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 microl 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.

FIG. 1.

Alignment of the HAT-PCR-OC target sequence within the 18S rRNA gene of the Trypanosomatidae parasites Trypanosoma brucei gambiense (T.b.g.) (GenBank accession number AJ009141), Trypanosoma brucei rhodesiense (T.b.r.) (GenBank accession number AJ009142), Trypanosoma cruzi (T.c.) (GenBank accession number AF303660), and Leishmania donovani (L.d.) (GenBank accession number X07773). Situation of the forward primer, 18S-F, the biotin-labeled reverse primer, 18S-R, and the T. brucei (T.b.) gold probe. Gaps in the sequence are represented by dots. Black, dark-gray, and light-gray shading indicates consensus among all four parasites, three parasites, or two parasites, respectively. The target regions of the 18S-F and 18S-R primers and the T. brucei gold probe are boxed.

FIG. 2.

HAT-PCR-OC test principle. Test side. In case of a positive sample, during migration, the T. brucei gold probes hybridize with the T. brucei amplicons that will accumulate on the Neutralite avidin line on the test side of the stick. Control side. During migration the IC gold probes hybridize with IC amplicons that will accumulate on the Neutralite avidin line on the control side of the stick (control for PCR). The unbound IC gold probes hybridize with the complementary migration control probes (control for migration).

FIG. 3.

Possible HAT-PCR-OC test result. a. Valid test, positive result. b. Valid test, negative result. c. Valid test, positive result of a sample with high T. brucei DNA content. d. Invalid test.

FIG. 4.

Analytical sensitivity and species specificity of the HAT-PCR-OC test. a. Serial dilution of T. b. gambiense DNA in water. Dipsticks 1 to 7: 500 pg, 50 pg, 5 pg, 500 fg, 50 fg, 5 fg, or 0.5 fg per PCR. b. Serial dilution of living T. b. gambiense bloodstream form parasites in naive human blood. Dipsticks 1 to 6: 10,000, 1,000, 100, 10, 1, or 0 parasites in 180 μl of blood. c. HAT-PCR-OC results obtained with DNA from T. b. gambiense (dipstick 1), T. b. rhodesiense (dipstick 2), Leishmania donovani (dipstick 3), Trypanosoma cruzi (dipstick 4), Mycobacterium tuberculosis (dipstick 5), Plasmodium falciparum (dipstick 6), or Schistosoma mansoni (dipstick 7).

FIG. 5.

Diagnostic sensitivity and specificity of the HAT-PCR-OC. a. HAT-PCR-OC results for 26 blood samples from confirmed T. b. gambiense sleeping sickness patients (dipsticks 1 to 26) and one negative control (−). b. HAT-PCR-OC results for one positive control (+) and for five blood samples from area-of-endemicity negative controls (dipsticks 1 to 5).