Evaluation of ITS1 rDNA primers for the detection and identification of African trypanosomes in mammalian hosts and tsetse flies

• Published in: Acta tropica 2024-10, Vol.258, p.107331, Article 107331
• Main Authors: Ofon, Elvis Amih, Metiadjoue, Mboo Cabrole Christelle, Kante, Sartrien Tagueu, Magang, Eugenie Melaine Kemta, Mewamba, Estelle Mezajou, Kamga, Rolin Mitterran Ndefo, Fogue, Soubgwi Pythagore, Simo, Gustave
• Format: Article
• Language: English
• Subjects: African trypanosomiasis; Internal transcribed-spacer 1 (ITS1); ITS1-A; Trypanosome species
• ISSN: 0001-706X; 1873-6254; 1873-6254
• DOI: 10.1016/j.actatropica.2024.107331

•Filling the gap of ITS1-primers comparatively sensitivity and concordance assessment.•Ten primers targeting ITS1 of trypanosomes were assessed using single-round and nested-PCR (eight) with archived DNA samples.•Little agreement levels (k = 0.05–0.52) were noticed between ITS1-primers used to detect african trypanosome species.•Fair sensitivity (11.9 %−50.5 %) of primers in detecting trypanosomes in samples of different epidemiological settings.•Primers targeting ITS1-A sequence of trypanosomes recorded the highest sensitivity (50.5 %) and concordance with ITS1-C (k = 0.52), thus most appropriate for trypanosomes detection. Although several primers targeted to the internal transcribed-spacer 1 (ITS1) of the ribosomal DNA (rDNA) have been designed to improve the detection of African trypanosomes, no study tried to compare their agreement level and ability to amplify different trypanosome species in tsetse flies and mammals in various epidemiological settings. This study was designed to fill this gap, by targeting tsetse-infested areas of Cameroon. For this, archived DNA samples reporting at-least one trypanosome species with species-specific PCR primers were reviewed. Ten sets of primers targeting different ITS1 rDNA sequences of trypanosomes were selected for assessment using single-round and nested-PCR method. Amplification rates (sensitivity) and agreement level of different ITS1 assays were compared using Cohen's-Kappa and McNemar's x2 statistic. Little agreement level (k = 0.05–0.52) were observed between different ITS1-primers PCRs detection of African trypanosome species despite significant (X2=54.3, p = 0.0001) high amplification rate 91.6 % (339/370). This sensitivity varied from quite low for T. simiae (11.9 %) and T. vivax (27.3 %) to fairly good for T. congolence (51.9 %), Trypanozoon (32.4 %) and T. theileri (40.3 %). Primers set targeting ITS1-A sequence of trypanosome species recorded the highest sensitivity (50.5 %) with fairly good agreement compared to 39.2 % for ITS1-C (k = 0.52), 32.4 % for ITS1-R (k = 0.47), 29.7 % for ITS1-N (k = 0.48) and 23.0 % for ITS1-KIN (k = 0.43) respectively. This study revealed a diversity in the sensitivity of different trypanosome species with different sets of ITS-primers enhancing the need to use the same sets of primers in different bio-ecological settings. The use of nested-PCR instead of single-round PCR enabled improvement of trypanosome infections detection in both tsetse and mammals. Among the sets