A Novel Cell-Based PCR Method for Accurate Characterization of African Trypanosomes.

Abstract

African trypanosomes exhibit significant genetic diversity but share similar morphological characteristics, making effective surveillance and diagnosis challenging with conventional microscopy and other detection methods. While polymerase chain reaction (PCR) offers enhanced specificity and sensitivity, it requires DNA extraction, making it time-consuming and costly, especially for resource-limited regions such as ours (Africa). Additionally, amplification using DNA from the host's whole blood often fails due to the presence of polymerase inhibitors or low parasite DNA, particularly during chronic infections. This study introduces a novel PCR method that overcomes these challenges by using purified trypanosome cells as templates, bypassing the need for DNA extraction. Trypanosoma brucei brucei cells were isolated from mice blood using DEAE-cellulose anion-exchange chromatography and directly used in PCR amplification of the ITS-1 ribosomal RNA gene. Successful amplification was achieved with as few as 1.25 × 10⁵ isolated trypanosome cells, while gDNA extracted from parasitized blood failed to amplify. This cost-effective and highly sensitive method improves species differentiation and enhances diagnostic capabilities, especially in cases of low parasitemia. The approach represents a significant advancement in trypanosome diagnosis, offering a faster, affordable, and reliable alternative to traditional methods. These findings support improved epidemiological surveillance and may contribute to the World Health Organization's goal of eliminating African trypanosomiasis, a disease that continues to present major economic and public health challenges in Africa. A graphical abstract of this study is presented in Figure 1. © 2026 Wiley Periodicals LLC. Basic Protocol 1: Isolation of Trypanosoma brucei brucei from parasitized blood Basic Protocol 2: PCR characterization of isolated T. brucei brucei.