fresh urine and urine stored at -20°C did not show any significant difference (P=0.13704) between the samples, meaning that urine storage has no effect on LAMP performance. The analysis of diluted urine sample with two volumes of absolute ethanol in a ratio of 1:2 (optimization) and stored at -20°C yielded consistent results for trypanosome DNA detection as compared to undiluted urine sample stored at -20°C, which yielded inconsistent results. Based on these results, dilution of urine appeared to optimize urine sample for utilization in LAMP. The difference between optimized and non-optimized sample was significant (P=0.00225) with optimized urine giving better results. The urine sample can be used as an alternative sample to blood which offers the unique advantage of being noninvasive. Based on this study, it is recommended that rat is a suitable model of HAT studies and urine samples provide a favourable non-invasive option in diagnosis of Trypanosomiasis.