Discovery Of Novel Babesia Infections And Identification Of Their Causative Agents

Backgrounds Babesiosis, caused by intraerythrocytic parasites of Babesia, belongs to Apicomplexa and is mainly transmitted through tick bite. Babesia was named after Victor Babes, the Hungarian pathologist and microbiologist, who firstly identified the intraerythrocytic microorganisms as the cause of febrile hemoglobinuria in cattle in 1888. Since the first human infection of Babesia was found in 1957, Babesia cases had been continuously reported around world. More than 100 Babesia species have been identified, which could be infective to wild and domestic animals, but only a few of them have been reported to be pathogenic to human being. Based on the morphological characteristics and phylogenetic analysis, Babesia pathogenic to human can be divided into four clades. The first clade is Babesia microti, which is the predominant Babesia species in the USA, being the causing agent for almost all human cases. The second one is Babesia duncani, which contribute to sporadic cases in the USA. The third is endemic in Europe, which includes Babesia divergens and Babesia venatorum. In 2007, Korean researchers identified a new Babesia species in a patient and nominated it as Babesia sp. “KO1”, which is the fourth.Before this study, several Babesia species have been discovered in mainland China, only a few of which were confirmed to be infective to human. By far, there were only sporadic cases of human infection ever reported in China. A total of 12 cases of B.microti in China, 8 of which were diagnosed in Yunnan province and the rest 2 were identified in Taiwan and Zhejiang province respectively. In 2011, 2 B.divergens infections were discovered in Shandong province and a child infected with B.venatorum was identified in 2014. There were another undescribed 14 Babesia cases reported in China, including Zhejiang, Shanxi and Yunnan province. Among these cases, co-infections with other pathogens and severe cases can always be found and health burden imposed by Babesia had increased strikingly. Its public significance is widely appreciated.All human babesiosis in mainland China were passively found and neglected by most clinicians, which often lead to misdiagnosis. However, predominant Babesia species and the distribution varied significantly, where Babesia infections have been found in ticks and natural hosts. Are they infective to human? Are there any other pathogenic Babesia species? The predominant Babesia genotype and features of natural foci in endemic areas still remain as an issue. A nation-wide investigation into Babesia is hardly applicable; therefore we planned to initiate the surveillance in Mudanjiang city. Mudanjiang, locating in northeastern China, a highly forestry area, with high diversity and complexity of tick-borne infectious disease, is the perfect site to initiate investigation into Babesia. Most of the local people are engaged in forestry works and agriculture, which imposes inevitable occupational exposure to tick bite, therefore local people should be critically monitored for Babesia infection.Objectives The objectives of this study were to detect Babesia cases, identify the predominant Babesia species and comprehend the pathogenicity and clinical characteristics. Additional aims were to understand the Babesia prevalence in ticks and natural hosts, and based on which health risks in local people imposed by Babesia can be estimated.Methods A sentinel hospital was setup in Mudanjiang Forestry Central Hospital, in which a hospital-based survey of Babesia infections in human with tick bite of last 2 months was conducted. A questionnaire was designed to obtain demographic and epidemiological information, while the clinical features were retrieved from medical records. The laboratory tests were based on PCR screening. Additionally, microscopic observation of blood smears and SCID(Severe Combined Immunodeficiency) mice inoculation were also applied to confirm the diagnosis. Fluorescence in situ hybridization(Fish) assays were conducted to support the diagnosis. Babesia species were identified based on 18 S r RNA gene sequence comparison and phylogenetic analysis. For those positives, we defined them into diagnosis of confirmed cases and probable cases, according to the diagnostic criteria designed by CDC of the USA. Meanwhile, we analyzed cytokines of cases, to validate the correlation between Babesia infection and cytokines. Host-seeking ticks and specimens of domestic animals were collected, and Babesia species were identified through 18 S r RNA gene comparison and phylogenetic analysis. Based on the molecular findings in local ticks and animals, we estimated the health risks imposed by Babesia in local ticks.Findings Via a five-year study, based on the sentinel hospital, a total of 3454 blood specimens were sampled and firstly identified northeastern China as the endemic area of two Babesia species, including B.venatorum and a novel Babesia. From 2011 to 2014, the first phase of the study, we found 48 B.venatorum cases in 2912 participants, which was the largest cluster of B.venatorum ever reported. According to the comparison of short fragment 18 S r RNA gene, we discovered the sequences from all 48 cases were identical to each other and also to the European B.venatorum sequence, indicating all 48 cases were B.venatorum infections. Subsequently, nearly all length of B.venatorum 18 S r RNA gene were obtained in four cases, which showed only 2 base pair difference from the European one, showing a striking similarity in the isolates collected in very different ecosystems. Besides the PCR assays, positive results in Microscopy, Fish assay and Animal inoculation could also serve as the conviction of B.venatorum infection. 48 cases were diagnosed as 32 confirmed cases and 16 probable cases, according to the Babesia diagnostic criteria developed by CDC of the USA. Among 32 confirmed cases, 21(66%) presented with a fever, 13(41%) with a headache, 12(38%) with myalgia or arthralgia, and three(9%) with chills. 14(44%) patients had fatigue, eight(25%) had dizziness, and eight(25%) had hypersomnia. Six(19%) patients had an erythematous non-pruritic rash around the tick-bite site and two(6%) had lymphadenopathy. Seven(22%) and four(13%) patients had anemia and thrombocytopenia, respectively, and seven(50%) of 14 patients with confirmed infection had increased hepatic transaminase concentrations. In the confirmed cases, concentrations of intercellular adhesion molecule 3(p<0.001), P-selectin(p<0.001), and platelet endothelial cell adhesion molecule 1(p<0.001) were significantly reduced, whereas tumour necrosis factor α(p<0.001) and vascular cell adhesion molecule 1(p<0.001) were signifi cantly increased. In probable cases, intercellular adhesion molecule 3(p<0.01) and platelet endothelial cell adhesion molecule 1(p<0.001) were significantly reduced, and vascular cell adhesion molecule 1(p<0.001) was significantly increased.Simultaneously, Ixodes persulcatus(n=2538) and Haemaphysalis concinna(n=553)ticks were collected from 2 sampling sites near Mudanjiang city, where 6 different Babesia species were revealed. The overall prevalence in I. persulcatus was 1.77 %, with 5 Babesia species including B.bigemina(n=7), B.divergens(n=3), B.microti(n=10), B.venatorum(n=17) and 1 novel Babesia, provisionally nominated as “Babesia sp. MDJ” which was most similar to B.crassa from sheep in Iran. Twelve(2.17%) H.concinna contained DNA of B.bigemina(n=1) and 2 genetic variants of Babesia. Besides “Babesia sp. MDJ” and B.bigemina, the other one presented by “Babesia sp. HLJ” was closely related to Babesia sp. MA#361-1 from Raccoons in Japan with 0.2% diversity.Based on the findings in ticks, we screened 542 samples collected in the sentinel hospital in 2015. Subsequently, 49 individuals infected with the novel Babesia were identified. Both phylogenetic analysis and sequence comparison is convincing that the Babesia identified in patients was a novel Babesia species, which was identical to the “Babesia sp. MDJ” found in ticks and confirmed as large Babesia through microscopic observation. According to the Babesia diagnostic criteria mentioned above, among 49 cases, 19 were confirmed cases while the rest were probable cases. The age of 49 cases ranged from 4 years to 72 years with a median of 51 years, and 37 were female. All had a recent tick-bite, and none had either received a blood transfusion or splenectomy. In 19 confirmed cases, dizziness(57.9%), headache(52.6%) and nausea or vomiting(52.6%) were the most common clinical manifestation. In addition, 7 patients(36.8%) had fever when they saw the doctor. Besides, 2(10.5%) reported a myalgia and 2(10.5%) reported fatigue. There were 2(10.5%) confirmed cases and 4(13.3%) probable cases manifesting lymphadenopathy. Alanine aminotransferase or aspartate aminotransferase, or both, increased in 6(14.3%) of 42 patients with serum samples available for hepatic function tests and simultaneously total bilirubin was also tested and 11 showed elevated level. Of the 11 patients admitted to hospital, 5 had increases in C-reactive protein. Then in a survey of Babesia in domestic animals, 5 out of 461 sheep were found infected with “Babesia sp. MDJ”, while there was no positive in goat, cattle and deer, indicating that sheep is the natural hosts for “Babesia sp. MDJ”. Combining with the findings in ticks, northeastern China is confirmed as natural foci of “Babesia sp. MDJ”.Conclusion We firstly identified the largest cluster of B.venatorum cases in the world and discovered a novel large Babesia pathogenic to human being, identifying northeastern China is the endemic area of the two Babesia species. Clinical features of cases revealed non-specific symptoms and immune-competent people were also at stake. Despite B.venatorum and “Babesia sp. MDJ”, another four Babeisa species were identified in local ticks, revealing the diversity and complexity of Babesia in ticks. Via pointed survey “Babesia sp. MDJ” in domestic animals, sheep were confirmed as one of the natural hosts for “Babesia sp. MDJ” and we firstly discovered northeastern China as the natural foci of the novel Babesia.Significance and innovation We firstly described the largest ever cluster of B.venatorum out of Europe and discovered a novel large Babesia(“Babesia sp. MDJ”) infective to human being, broadening the horizon into babesiosis and confirming northeastern China is the endemic area for these two Babesia species. And firstly,two Babesia species were approved to be infective to immune-competent individuals. Because of the non-specific symptoms the Babesia cases manifested, misdiagnosis is inevitable. Based on this study and previous research, confirmed the babesiosis are more common than ever thought. Along with development and popularization of babesial detection, those previously thought to be non-pathogenic to human could be possibly proved to be infective to human being, which will raise the awareness of babesiosis in clinicians, public health staffs and especially in those with occupational exposure to tick bite.Babesia is a potential risk for not only immune-compromised people but also immune-competent individuals. Though symptoms of Babesia infection in immune-competent individuals were not severe, the epidemiological significance still cannot be ignored. Firstly, subclinical Babesia cases or those with moderate symptoms can deteriorate into severe Babesia illness when have undergone splenectomy, received immune-suppressing drugs and fell in cancer. Secondly, subclinical Babesia cases or those with moderate symptoms can ignorantly transfer Babesia to blood recipients through blood-transfusion. Because recipients requiring blood-transfusion have already suffered from underlying disease or suppressing immune status, therefore blood-transfusion associated Babesia infection are commonly more critical. This study set the alarm for safety of blood-transfusion and potential risks in endemic areas.Briefly speaking, this study deepened recognitions into Babesia in different aspects, including geographic features, clinical and epidemiological characteristics. A systematic survey into Babesia in local ticks and domestic animals were conducted and firstly confirmed babesial epidemic area. This study significantly raises awareness of this emerging intraerythrocytic protozoan in the clinicians in tick-borne epidemic regions, especially where Lyme disease and tick-borne encephalitis exist, reducing misdiagnosis, fail to diagnose, and facilitate the drug-usage. Moreover, the safety of blood-transfusion and the related potential risks were revealed in this study, which provides new research directions into focused tick-borne infectious diseases, and raising the recognition of health burden imposed by Babesia.