Loop Mediated Isothermal Amplification for Detection of Trypanosoma brucei gambiense in Urine and Saliva Samples in Nonhuman Primate Model.

Maina Ngotho,Simon Muturi Karanja,John Maina Kagira,Maxwell Wambua Waema,Dawn Nyawira Maranga,Beatrice Muthoni Gachie,Naomi Wangari Maina

doi:10.1155/2015/867846

Abstract

Human African trypanosomiasis (HAT) is a vector-borne parasitic zoonotic disease. The disease caused by Trypanosoma brucei gambiense is the most prevalent in Africa. Early diagnosis is hampered by lack of sensitive diagnostic techniques. This study explored the potential of loop mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR) in the detection of T. b. gambiense infection in a vervet monkey HAT model. Six vervet monkeys were experimentally infected with T. b. gambiense IL3253 and monitored for 180 days after infection. Parasitaemia was scored daily. Blood, cerebrospinal fluid (CSF), saliva, and urine samples were collected weekly. PCR and LAMP were performed on serum, CSF, saliva, and urine samples. The detection by LAMP was significantly higher than that of parasitological methods and PCR in all the samples. The performance of LAMP varied between the samples and was better in serum followed by saliva and then urine samples. In the saliva samples, LAMP had 100% detection between 21 and 77 dpi, whereas in urine the detection it was slightly lower, but there was over 80% detection between 28 and 91 dpi. However, LAMP could not detect trypanosomes in either saliva or urine after 140 and 126 dpi, respectively. The findings of this study emphasize the importance of LAMP in diagnosis of HAT using saliva and urine samples.

Highlights

Human African trypanosomiasis (HAT) is a tropical disease that is endemic in several countries in sub-Saharan Africa.Control of sleeping sickness relies on passive case detection and it is considered to be the most cost-effective when compared to active case detection [1]
Screening of the population at risk is done by antibody detection with the Card Agglutination Test for Trypanosomiasis (CATT) and confirmed by parasitological methods
There was no amplification in any cerebrospinal fluid (CSF) sample with either loop mediated isothermal amplification (LAMP) or polymerase chain reaction (PCR)

Summary

Introduction

Human African trypanosomiasis (HAT) is a tropical disease that is endemic in several countries in sub-Saharan Africa. False negatives (CATT negative) but parasitemic cases have been reported [4] These limitations imply the need for more sensitive and specific diagnosis. Loop mediated isothermal amplification (LAMP) is performed under isothermal conditions and relies on autocycling strand displacement DNA synthesis [11] It requires a simple heating device and is rapid and results are viewed by several detection formats. LAMP uses four to six specially designed primers recognizing six to eight regions of the target DNA sequence resulting in a high specificity. It has been used in detection of the Trypanozoon subgenus [12], T. b. The performance of LAMP based on the TgsGP gene was assessed in detection of T. b gambiense in serum, CSF, saliva, and urine

Materials and Methods

Results

Methods

Parasitological methods

Discussion