Rapid detection of Babesia motasi responsible for human babesiosis by cross-priming amplification combined with a vertical flow

Jinming Wang,Jianxun Luo,Youquan Li,Hong Yin,Junlong Liu,Shandian Gao,Shangdi Zhang,Guiquan Guan,Xin He,Aihong Liu,Guangyuan Liu

doi:10.1186/s13071-020-04246-4

Abstract

BackgroundBabesia motasi is known as an etiological agent of human and ovine babesiosis. Diagnosis of babesiosis is traditionally performed by microscopy, examining Giemsa-stained thin peripheral blood smears. Rapid detection and accurate identification of species are desirable for clinical care and epidemiological studies.MethodsAn easy to operate molecular method, which requires less capital equipment and incorporates cross-priming amplification combined with a vertical flow (CPA-VF) visualization strip for rapid detection and identification of B. motasi.ResultsThe CPA-VF targets the 18S rRNA gene and has a detection limit of 50 fg per reaction; no cross reaction was observed with other piroplasms infective to sheep or Babesia infective to humans. CPA-VF and real-time (RT)-PCR had sensitivities of 95.2% (95% confidence interval, CI 78.1–99.4%) and 90.5% (95% CI 72–97.6%) and specificities of 95.8 (95% CI 80.5–99.5%) and 97.9 (95% CI 83.5–99.9%), respectively, versus microscopy and nested (n) PCR combined with gene sequencing. The clinical performance of the CPA-VF assay was evaluated with field blood samples from sheep (n = 340) in Jintai county, Gansu Province, and clinical specimens (n = 492) obtained from patients bitten by ticks.ConclusionsOur results indicate that the CPA-VF is a rapid, accurate, nearly instrument-free molecular diagnostic approach for identification of B. motasi. Therefore, it could be an alternative technique for epidemiological investigations and diagnoses of ovine and/or human babesiosis caused by B. motasi, especially in resource-limited regions.

Highlights

Babesia motasi is known as an etiological agent of human and ovine babesiosis
Optimization of the Cross-priming amplification (CPA) primers, reaction temperature and time Sequence alignment of the 18S rRNA genes of piroplasms infective to sheep and goats available in NCBI showed that two regions are conserved intra-species and variable among species
Changes in amplification temperature had a slight impact on the brightness of the bands, indicating that incubation temperature is significant for the CPA reaction

Summary

Introduction

Babesia motasi is known as an etiological agent of human and ovine babesiosis. Diagnosis of babesiosis is traditionally performed by microscopy, examining Giemsa-stained thin peripheral blood smears. Babesiosis, caused by protozoan pathogens of the genus Babesia infective to humans, domestic and wild animals, is one of the emerging and re-emerging tick-borne disease in the tropical and subtropical regions of the world [1]. It causes a wide spectrum of clinical signs which range from mild fever to serve anemia, haemoglobinuria and even death. Two newly emerging Babesia species, named as B. motasi and B. crassa, which were previously reported as causative agents of ovine babesiaosis, have been sporadically reported in cases of human babesiosis in Asia [6,7,8,9,10]. A 70-year-old man in Korea was diagnosed as infected with B. motasi [7]

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