Dynamic Changes Of Viral Loads And Antibodys In Patients With Severe Fever With Thrombocytopenia Syndrome

BACKGROUNDSevere fever with thrombocytopenia syndrome(SFTS) is an emerging hamorrhagic fever that was first described in rural areas of China. The causative agent, SFTS virus(SFTSV) is a novel phlebovirus in the Bunyaviridae family. Since the first report in 2010, SFTS has been found in 19 provinces of China.The disease was also reported in Japan and Korea.Heartland virus, another phlebovirus genetically closely related to SFTSV, was isolated from two patients in the USA.SFTS incubation period is generally 5-12 days.The disease is characterized by acute fever(mostly high fever,≥ 40℃),leukocytopenia and thrombocytopenia etc.The majority of patients have a good prognosis.Patients with older age, with chronic diseases, with immune depression, and long delay in receiving therapy were found to have adverse prognosis.A few severe cases can develop multiple organ injury and even death.The mortality rate ranged between 6.3% and 30%.At present, there is no effective treatment for this disease.The correlation between higher viral load and adverse clinical outcome has explained partly that the viral loads played an important role in the prognosis. However, the study on the dynamic pattern of viral load in SFTS patients during the whole period of hospitalization was scarce. On the other hand, it remains unclear as to whether a robust or depressed immunological response is associated with more severe disease course after SFTSV infection.The data on the relationship between immune response and clinical outcomes are lacking.OBJECTIVETo acquire the dynamic pattern of viral excretion in SFTSV infected patients during the whole period of hospitalization, and to explore the relationship between the factors affecting viral load and clinical prognosis. To characterize the immune response in SFTS patients,and to analyze the relationship between immune response and prognosis of patients.METHODSFor patients who meet the inclusion criteria of SFTS, the epidemiological questionnaires and the clinical data were collected.Collection of serum samples(acute phase serum and continuous serum) were used for nucleic acid extraction.Real time Polymerase chain reaction (RT-PCR) was used to detect the SFTSV, and the viral loads of the positive samples were quantified by the plasmid standard curve.For patients who meet the inclusion criteria of the follow-up cases registered follow-up questionnaires.At each visit, blood samples were collected for Ig M, Ig G antibody titer quantification and routine blood tests. From the cohort, we selected patients who agreed to have additionally 5-ml EDTA anti-coagulated blood samples collected.The samples were transferred to our laboratory within 6 hours of collection for peripheral blood mononuclear cell(PBMC) separation, which was used for lymphocytes and lymphocyte subsets evaluation.RESULTSFrom April to November 2013, a total of 208 patients with confirmed SFTSV infection were included into the study.The initial viral loads averaged to be 3.79±2.38 Log10 copies/m L at day 3 post-infection, which sharply increased, reaching maximum level of 4.29±2.59 Log10 copies/m L at day 6, and returned to 2.13±2.16 Log10 copies/m L at day 15 post infection. The PCR positive rate for SFTSV was calculated to be 44.4%(4/9) on one day after disease onset, peaking at 82.5%(33/40) on three days, reducing gradually to 78.5%(117/149) on six days, 57.5%(103/179) on 12 days, and 55.6%(25/45) on 18 days after disease onset.The nadir White blood cell(WBC) count was detected close to that of the peak viral load, both occurring at day 6 after disease onset. The nadir Platelet(PLT), the peak Aspartate aminotransferase(AST), Lactate dehydrogenase(LDH) and Creatine kinase(CK) level lagged behind the viral load peaking by three days, while peak Alanine transaminase(ALT) level lagged behind viral peak by six days. Altogether 34.3%(47/137) samples which had PLT returned to normal level were detected to be positive for SFTSV. For 55.6%(80/144) samples which had WBC restored to be normal was shown have detectable SFTSV, this prevalence was calculated to be 30.8%(20/65).34.8%(16/46) and 50.7%(76/150) for AST, LDH and CK respectively. There were still 30.8%(20/65) patients detected to be positive for SFTSV when both PLT and WBC evaluations were restored to be within normal range.From 2011 to 2014, altogether 298 laboratory-confirmed SFTS cases were analyzed, from whom 55 patients were followed after convalescence and 438 samples were collected. Three(5.9%), 4(7.8%) and 1(2.0%) patients experienced thrombocytopenia, leukocytopenia and elevated LDH, respectively, which was observed on M6 after disease onset. SFTSV specific Ig M antibody could be detected at 4 days, surged to peak levels by 4 weeks, and remained persistent until 6 months after disease onset. The positive rates peaked at week 4(79.4%, 27/34) and decreased dramatically thereafter with 35.0%(14/40) tested positive at M3,10.9%(6/51) positive at M6. None of the samples collected after M9 were detected to be positive. For SFTSV specific Ig G antibody peaked at M6.The positive rates peaked at M6(89.1%, 49/55) and decreased thereafter with positive rates of 87.3%(48/55) at M9, 87.2%(41/47) at M18 and 84.0%(21/25) at M30. SFTS patients were evaluated for the peripheral lymphocyte subsets, from whom experienced significant T, B and NK cells loss in the peripheral blood during the first week of infection. Severe SFTS patients had even lower T cell counts than other SFTS patients, with median absolute CD3 T cell counts reduced to 176.8 cells/mm3,CD4 and CD8 counts reduced to as low as 66 and 63 cells/mm3, respectively. Sequential evaluation showed a rapid and significant increase of all the evaluated lymphocyte subsets on 2 weeks after the onset of illness, which were restored to normal levels at M6 after the disease onset.CONCLUSIONS1.The prospective study of viral load in SFTS patients during hospitalization.(1) The viral RNA is undetectable in 55.6% of the patients on admission into the hospital. The result suggests that only one RT-PCR test is not appropriate to exclude SFTSV infection. The diagnosis of SFTSV infection based on PCR test should be performed at least three days after disease onset. Its clinical reference value is more reliable, but also can avoid the occurrence of misdiagnosis or missed diagnosis.(2) The Peaking viral loads are attained around six days after disease, at which point posing a highest risk of human-to-human transmission.(3) The Initial and maximum SFTSV RNA concentrations in severe patients is much higher than that of mild patients. Especially female Patients with older age(long delay before hospitalization of ≥5 days), with higher viral load, virus excretion rate is relatively slow. And 5 death cases of peak viral load is greater than 107copies/ml. this level can occur as a threshold to predict the risk of death. It is suggested that clinicians take timely clinical intervention measures.(4) Abnormal laboratory parameters of PLT, WBC, LDH, AST and CK were shown to be significantly associated with viral loads. This result can be used as a reference for clinical diagnosis and analysis of the disease.2. The prospective study on immune characteristics in SFTS patients.(1) SFTS patients experienced obvious T cell, B cell and NK cells loss during the first week of infection, suggesting that the remarkably and consistently depressed immunity, which has been illustrated by both significantly lower lymphocyte subsets level and Ig M/Ig G antibody level during early infection, which further aggravate the deterioration of the patient’s condition. Therefore, these findings indicate that changes on peripheral lymphocyte may contribute to the pathogenesis during the early stages of the SFTSV infection and their normalization may predicate disease alleviation.(2) Ig M and Ig G antibody magnitude have important application in diagnosis. SFTSV-specific Ig M antibody can be detected at medium of 9 days after disease onset; surged to peak levels by the early 4 weeks and became negative on 6 months after disease onset. The period that Ig M antibody test can be used in determining whether a patient has recently been infected with SFTSV. Ig G could be detected at a medium of 6 weeks after disease onset; surged to peak levels by 6 months and remained detectable in most of the patients as long as 3 years after disease. The duration of Ig G antibody persistent over 3 years is a useful indicator in the protection of reinfection in cases of re-exposure to SFTSV, and therefore can have important implications in developing vaccination strategy.(3) Patients with a high initial viral load or impaired immune system, such as those who are older and/or with underlying diseases, are severely depressed.The innate immune system in these patients might be suppressed to a more severe degree, leaving insufficient time for activation of adaptive immunity. This has led to the ineffectiveness of viral clearance. Based on this observation, we propose that remarkable clinical deterioration is related to depressed cellular and humoral responses to SFTSV, which might be due to the ability of SFTV to disable the host immune response by attacking and manipulating cells that initiate the antiviral response.