Investigation Of Spotted Fever Group Rickettsiae In The Epidemic Region Of Severe Fever With Thrombocytopenia Syndrome

BACKGROUNDIn recent years,emerging tick-borne infectious diseases have been discovered increasingly,causing serious threat to human health.Due to the geographical distribution of ticks,it is easy to be bitten by ticks when entering the natural foci,which causes infectious disease.There are diverse agents which ticks carried,including several major species,such as Spotted Fever Group Rickettsiae(SFGR),virus,bacteria and protozoa.Co-infection may occur in humans either through the bite of 1 tick co-infected with multiple pathogens or bites of multiple ticks.Severe fever with thrombocytopenia syndrome(SFTS)caused by a novel-phlebovirus in the Bunyaviridae family which named SFTS virus(SFTSV),and was mostly transmitted by ticks.Since the discovery of SFTS patients in 2009,25 provinces in China have reported cases of the disease,within which Henan and Shandong are of high-risk.The disease usually manifests with fever,thrombocytopenia,leukopenia,and gastrointestinal symptoms.The clinical symptoms are non-specific and need to be differentially diagnosed with human granulocytic anaplasmosis and hemorrhagic fever with renal syndrome.The peaking of SFTS is from May to July of each year,showing an obvious seasonality,which is mainly related to habits of ticks.Haemaphysalis longicornis ticks are most abundant in hills and mountainous areas in the eastern of China.Most SFTS patients are the elderly farmers.The elderly engage in agricultural activities,especially tea picking activity,and are easily bitten by ticks.SFGR infections are emerging tick-borne infectious diseases,gradually attracted worldwide attention.The typical symptoms of the infectious of SFGR usually included fever,headache,eschar and rash following,while a few of them can cause severe complications such as multiple organ dysfunction syndrome.SFGR was frequently detected in multiple tick species,whereas human infection remains scarcely studied.Especially emerging infectious disease is easily ignored in developing countries.In East Asia,Rickettsia japonica caused human SFGR infections in Japan;R.conorii,R.japonica,and R.felis were discovered in South Korea,while more than ten species of SFGR was discovered and identified in the mainland of China,incuding R.sibirica,R.hulinii,R.raoultii,R.heilongjiangensis,R.monacensis,etc.The previous surveillance of tick-borne pathogens in a sentinel hospital in Xinyang,Henan Province,reported 56 cases infected with Candidatus Rickettsia tarasevichiae(CRT).This is the largest reported number of cases of CRT infection,which was found in the SFTS endemic area in central China.Among them,37 were coinfected with SFTSV and CRT.In recent years,many patients have been detected by molecular and serologic tests and showed negative results for SFTSV infection,which indicating that there would exist varied genotypes of SFGR in the disaster areas with active ticks.Therefore,the disaster areas with active ticks is the major surveillance area for SFGR.OBJECTIVEThe surveillance study was performed in two hospitals from 2015 to 2017: the People's Liberation Army 154 hospital in Xinyang City and the Affiliated Hospital of Taishan Medical University in Tai'an City.Through field epidemiological investigation,samples collection,and surveillance,the aims of our research is to find out the laboratory-confirmed cases of SFGR infection,to clarify the etiological characteristics,the clinical spectrum,and the abnormal changes in laboratory indicators of SFGR.In addition,a retrospective study of SFTS patients from 2013 to 2015 was conducted,to expose the co-infection issues of SFGR in the epidemic focus of SFTS.METHODPatients were recruited in the study according to the case enrolled criteria,and then their samples,clinical data and epidemiological information were collected.Polymerase chain reaction(PCR)followed by sequencing was performed to detect the SFGR infection and determine the species of the pathogen,and immunofiltration assay(IFA)was applied to detect antibody level of Ig G against SFGR.The clinical characteristics and the abnormal changes in laboratory indicators of the cases was analysed.In addition,a retrospective study of SFTS cases was conducted to analyze the epidemiological characteristics and clinical features of the co-infection of SFGR and SFTSV.All analyses were performed by SPSS software,version 17.0.All P values were 2-tailed and P<0.05 was considered statistically significant.RESULTSThe samples collected from the sentinel hospital were screened for the SFGR.We found that R.raoultii and R.japonica could cause human infections,and also report 3 patients infected with a novel SFGR,temporarily designated as Candidatus Rickettsia xinyangensis.The first chapter results : from 2015 to 2016,a total of 1016 subjects were screened in 2 sentinel hospitals in the study,through polymerase chain reaction(PCR),followed by sequencing,and confirmed serologically.As a result,fifteen subjects were determined to have R.raoultii infection,including 11 with mild to moderate illness,and 4 with severe illness.Common nonspecific manifestations in the 15 patients with mild to moderate or severe illness included fever(100%),malaise(100%),myalgia(66.7%),lymphadenopathy(60%),and nausea(53.3%).Only 6.7% of them had rash,and 13.3% had eschar.Scalp eschar and neck lymphadenopathy after a tick bite syndrome was only seen in 2 patients.Of the 4 patients with severe complications,3 developed pulmonary edema,and 1 developed clouding of consciousness and lethargy.While frequent abnormalities of laboratory testing included leukopenia,thrombocytopenia,lymphopenia,neutropenia,hypoproteinemia,and elevated levels of total bilirubin,hepatic aminotransferases,lactate dehydrogenase,and creatine kinase.All the 15 patients recovered without sequelae after receiving doxycycline treatment.The second chapter results: from 2015 to 2017,a total of 1803 febrile patients were enrolled in the People's Liberation Army 154 Hospital in Xinyang,Henan Province.We determine 20 patients with R.japonica infection by molecular and serological methods,including 6 patients were found to be coinfected with SFTSV.The major clinical manifestations of single infection were fever,asthenia,myalgia,rash,and anorexia.The laboratory abnormalities included thrombocytopenia and elevated hepatic aminotransferase concentrations.Three patients had mild multiple organ dysfunction and no death occurred.The third chapter results: from March through November 2015,a total of 221 febrile patients were recruited at the People's Liberation Army 154 Hospital in Xinyang,Henan Province.SFGR gene fragment amplified by nested PCR was positive and sequenced.Three patients were found to be infected with a novel genotype of SFGR,according to the gene sequence-based criteria for identification a new Rickettsia,a Candidatus status could be assigned,and we temporarily named it Candidatus Rickettsia xinyangensis.Serum samples were tested by IFA for Ig G antibodies against R.rickettsii.Two patients showed seroconversion and one showed a 4-fold titer increase of Ig G antibody.Two of the 3 patients had reported history of tick-bite and 1 reported animal contact.All 3 patients manifested fever,asthenia,and anorexia.Two patients had eschar,1had lymphadenopathy,and none had rash.None of the 3 patients developed any severe complications(i.e.hemorrhagic sings or neurologic symptoms).The fourth chapter results: based on the three types of SFGR detected during surveillance study in the SFTS epidemic between 2015 and 2017,by performing a retrospective investigation in the SFTS-endemic region,we identified coinfection of SFGR in about 8.5% of the 823 SFTS patients,and observed higher frequency of fatal outcome and delayed recuperation in the coinfected patients.The commonly seen clinical symptoms,such as influenza like illness and gastrointestinal manifestations,although characterizing infection of each pathogen,appeared not synergized in coinfected patients.In contrast,the hemorrhagic signs,especially gastrointestinal hemorrhages,were apparently exacerbated in coinfected patients.The slow recovery of platelet counts and the prolonged prothrombin time in co-infections.These findings highlight the importance of considering differential diagnosis of SFGR in SFTS-endemic regions.CONCLUSIONThis study found that SFGR concomitantly exist in the infectious focus of SFTS.Rickettsia raoultii infections were identified in two regions that host different ecosystems,but the predominant tick species were Haemaphysalis Iongicornis which have been with R.raoultii infection.The tick have a wide geographic distribution in China mainland,possibly indicating a wider distribution of R.raoultii in endemic regions.This is the first time that R.raoultii infections has been found in the central and eastern regions of China,and the number of cases has been found to be the highest.While cases infected with R.japonica were first reported in the central regions of China.Human infection with these two SFGR species has a wide clinical spectrum that ranged from subclinical infection to severe illness,but no deaths.The treatment of doxycycline was effective,while It is presumed that they might be largely misdiagnosed or underdiagnosed as the non-specificity of clinical symptoms.In addition,a novel SFGR was detected in sentinel hospitals by molecular biology and serological experiments,which was temporarily proposed the name as Candidatus Rickettsia xinyangensis due to the failure to isolate and cultivate.But the causal relationship with clinical disease could be derived by the molecular biology and serological evidence,indcating it is of significance for public health.But only 3 patients of Candidatus Rickettsia xinyangensis infection have been reported,the clinical characteristics of the infections needed to be assessed in further investigation.In the current study,by performing a retrospective investigation in the SFTS-endemic region,we identified coinfection of SFGR in about 8.5% of the 823 SFTS patients,and observed higher frequency of fatal outcome and delayed recuperation in the coinfected patients.These findings highlight the importance of considering differential diagnosis of SFGR in SFTS-endemic regions.Doxycycline is currently recommended therapeutic regimen for Rickettsia infection,which might be administered in cases of recognizing SFGR infection in SFTS patients.From the public health perspective,recognition of the SFTSV-SFGR coinfection should be made with intensified efforts in those regions where H.longicornis are predominant and carry both SFTSV and SFRG.SIGNIFICANCE AND INNOVATIONBecause emerging SFGR infection was associated with a wide clinical spectrum ranging from asymptomatic infection to mild illness and even severe complications,It is easy to be largely and frequently misdiagnosed or underdiagnosed.This study further confirms the wider distribution of SFGR and SFTFV-SFGR co-infections in the epidemic region of Severe Fever with Thrombocytopenia Syndrome,and physicians need to be aware of the high potential and clinical complexity of SFGR infection,as well as the high incidence of co-infections,to ensure appropriate testing and treatment in endemic regions.