Molecular Dipstick Test for Diagnosis of Sleeping Sickness

Abstract

Human African trypanosomiasis (HAT) or sleeping sickness is a neglected disease that affects poor rural populations across sub-Saharan Africa. Confirmation of diagnosis is based on detection of parasites in either blood or lymph by microscopy. Here we present the development and the first-phase evaluation of a simple and rapid test (HAT-PCR-OC [human African trypanosomiasis-PCR-oligochromatography]) for detection of amplified Trypanosoma brucei DNA. PCR products are visualized on a dipstick through hybridization with a gold-conjugated probe (oligochromatography). Visualization is straightforward and takes only 5 min. Controls both for the PCR and for DNA migration are incorporated into the assay. The lower detection limit of the test is 5 fg of pure T. brucei DNA. One parasite in 180 l of blood is still detectable. Sensitivity and specificity for T. brucei were calculated at 100% when tested on blood samples from 26 confirmed sleeping sickness patients, 18 negative controls (nonendemic region), and 50 negative control blood samples from an endemic region. HAT-PCR-OC is a promising new tool for diagnosis of sleeping sickness in laboratory settings, and the diagnostic format described here may have wider application for other infectious diseases.

Controlling sleeping sickness – a review

“…Progress is being made in detecting trypanosomes in blood and cerebro-spinal fluid based on a variety of techniques including oligochomatography (Deborggraeve et al 2006) and isothermal tests (Njiru et al 2008) -the first steps towards a simplified molecular test. There are also several promising developments in disease biomarkers (Papadopoulos et al 2004 ;Agranoff et al 2005).…”

The role of nanogold in human tropical diseases: research, detection and therapy

“…Deborggraeve et al [31] developed a simple and rapid test for detection of amplified T. brucei DNA, the human African trypanosomiasis-polymerase chain reactionoligochromatography (HAT-PCR-OC). In this case, gold was used essentially as a colorimetric indicator.…”

Human African Trypanosomiasis

“…110 Present research eff orts focus on several areas: (i) recombinant or native trypanosome antigens that could be used to develop an improved serological test; (ii) methods to detect parasite antigens in blood or CSF; (iii) proteomic fi ngerprinting; (iv) low-tech PCR methods such as loop-mediated isothermal amplifi cation or oligochromatography; and (v) new blood or CSF markers of second-stage disease. [111][112][113][114][115] …”