Babesiosis, caused by bites from ticks infected with the genus Babesia or transfusion of blood products, is one of the most prevalent protozoan disease in human and animals worldwide [26]. The first case of babesiosis was described in Rameses in 1888, when a Hungarian pathologist investigated the febrile hemoglobinuria in cattle [27]. It was caused by infection with B. bovis. Shortly afterwards, an organism that was similar to B. bovis was also described in cattle in Texas. Currently, more than 100 Babesia spp. have been identified in humans and other animals across the world [28]. However, only few Babesia spp. have been reported to be responsible for human babesiosis, including B. microti, B. divergens, B. duncani, B. venatorum, B. crass, and Babesia sp. XXB/HangZhou [29, 30, 31, 32, 33]. As a causative agent responsible for human babesiosis, the first case caused by B. motasi-like was reported in Korea in 2005 [31]. Recently, infected with B. motasi was diagnosed in a 70-year-old man in Korea [34]. Moreover, it is the most prevalent pathogen among vector ticks and host animals on the basis of serological and molecular epidemiological investigations conducted in China[24]. However, till now there was no report of human babesiosis caused by B. motasi in China.
As clinical signs and symptoms of human babesiosis are viral-like and often overlap with those of several other illnesses, it can be challenge to discriminate human babesiosis. However, the increasing cases of human babesiosis has led to an urgent need to identify Babesia species. The rapid and accurate diagnosis of Babesia infection can facilitate clinical managements and provide probable treatment. Several molecular methods have been developed with high specificity and sensitivity for detection of the B. motasi infection, including nPCR, RT-PCR, RLB [14, 15, 25, 35]. However, those approaches have intrinsic disadvantages, such as time-consuming, and requiring expensive thermal cycle instruments and/or manipulation of amplified products, which have limited their application in field-site and low-resource laboratories. When compared with PCR, RT-PCR and RLB, isothermal amplification technique is a powerful tool to overcome these drawbacks. Furthermore, CPA-VF is a novel isothermal amplification technique developed in recent years and the results can be visualized by naked eye, needing no additional instruments. To provide an effective diagnostic tool, a CPA method targeting the 18S rRNA sequences of B. motasi was successfully developed for rapid detecting and discriminating B. motasi infection. The CPA assay could detect four strains of B. motasi, B. motasi Lintan, B. motasi Tianzhu, B. motasi Hebei, and B. motasi Ningxian. In addition, cross reaction was not observed with piroplasm infective to sheep (Babesia sp. Xinjiang, T. uilenbergi, T. luwenshuni, T. ovis, A. ovis) and humans (B. duncani, B. divergens, B. microti, and B. crassa). These results indicated that this CPA assay has good specificity. Further studies should be performed to investigate potential cross reaction with other pathogens infective to humans using the CPA-VF approach developed herein.
The CPA assay was high sensitivity and could detect as few as 50 fg of genomic DNA from B. motasi per reaction, which was equal to approximately 50 µl of 0.000005% parasitic erythrocytes. The process of CPA reaction does not require expensive equipment and can be performed in a constant temperature block to maintain reaction temperature of 61 °C for 60 min. Furthermore, generated products by the CPA amplification can be detected using a VF strip, which only need 2–5 min and is visible to the naked eye. Thus, the CPA assays are suitable for rapid, simple, and sensitive detection of B. motasi infection in limited-resource setting in endemic regions.
To access its suitability for clinical use, we conducted the first diagnostic study in clinical specimens and host animals with CPA-VF, comparing it with microscopy, RT-PCR, and nPCR combined with gene sequencing. The results from studies of a positive and negative panel revealed that CPA-VF has better sensitivity than that of RT-PCR. Because of its sensitivity, CPA-VF could be useful for the preliminary screening of low-level parasitemia. Furthermore, 340 field blood samples from animals and 492 clinical specimens from patients were used to evaluate analytic performance regarding B. motasi identification. Our results demonstrate that excellent sensitivity was observed in CPA-VF approach in comparison to that of RT-PCR. False positives that were needed to be confirmed by microscopy should be noted with CPA-VF assay. To avoid a risk of contamination, genomic DNA extraction, CPA amplification and its products analysis with VF strips were performed in separated rooms, so contamination was not observed in our study. Three samples determined to be negative of piroplsam infection by nPCR were shown to present B. motasi infections by CPA-VF analysis. This could be explained by false positive or more sensitivity of CPA analysis, compared with nPCR. A proposed algorithm for B. motasi identification in patients suspected babesiosis, caused by B. motasi, is presented in Fig. 5. However, further studies are needed to explore this issue. One limitation of this study was the small number of positive specimens, which were used to evaluate clinical performance. Further studies are needed regarding the implementation of this approach into clinical practice.