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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.

SUBMITTER: Deborggraeve S

PROVIDER: S-EPMC1594664 | biostudies-literature | 2006 Aug

REPOSITORIES: biostudies-literature

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Molecular dipstick test for diagnosis of sleeping sickness.

Deborggraeve S S Claes F F Laurent T T Mertens P P Leclipteux T T Dujardin J C JC Herdewijn P P Büscher P P

Journal of clinical microbiology 20060801 8

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 thro ...[more]

PMID: 16891507

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Project description:ObjectivesRecently, improvements have been made to diagnostics for gambiense sleeping sickness control but their performance remains poorly documented and may depend on specimen processing prior to examination. In a prospective study in the Democratic Republic of the Congo, we compared the diagnostic performance of several parasite detection techniques, immune trypanolysis and of m18S PCR on whole blood stored in a stabilisation buffer or dried on filter paper.MethodsIndividuals with CATT whole blood (WB) titer ?1?4 or with clinical signs indicative for sleeping sickness were examined for presence of trypanosomes in lymph node aspirate (LNA) and/or in blood. Blood was examined with Capillary Centrifugation Technique (CTC), mini-Anion Exchange Centrifugation Technique (mAECT) and mAECT on buffy coat (BC). PCR was performed on whole blood (i) stored in guanidine hydrochloride EDTA (GE) stabilisation buffer and (ii) dried on filter paper, and repeatability and reproducibility were assessed. Immune trypanolysis (TL) was performed on plasma.ResultsA total of 237 persons were included. Among 143 parasitologically confirmed cases, 85.3% had a CATT-WB titre of ?1/8, 39.2% were positive in LNA, 47.5% in CTC, 80.4% in mAECT-WB, 90.9% in mAECT-BC, 95.1% in TL and up to 89.5% in PCR on GE-stabilised blood. PCR on GE-stabilised blood showed highest repeatability (87.8%) and inter-laboratory reproducibility (86.9%). Of the 94 non-confirmed suspects, respectively 39.4% and 23.4% were TL or PCR positive. Suboptimal specificity of PCR and TL was also suggested by latent class analysis.ConclusionThe combination of LNA examination with mAECT-BC offered excellent diagnostic sensitivity. For PCR, storage of blood in stabilisation buffer is to be preferred over filter paper. TL as well as PCR are useful for remote diagnosis but are not more sensitive than mAECT-BC. For TL and PCR, the specificity, and thus usefulness for management of non-confirmed suspects remain to be determined.

Project description:The number of imported Human African Trypanosomiasis (HAT) cases in non-endemic countries has increased over the last years. The objective of this analysis is to describe the clinical presentation of HAT in Caucasian travelers. Literature was screened (MEDLINE, Pubmed) using the terms "Human African Trypanosomiasis", "travelers" and "expatriates"; all European languages except Slavic ones were included. Publications without clinical description of patients were only included in the epidemiological analysis. Forty-five reports on Caucasians with T.b. rhodesiense and 15 with T.b. gambiense infections were included in the analysis of the clinical parameters. Both species have presented with fever (T.b. rhodesiense 97.8% and T.b. gambiense 93.3%), headache (50% each) and a trypanosomal chancre (T.b. rhodesiense 84.4%, T.b. gambiense 46.7%). While sleeping disorders dominate the clinical presentation of HAT in endemic regions, there have been only rare reports in travelers: insomnia (T.b. rhodesiense 7.1%, T.b. gambiense 21.4%), diurnal somnolence (T.b. rhodesiense 4.8%, T.b. gambiense none). Surprisingly, jaundice has been seen in 24.2% of the Caucasian T.b. rhodesiense patients, but has never been described in HAT patients in endemic regions. These results contrast to the clinical presentation of T.b. gambiense and T.b. rhodesiense HAT in Africans in endemic regions, where the presentation of chronic T.b. gambiense and acute T.b. rhodesiense HAT is different. The analysis of 14 reports on T.b. gambiense HAT in Africans living in a non-endemic country shows that neurological symptoms such as somnolence (46.2%), motor deficit (64.3%) and reflex anomalies (14.3%) as well as psychiatric symptoms such as hallucinations (21.4%) or depression (21.4%) may dominate the clinical picture. Often, the diagnosis has been missed initially: some patients have even been hospitalized in psychiatric clinics. In travelers T.b. rhodesiense and gambiense present as acute illnesses and chancres are frequently seen. The diagnosis of HAT in Africans living outside the endemic region is often missed or delayed, leading to presentation with advanced stages of the disease.

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Molecular dipstick test for diagnosis of sleeping sickness.

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Molecular dipstick test for diagnosis of sleeping sickness.

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