To Study A Cluster Of Severe Fever With Thrombocytopenia Syndrome(SFTS) And The Relationship Between SFTSV Load And Patient’s Condition

BackgroundTo study a cluster of six cases of SFTSV infection and a correlational study of the relationship between SFTSV load and patient’s condition Fever with Thrombocytopenia Syndrome(SFTS)is a new viral infectious disease first found in China in 2009,with a fatality rate reach up to 30%.Severe fever with thrombocytopenia syndrome(SFTS)is considered as a result of Severe feverwith thrombocytopenia syndrome virus(SFTSV)infection.The main clinical manifestations are fever,thrombocytopenia,leukopenia and gastrointestinal symptoms etc.Multiple organ damage could be the cause of death of a few critically ill patients.Currently 5352 cases are reported in 23 provinces all over our country,among all these cases,a total of 2 750 cases of laboratory diagnosis cases werereported in 16 provinces and 221 counties,cases are highly prevalent in Henan,Hubei,Shandong etc.Similar cases were also reported in Japan,South Korea and the United States.SFTSV could be transmitted through tick bite.SFTSV may also transmit from person to person.Clusters caused by the transmission among humans has been described from 2007 to 2015 in the epidemic provinces in China,including Henan,Jiangsu,Liaoning,Shandong,Anhui,and Hubei,as well as in South Korea.The major risk factor was direct blood exposure without personal protection.Other possible contacts such as urine,feces,and respiratory secretions were evaluated to be not significantly associated.In recent years,asymptomatic infections have been noted in a few cluster reports of SFTSV infection,suggesting that SFTSV infection could be asymptomatic via person-to-person transmission,which,however,needs additional attentions.SFTSV can affect multiple organ such as liver,lung,kidney etc.SFTS is a disease with deverse clinical manifestation and multiple organ involvement.SFTSV are commonly started with a sudden fever onset,characterized by loss of appetite,nausea,vomiting,abdominal pain,diarrhea,thrombocytopenia,leukopenia etc.SFTS can also display biochemical and other laboratory abnormalities,such as serum ALT,AST,LDH,CK and CK-MB over-expression,critically ill patients can display multiple organ failure and DIC.Common central nervous system symptoms are indifference,drowsiness,tremors,convulsions,coma is often appeared in the terminal stage.Bleeding manifestations including petechiae,ecchymoses,injection site hematoma,vaginal bleeding,hematemesis,hematuria and bloody.Reports on SFTSV existence time limit in the body,correlation between viral load and the severity of SFTSV,correlation between viral load and the prognosis of SFTSV are rare.This research mainly separated two parrts:1.To study a cluster of six cases of SFTSV infection;2.A correlational study of the relationship between SFTSV load and patient’s condition.To further understand SFTS transmission mechanism,improve the clinician awareness of the disease,in order to guide clinical diagnosis and treatment of SFTS and protection.Part1 To Study a Cluster of Six Cases of SFTSV InfectionObjective1.To evaluated the potential contacts responsible for person-to-person transmission of SFTSV among the patients.2.To further study the contacts responsible for asymptomatic infection of SFTSV,in order to make guidance for clinical diagnosis and treatment of SFTS and protection.Methods1.A cluster of 6 patients(case A,caseB,caseC,caseD,caseE and caseF)with suspected SFTS in Shandong province in July 2014 were enrolled,patients’clinical data and samples were collected for further investigation.Epidemiological investigations were conducted to inquire information including the living surroundings,history of tick bites and animal contacts,the routes of possible exposure to the risk factors,and the use of protective devices as well as protective behaviors.2.Total RNA were extracted from blood smear of sample A for real-time PCR.The remain 5 blood sample were centrifuged for serum,serum were stored in-70 ℃for viral RNA extraction.3.Extract serum total RNA using Trizol reagent.4.Reverse transcription syntheses of cDNA were performed and cDNA were stored in-80℃.5.Two pairs of primers were designed for S,M and L segments of the SFTSV gene for nested PCR to amplify nucleotide fragments of SFTSV S,M and L segments.6.Southern blot were performed using PCR products,results were observed using agarose gel electrophoresis and DNA were recollected.7.PCR fragments were purified and sequenced,results were stitched using seqman in DNAStar software,SFTSV infection was confirmed by sequence comparison analysis.8.Serum samples confirmed with positive SFTSV infection were used to culture DH82 cells,culture supernatant were harvested on each generation,and cell debris were divided through centrifuge and discarded.Culture supernatants were stored at-70 ℃.9.Total RNA were extracted from culture supernatant and reverse transcripted into cDNA,virus nucleotide fragments were amplified and detected.10.1ml of positive samples from PCR was used to culture DH82 cells,IFA were used to detect viral protein expression.11.Genome Sequencing was performed for extracted virus.12.IFA and ELISA were performed to detect SFTSV specific antibody IgM and IgG expression level in sample serum.13.Detect antibody expression level through antibody neutralization assay.14.Cloning and sequencing were performed for purified positive PCR product,results were stitched using seqman in DNAStar software,sequence alignment were performed using MegAlign in DNAStar,Phylogenetic Trees were reconstructed using Mega 5.0.PCR product nucleotide sequences and purified viral genome sequence were aligned and evolutionary analyses were performed to analyze possibility of SFTS transition from human to human from a genetics perspective.Results1.Case A died of multiple organ dysfunction on the second day of admission.With supportive therapy and infusion with blood platelets(PLT)and human granulocyte colony-stimulating factor(G-CSF),case C’s condition deteriorated and died on the 10th day of the disease.Case C was recovered after supportive treatment,and was discharged from the hospital for 14 days of onset.None of cases D,E and F developed disease..Cases A,B and C have fever accompanied by thrombocytopenia,with cases A and B accompanied by leukopenia.2.Case B and case C have direct contact with blood.and their coats were contaminated when case A was vomiting blood.Case B wiped blood off A’s mouth with his bare hands.After they arrived home,case C threw the bloody coat away,washed his hands,and took a shower,while case B was still wearing the bloody coat.Case D have close contacted with case B,case E have close contacted with case C and case F have close contacted with case B and case C.They all contacted with the patient’s skin,mucosa and fluids.Neither of case D and case E had protective precautions.Case F wore masks and then washed his hands.So the potential transmission routes among the cases in the cluster were from the index patient(case A)to the secondary patients(cases B and C),then from case B to cases E and F,and from case C to cases D and F.3.RNA fragments of the SFTSV S(1160-1513 nt)and L segments(589-806 nt)were detected in the blood smear of case A and the serum sample of case B but not of case C by RT-PCR(Table 1).Case D was found negative for SFTSV RNA by her serum sample;while the SFTSV L segment was detected in the serum samples of cases E and F.4.Different levels of IgM response were detected in the sera samples of cases B,C,D,E,and F by cELISA;while the sera of cases E and F were detected positive for IgM by IFA.5.Cases B and C had low levels of IgG response detected by cELISA;while IFA showed that the serum sample of case B was positive for IgG.6.MNA showed that QD7 could be neutralized by the sera samples of cases E and F.7.The sequence of PCR products of S and L segments from case B(354 bp and 218 bp,respectively)were identical to the isolate QD7.The sequence of PCR products of S segment from case A(199 bp)shared 99.5%nucleotide identity to case B;while the sequence of PCR products of L segment from case A(218 bp)was identical to case B.8.Phylogenetic analysis showed that the sequences of S segment from the isolated virus clustered with sequences of the PCR products from cases A and B.Furthermore,the sequence of PCR products of L segment from cases E and F(218 bp)were identical to cases A and B as well as the isolated virus.The results suggested that the viral sequences detected from cases A,B,E,F and the isolated virus might share the same origins,which provided genetic evidence for the possible transmission routes of SFTSV in the cluster.’Conclusions1.SFTSV was transmitted from the index patient to the secondary patients via direct blood exposure。2.the asymptomatic infections had recent SFTSV infection most likely due to the close contact with the body fluids and mucosa of the secondary patients.Part 2 A Correlational Study of the Relationship Between SFTSV Load and Patient’s ConditionObjective1.To explore the SFTSV existence time limit in the body.2.To explore the correlation of viral load and the severity and prognosis of disease.Methods1.125 cases of blood samples of SFTS patients were collected in the infectious diseases department of weihai central hospital in shandong province,from May 2015 to October 2016.Specimen after centrifugal suck serum that save in the supernatant fluid-70 ℃ and used to extract RNA virus.Blood samples of 46 cases were collected continuously during admission.2.Collected the clinical data including the presence of complications,including nervous system symptoms(including indifference,drowsiness,coma,Muscle tremor,convulsions,etc.),bleeding tendency(including skin petechiae,pulmonary hemorrhage,tarry stool,diffuse intravascular coagulation),severe myocardial damage(CK-MB>2 times the upper limit of normal)and the incidence of pneumonia.3.The laboratory indexes of patients were collected,including leukocyte,platelet,liver function,renal function,myocardial enzyme,serum electrolyte levels and blood coagulation routine.4.The viral RNA was extracted from blood samples and collected into sterile EP tubes.5.A real-time fluorescent quantitative PCR method was used to detect the SFTSV load of the extracted virus RNA.6.SFTSV RNA of 46 cases were detected dynamically to observe the time limit of SFTSV RNA in the human body and its correlation with the disease.7.It were divided into viral load<104 copies/mL group(81 cases)and viral load>104 copies/mL group(44 cases).The incidence of complications laboratory biochemical index difference and prognosis of correlation of two groups were compared.8.The variance was tested by t-test,and heterogeneity of variance was tested by rank sum test.Results1.Of the 125 cases,64 were male,61 were female,the mean age(59.0±3.6),and 101 cases were cured,and 24 were fatal.2.SFTSV RNA loads of low viral load group(81 cases)were(3.08 + 1.01)copies/mL,and it of high viral load group(44 cases)were(5.69±0.99)copies/mL.Comparing SFTSV RNA loads of the two groups have significant difference(t=11.78,P<0.05).3.Viral load of most patients was gradually reduced after 1 week,and cleared 23 days later by detecting SFTSV RNA of 46 cases dynamically.4.The incidence of two groups of neurological symptoms,bleeding tendency,severe myocardial damage and pneumonia were 4.76%,0%,0%,2.47%and 90.9%,40.9%,70.5%,40.9%(χ2=92.987,38.711,75.889,54.680,respectively;all P<0.05).The dead patient’s viral load varied from 1.06×104copies/mL to 5.78x 107copies/mL.5.Comparing the WBC count of two groups have no significant difference(t=0.181,P>0.05).6.Comparing the platelet count of two groups have significant difference(t=2.869,P<0.05).7.Comparing ALT,BUN,Cr and UA of two groups have no significant difference(Z/t=-1.019,-0.702,-1.212,1.892,respectively;all P>0.05).Comparing AST and GGT of two groups have significant difference(Z/t=-2.612,-2.015,respectively;all P<0.05).8.Comparing CK,CK-MB,LDH and HBDH of two groups have significant difference(Z=-1.985,一3.279,一3.126,-2.152,respectively;all P<0.05)。9.Comparing serum sodium,glucose,blood calcium of two groups have significant difference(Z/t=2.666、2.612、-2.321,均P<0.05).Comparing serum potassium and serum chlorine of two groups have no significant difference(Z/t=1.498,0.287,respectively;all P>0.05)。10.Comparing APTT of two groups have significant difference(t=5.623,P<0.01).Comparing PT,Fbg and TT of two groups were compared have no significant difference(t=2.232,0.408,1.413,respectively;all P>0.05).ConclusionsAfter the onset of SFTSV infection,the virus existence in the body may not be more than 4 weeks.Viral load are closely associated with disease severity and prognosis,the higher the viral load,organ dysfunction is heavier,the higher the mortality.