Trypanosome spliced leader RNA for diagnosis of acoziborole treatment outcome in gambiense human African trypanosomiasis: A longitudinal follow-up study.

BACKGROUND: Detection of spliced leader (SL)-RNA allows sensitive diagnosis of gambiense human African trypanosomiasis (HAT). We investigated its diagnostic performance for treatment outcome assessment. METHODS: Blood and cerebrospinal fluid (CSF) from a consecutive series of 97 HAT patients, originating from the Democratic Republic of the Congo, were prospectively collected before treatment with acoziborole, and during 18 months of longitudinal follow-up after treatment. For treatment outcome assessment, SL-RNA detection was compared with microscopic trypanosome detection and CSF white blood cell count. The trial was registered under NCT03112655 in clinicaltrials.gov. FINDINGS: Before treatment, respectively 94.9% (92/97; CI 88.5-97.8%) and 67.7% (65/96; CI 57.8-76.2%) HAT patients were SL-RNA positive in blood or CSF. During follow-up, one patient relapsed with trypanosomes observed at 18 months, and was SL-RNA positive in blood and CSF at 12 months, and CSF positive at 18 months. Among cured patients, one individual tested SL-RNA positive in blood at month 12 (Specificity 98.9%; 90/91; CI 94.0-99.8%) and 18 (Specificity 98.9%; 88/89; CI 93.9-99.8%). INTERPRETATION: SL-RNA detection for HAT treatment outcome assessment shows ≥98.9% specificity in blood and 100% in CSF, and may detect relapses without lumbar puncture. FUNDING: The DiTECT-HAT project is part of the EDCTP2 programme, supported by Horizon 2020, the European Union Funding for Research and Innovation (grant number DRIA-2014-306-DiTECT-HAT).

Single nucleotide polymorphisms and copy-number variations in the Trypanosoma brucei repeat (TBR) sequence can be used to enhance amplification and genotyping of Trypanozoon strains.

The Trypanosoma brucei repeat (TBR) is a tandem repeat sequence present on the Trypanozoon minichromosomes. Here, we report that the TBR sequence is not as homogenous as previously believed. BLAST analysis of the available T. brucei genomes reveals various TBR sequences of 177 bp and 176 bp in length, which can be sorted into two TBR groups based on a few key single nucleotide polymorphisms. Conventional and quantitative PCR with primers matched to consensus sequences that target either TBR group show substantial copy-number variations in the TBR repertoire within a collection of 77 Trypanozoon strains. We developed the qTBR, a novel PCR consisting of three primers and two probes, to simultaneously amplify target sequences from each of the two TBR groups into one single qPCR reaction. This dual probe setup offers increased analytical sensitivity for the molecular detection of all Trypanozoon taxa, in particular for T.b. gambiense and T. evansi, when compared to existing TBR PCRs. By combining the qTBR with 18S rDNA amplification as an internal standard, the relative copy-number of each TBR target sequence can be calculated and plotted, allowing for further classification of strains into TBR genotypes associated with East, West or Central Africa. Thus, the qTBR takes advantage of the single-nucleotide polymorphisms and copy number variations in the TBR sequences to enhance amplification and genotyping of all Trypanozoon strains, making it a promising tool for prevalence studies of African trypanosomiasis in both humans and animals.

Trypanosome SL-RNA detection in blood and cerebrospinal fluid to demonstrate active gambiense human African trypanosomiasis infection.

BACKGROUND: Spliced Leader (SL) trypanosome RNA is detectable only in the presence of live trypanosomes, is abundant and the Trypanozoon subgenus has a unique sequence. As previously shown in blood from Guinean human African trypanosomiasis (HAT) patients, SL-RNA is an accurate target for diagnosis. Detection of SL-RNA in the cerebrospinal fluid (CSF) has never been attempted. In a large group of Congolese gambiense HAT patients, the present study aims i) to confirm the sensitivity of SL-RNA detection in the blood and; ii) to assess the diagnostic performance of SL-RNA compared to trypanosome detection in CSF. METHODOLOGY/PRINCIPAL FINDINGS: Blood and CSF from 97 confirmed gambiense HAT patients from the Democratic Republic of Congo were collected using PAXgene blood RNA Tubes. Before RNA extraction, specimens were supplemented with internal extraction control RNA to monitor the extraction, which was performed with a PAXgene Blood RNA Kit. SL-RNA qPCR was carried out with and without reverse transcriptase to monitor DNA contamination. In blood, 92/97 (94.8%) HAT patients tested SL-RNA positive, which was significantly more than combined trypanosome detection in lymph and blood (78/97 positive, 80.4%, p = 0.001). Of 96 CSF RNA specimens, 65 (67.7%) were SL-RNA positive, but there was no significant difference between sensitivity of SL-RNA and trypanosome detection in CSF. The contribution of DNA to the Cq values was negligible. In CSF with normal cell counts, a fraction of SL-RNA might have been lost during extraction as indicated by higher internal extraction control Cq values. CONCLUSIONS/SIGNIFICANCE: Detection of SL-RNA in blood and CSF allows sensitive demonstration of active gambiense HAT infection, even if trypanosomes remain undetectable in blood or lymph. As this condition often occurs in treatment failures, SL-RNA detection in blood and CSF for early detection of relapses after treatment deserves further investigation. TRIAL REGISTRATION: This study was an integral part of the diagnostic trial "New Diagnostic Tools for Elimination of Sleeping Sickness and Clinical Trials: Early tests of Cure" (DiTECT-HAT-WP4, ClinicalTrials.gov Identifier: NCT03112655).