Performance of parasitological and molecular techniques for the diagnosis and surveillance of gambiense sleeping sickness.

OBJECTIVES: Recently, 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. METHODS: Individuals 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. RESULTS: A 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. CONCLUSION: The 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.

Diagnostic accuracy of loopamp Trypanosoma brucei detection kit for diagnosis of human African trypanosomiasis in clinical samples.

BACKGROUND: Molecular methods have great potential for sensitive parasite detection in the diagnosis of human African trypanosomiasis (HAT), but the requirements in terms of laboratory infrastructure limit their use to reference centres. A recently developed assay detects the Trypanozoon repetitive insertion mobile element (RIME) DNA under isothermal amplification conditions and has been transformed into a ready-to-use kit format, the Loopamp Trypanosoma brucei. In this study, we have evaluated the diagnostic performance of the Loopamp Trypanosoma brucei assay (hereafter called LAMP) in confirmed T.b. gambiense HAT patients, HAT suspects and healthy endemic controls from the Democratic Republic of the Congo (DRC). METHODOLOGY/PRINCIPAL FINDINGS: 142 T.b. gambiense HAT patients, 111 healthy endemic controls and 97 HAT suspects with unconfirmed status were included in this retrospective evaluation. Reference standard tests were parasite detection in blood, lymph or cerebrospinal fluid. Archived DNA from blood of all study participants was analysed in duplicate with LAMP. Sensitivity of LAMP in parasitologically confirmed cases was 87.3% (95% CI 80.9-91.8%) in the first run and 93.0% (95% CI 87.5-96.1%) in the second run. Specificity in healthy controls was 92.8% (95% CI 86.4-96.3%) in the first run and 96.4% (95% CI 91.1-98.6%) in the second run. Reproducibility was excellent with a kappa value of 0.81. CONCLUSIONS/SIGNIFICANCE: In this laboratory-based study, the Loopamp Trypanosoma brucei Detection Kit showed good diagnostic accuracy and excellent reproducibility. Further studies are needed to assess the feasibility of its routine use for diagnosis of HAT under field conditions.

High failure rates of melarsoprol for sleeping sickness, Democratic Republic of Congo.

A retrospective chart review of 4,925 human African trypanosomiasis patients treated with melarsoprol in 2001-2003 in Equateur Nord Province of the Democratic Republic of Congo showed a treatment failure rate of 19.5%. This rate increased over the 3 years. Relapse rates were highest in the central part of the province.

Trypanosomiasis control, Democratic Republic of Congo, 1993-2003.

In the Democratic Republic of Congo (DRC), human African trypanosomiasis (HAT) reached unprecedented levels in the 1990s. To assess recent trends and evaluate control efforts, we analyzed epidemiologic and financial data collected by all agencies involved in HAT control in DRC from 1993 to 2003. Funds allocated to control populations, as well as to the population screened, doubled from 1993 to 1997 and from 1998 to 2003. The number of cases detected decreased from 26,000 new cases per year in 1998 to 11,000 in 2003. Our analysis shows that HAT control in DRC is almost completely dependent on international aid and that sudden withdrawal of such aid in 1990 had a long-lasting effect. Since 1998, control efforts intensified because of renewed donor interest, including a public-private partnership, and this effort led to a major reduction in HAT incidence. To avoid reemergence of this disease, such efforts should be sustained.