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

Abstract

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 JXT (EO) 19 AUG 2021: 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).

A review on the diagnosis of animal trypanosomoses

“…Spliced Leader trypanosome RNA (SL-RNA) detection was recently developed for the diagnosis of living T. b. gambiense in humans, but this technique has not been evaluated in animals so far [ 151 ].…”

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

“…The detailed procedures for HAT diagnosis have been described previously. 21 Briefly, in all HAT patients, the parasite presence was confirmed by direct lymph examination, by blood examination using the mini-anion exchange centrifugation technique on buffy coat (mAECT-BC) or the mini-haematocrit centrifugation technique (mHCT), or by observation of trypanosomes in the CSF, during the WBC count or after modified single centrifugation. A patient was classified as early or intermediate stage with absence of trypanosomes in the CSF and a WBC count in the CSF of respectively ≤5 or 6–20 WBC/μl.…”

“… 20 In addition, sensitivity of SL-RNA detection in blood outperforms all traditional techniques for diagnosis of HAT, while in CSF, SL-RNA detection compares in sensitivity to the single modified CSF centrifugation. 21 The detection of SL-RNA in blood could thus offer potential advantages for the non-invasive and earlier detection of treatment failure.…”

“…We recently performed SL-RNA detection in the blood and CSF of HAT patients in the Democratic Republic of the Congo (DRC). 21 Here, we evaluate the diagnostic performance of SL-RNA detection for gambiense HAT treatment outcome assessment in blood and CSF collected at different time points during the 18 months follow-up of these patients, which were treated with a single dose of acoziborole. 22…”

Gambiense human African trypanosomiasis: the bumpy road to elimination

“…For a posteriori diagnostic confirmation too, there have been some promising innovations. A highly specific inhibition ELISA, as well as a molecular test, are currently being validated for use in regional hospitals [ 53 , 54 ▪ ].…”