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Infection with a novel virus causes hemorrhagic fever in China

  • Wei Li
    Affiliations
    Department of Infectious Diseases, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China
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  • Yong-Wen He
    Correspondence
    Corresponding author. Tel.: +86 27 85726135; fax: +86 27 85356369.
    Affiliations
    Department of Infectious Diseases, the Affiliated Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430022, China
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Open AccessPublished:April 22, 2013DOI:https://doi.org/10.1016/j.ijid.2013.03.007

      Summary

      Background

      Patients have been identified with hemorrhagic fever (HF) caused by Huaiyangshan virus (HYSV) infection since 2009. This study aimed to investigate the characteristics of clinical symptoms, laboratory examinations, epidemiological factors, and therapeutic responses in patients with Huaiyangshan hemorrhagic fever (HYSHF).

      Methods

      A total of 57 patients with a suspected HF were admitted to the Department of Infectious Diseases, the Affiliated Union Hospital of Tongji Medical College between June 2009 and October 2010. A potential infection with HYSV was determined by indirect immunofluorescent assay and reverse-transcription (RT)-PCR. The clinical symptoms, epidemiological characteristics, laboratory examinations, and therapeutic responses of these patients were evaluated.

      Results

      Forty-eight out of 57 patients were diagnosed with HYSHF. They displayed diverse clinical symptoms, such as an acute febrile flu-like illness, and progressed to proteinuria, hemorrhagic manifestations, and encephalopathy. Some patients exhibited progressive leukopenia, thrombocytopenia, liver and kidney dysfunction, and systemic cell injury. Following symptom-specific treatment, 35 patients recovered completely and 13 patients died from severe complications, including central nervous system manifestations.

      Conclusions

      Patients with HYSHF displayed diverse clinical symptoms and laboratory findings. Such patients should be treated immediately and closely monitored to prevent severe complications.

      Keywords

      1. Introduction

      Viral hemorrhagic fevers (VHFs) in animals and humans are caused by infections with RNA viruses from the families Arenaviridae, Filoviridae, Bunyaviridae, and Flaviviridae.
      • Nichol S.T.
      • Beaty B.J.
      • Elliott R.M.
      • Goldbach R.
      • Plyusnin A.
      • Schmaljohn C.S.
      • et al.
      Family Bunyaviridae.
      • Fauquet C.
      Virus taxonomy: classification and nomenclature of viruses: eighth report of the International Committee on the Taxonomy of Viruses.
      Hantavirus, Crimean-Congo hemorrhagic fever (CCHF) virus, and Rift Valley fever (RVF) virus, in the Bunyaviridae family, can cause severe HF in Asians and remain serious threats to human health. These, together with new more recently discovered viruses, are a great challenge to the prevention and treatment of VHF. In addition, patients infected with some of these viruses can develop severe complications that have a high mortality. Therefore, recognition of new VHF-related viruses and their pathogenesis in humans is of great significance.
      Previous studies have reported a new disease known as Huaiyangshan hemorrhagic fever (HYSHF), also known as severe fever with thrombocytopenia syndrome (SFTS), in the Huaiyangshan mountain region of Hubei and Henan, China.
      • Zhang Y.Z.
      • Zhou D.J.
      • Xiong Y.
      • Chen X.P.
      • He Y.W.
      • Sun Q.
      • et al.
      Hemorrhagic fever caused by a novel tick-borne bunyavirus in Huaiyangshan, China.
      • Zhang Y.Z.
      • Zhou D.J.
      • Qin X.C.
      • Tian J.H.
      • Xiong Y.
      • Wang J.B.
      • et al.
      The ecology, genetic diversity, and phylogeny of Huaiyangshan virus in China.
      HYSHF is caused by the Huaiyangshan virus (HYSV, also known as the severe fever with thrombocytopenia virus (SFTSV)), a novel member of the Bunyaviridae family that is closely related to the viruses of the genus Phlebovirus.
      • Zhang Y.Z.
      • Zhou D.J.
      • Xiong Y.
      • Chen X.P.
      • He Y.W.
      • Sun Q.
      • et al.
      Hemorrhagic fever caused by a novel tick-borne bunyavirus in Huaiyangshan, China.
      • Zhang Y.Z.
      • Zhou D.J.
      • Qin X.C.
      • Tian J.H.
      • Xiong Y.
      • Wang J.B.
      • et al.
      The ecology, genetic diversity, and phylogeny of Huaiyangshan virus in China.
      There has been no systematic analysis of the clinical and laboratory characteristics of patients with HYSHF. Furthermore, it is unclear what the epidemiological features of HYSV infection are in China.
      This study aimed to investigate the epidemiological characteristics that contribute to HYSV infection, clinical manifestations, laboratory findings, and therapeutic responses of patients with HYSHF in a Chinese population. We discuss the implications of our findings.

      2. Methods

      2.1 Cases

      A total of 57 patients with a suspected tick-borne disease were referred to the Department of Infectious Diseases, the Affiliated Union Hospital of Tongji Medical College, Wuhan, China between June 2009 and October 2010. These patients had unexplained fever (>37.5 °C), leukopenia (<4.0 × 109/l), thrombocytopenia (<100 × 109/l), and multiple organ dysfunctions, but they had no evidence of infection with hantaviruses, Leptospira, dengue virus, human Ehrlichia, or bacterium typhosum. All 57 suspected cases fulfilled the prevention and care guidelines for SFTS of the Ministry of Health, the People's Republic of China (MOHC; 2010).
      Ministry of Health, PR China
      Prevention guideline for severe fever with thrombocytopenia syndrome (2010).
      Individuals with fever, leukopenia, or thrombocytopenia alone were excluded from this study.
      Written informed consent was obtained from conscious patients or the relatives of unconscious patients. The experimental protocols were established in accordance with the medical research regulations of the Ministry of Health, China, and were approved by the Ethics Committee of the Affiliated Union Hospital of Tongji Medical Colleges.

      2.2 Laboratory tests

      Peripheral blood samples were obtained from individual patients, and their blood cell counts with differential were routinely examined at the Clinical Diagnosis Laboratory of the Union Hospital. Plasma levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and creatine kinase (CK) in individual patients were tested at the Biochemistry Laboratory of the Union Hospital. Furthermore, the activated partial thromboplastin time (APTT) and thrombin time (TT) of fresh plasma samples from individual patients were measured at the Clinical Diagnosis Laboratory of the Union Hospital.
      HYSV-specific immunoglobulin IgM and IgG antibodies and HYSV-specific RNA in individual subjects were characterized by immunofluorescent assay (IFA) using HYSV-infected Vero-E6 cells as antigens,
      • Zhang Y.Z.
      • Zhou D.J.
      • Xiong Y.
      • Chen X.P.
      • He Y.W.
      • Sun Q.
      • et al.
      Hemorrhagic fever caused by a novel tick-borne bunyavirus in Huaiyangshan, China.
      and reverse-transcription (RT)-PCR,
      • Zhang Y.Z.
      • He Y.W.
      • Dai Y.A.
      • Xiong Y.
      • Zheng H.
      • Zhou D.J.
      • et al.
      Hemorrhagic fever caused by a novel bunyavirus in China: pathogenesis and correlates of fatal outcome.
      respectively, at the National Institute for Communicable Disease Control and Prevention, China Center for Disease Control and Prevention. The levels of plasma IgM and IgG antibodies against Ehrlichia chaffeensis were determined by IFA using E. chaffeensis (Arkansas strain)-infected DH82 cells as antigens, as described previously.
      • Olano J.P.
      • Walker D.H.
      Human ehrlichioses.
      Similarly, hantavirus, Leptospira, dengue virus, CCHF virus, and typhoid bacillus-specific antibodies in individual subjects were analyzed by IFA.
      • Zhang Y.Z.
      • Dong X.
      • Li X.
      • Ma C.
      • Xiong H.P.
      • Yan G.J.
      • et al.
      Seoul virus and hantavirus disease, Shenyang, People's Republic of China.

      2.3 Treatments

      All patients with HYSHF were treated with the same basic protocol. First, the patients were provided with supportive therapy daily to maintain the balance of the internal environment, including vitamins and energy. Patients with severe neutropenia (<2.0 × 109/l) were treated with 100 μg granulocyte colony-stimulating factor (G-CSF) for 1–3 days. Some patients were positively transfused with platelets and treated with clotting factors. All patients were treated with antibiotics (piperacillin 3.0–5.0 g per day, levofloxacin 0.6 g per day, or cephalosporins 2–4 g per day) to prevent secondary bacterial infections. All patients were provided with doxycycline (0.2–0.4 g per day) for at least 5 days to treat potential infections with monocytic ehrlichiosis before the diagnosis. Some patients were treated with appropriate hemostatic medicines, such as vasopressin, somatostatin, oxytocin, and others, to terminate bleeding and to prevent disseminated intravascular coagulation (DIC). Moreover, some patients received oxygen masks with positive pressure ventilation through a breathing machine if they had shortness of breath, rapidly dropping oxygen saturation, pulmonary edema, acute respiratory distress syndrome (ARDS), or a concurrent lung infection. In addition, patients with ARDS were treated with extracorporeal membrane oxygenation (ECMO) therapy. Patients with heart failure were treated with oxygen, nutrition, cardiac drugs, and digitalis, and patients with kidney dysfunction were treated with diuretic drugs, improved renal microcirculation, and kidney dialysis. Finally, a few patients with intracranial hemorrhages, convulsion, and coma were treated with supportive medicines.

      2.4 Data collection and analysis

      Demographic and clinical data, medication/treatment history, clinical course (including the number of days before seeking medical attention), and clinical symptoms (such as fever, headache, myalgia, nausea, malaise, pulmonary and hepatic dysfunction, renal dysfunction, and neurological involvement), were collected and evaluated. Physical findings, laboratory data, time of defervescence, treatment, and mortality were observed. Data are expressed as the number of cases and percentage, or the median and range. These patients were stratified on the basis of some of the measures for the analysis of mortality by Fisher's exact test; SPSS version 17.0 was used for the analysis (SPSS Inc., Chicago, IL, USA). A two-tailed p-value of less than 0.05 was considered statistically significant.

      3. Results

      3.1 Diagnosis of patients with HYSVF

      From June 2009 to October 2010, a total of 57 patients with suspected VHF were referred to our hospital. Analysis for a potential infection with HYSV revealed that 48 (84.2%) out of 57 subjects displayed positive serum anti-HYSV IgM; 37 out of 48 patients were positive for HYSV-specific RNA detection. However, no blood samples from the patients were positive for serum antibodies against E. chaffeensis, hantaviruses, Leptospira, dengue virus, CCHF virus, or bacterium typhosum. These 48 patients were diagnosed with HYSHF. Interestingly, among the remaining nine patients with negative detection of HYSV, three were diagnosed with lymphoma, four with Gram-negative bacterial septicemia, and for two there was positive detection of blood cell coxsackie virus RNA (data not shown).

      3.2 Demographic characteristics and clinical epidemiology

      There were 29 female patients (60%) and 19 male patients (40%), and their mean age was 53.7 ± 9.1 years. With the exception of two cases, all of the patients were older than 40 years of age; no patient was younger than 20 years of age. Analysis of the distribution of these cases revealed that the patients came from an area approximately 200 km around Wuhan City, Hubei Province, China. These patients were transferred from local clinics in Caidian-qu District of Wuhan City (one case), Xiaogan City (three cases), Suizhou City (seven cases), Huanggang City (17 cases), Dawu County (four cases) and Xianning City (two cases) (Hubei Province), and Xinyang City (14 cases) (Henan Province), which are geographically connected. The majority of cases occurred sporadically. The disease appeared in May, and the numbers of cases reached a peak in June or July, decreasing in August. All cases happened in rural hilly areas, and many patients were elderly and young men with houses surrounded by the bush. Forty-six out of 48 patients (95%) were farm workers. The majority of patients worked in farming fields picking tea in the mountains, or herding sheep, planting mushrooms, farming, etc. Almost all patients had a history of animal contact or a recent tick bite. There was no infection among attending health care workers and the close relatives of patients. It would appear that the disease occurs sporadically in a local region of China.

      3.3 Clinical findings and laboratory examination

      The incubation period (from tick bite to the appearance of clinical symptoms) of the disease in these patients ranged from 2 to 14 days (average 7–9 days). Most patients with the disease had no symptoms during the incubation period, except for seven patients who felt weak and tired. The disease began abruptly with fever (100%), headache (75%), anorexia (77%), muscle aches (33%), nausea and vomiting (23%), diarrhea (31%), and other nonspecific signs and symptoms. Fifteen patients (31%) exhibited inguinal lymphadenectasis with incrustation and defined borders, but no rupture or necrosis.
      Within a few days of onset, most patients exhibited coagulation defects and mild hemorrhagic symptoms, such as hematuria and ecchymosis (Figure 1), and severe cases had massive bleeding from the gastrointestinal tract (10%) to other organs. Some patients displayed arrhythmias, proteinuria (98%), hepatic dysfunction (95%), and encephalopathy (48%), even with trembling, astasia, and coma (8.3%). Thirty-five patients (73%) had hemorrhagic manifestations, including one patient (2.1%) with epistaxis, bleeding gums, and petechiae; two patients (4.2%) with vaginal hemorrhage, hematemesis, and hemoptysis; 19 patients (39.6%) with ecchymoses; 10 patients (20.8%) with melena; and 21 patients (43.8%) with hematuria. Five patients (10.4%) suffered confusion, 17 patients (35.4%) suffered drowsiness, and 18 patients (37.5%) had a tremor. Therefore, most patients displayed manifestations involving multiple organs. Of the 48 patients, many had abnormal levels of ALT (95%) and AST (92%), but only a few patients displayed abnormal levels of plasma total bile acid (15%). During the course of the disease, many patients showed higher levels of plasma CK (95%) and LDH (100%), indicative of cell death and organ dysfunction. Almost all of the patients had a prolonged APTT (88%) and TT (88%). More than half of the patients had elevated blood glucose (56%), indicative of pancreatic lesions. In addition, 14 patients (29.1%) showed an abnormal electrocardiogram of sinus bradycardia, abnormal ST segments, and/or abnormal T waves, but no patient had abnormal findings on echocardiography.
      Figure thumbnail gr1
      Figure 1Bleeding visible on the back of one patient infected with Huaiyangshan virus.
      The diversity of laboratory findings in five patients who were admitted to the hospital during the early stage of the disease process and who recovered fully from the illness, is shown in Table 1. Clearly, their laboratory results changed on days 5–8 post disease onset (p < 0.05) as compared to days 2–4 post onset. Some of the laboratory results improved slightly on days 9–12 post onset as compared with days 5–8 post onset (p < 0.05).
      Table 1Laboratory findings of five patients admitted to the hospital during the early phase of the illness
      Laboratory testDays 2–4 after onset, median (range)Days 5–8 after onset, median (range)Days 9–12 after onset, median (range)Normal ranges
      AST (U/l)45 (32–86)231 (89–465)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      145 (71–239)8–40
      ALT (U/l)27 (21–57)119 (55–358)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      78 (52–204)5–40
      Leukocytes (× 109/l)4.42 (2.92–7.22)2.29 (0.89–3.23)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      4.61 (2.58–6.47)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      4–10
      Platelets (× 109/l)91 (75–98)33 (18–62)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      59 (31–87)100–300
      CK (U/l)102 (77–180)942 (244–3822)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      203 (127–412)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      38–174
      LDH (U/l)73 (47–105)875 (295–3054)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      292 (195–679)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      109–225
      APTT (s)42.2 (33.7–47.2)60.4 (49.4–71.3)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      22.8 (24.1–19.3)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      23.5–43.5
      TT (s)14.5 (11.7–17.6)25.5 (21.1–34.2)
      p<0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      18.4 (15.6–21.7)16–20
      ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactate dehydrogenase; APTT, activated partial thromboplastin time; TT, thrombin time.
      a p < 0.05 vs. the first 2–4 days, as determined by the Mann–Whitney test.
      Laboratory examinations of all 48 patients with HYSV infection on day 1 post admission are shown in Table 2. Although we knew that the five patients who had come to the hospital during the early stage of disease had some nonspecific symptoms within the initial 3–4 days, the laboratory test values of these patients were significantly different from those of the 28 patients who arrived at the hospital on days 5–8 post disease onset (p < 0.05). However, there was no significant difference in the laboratory test values between the 15 patients admitted at the later stage of disease and the 28 patients admitted during the intermediate stage.
      Table 2Laboratory results of 48 patients with Huaiyangshan virus-related hemorrhagic fever on admission
      Data shown are the ratios of cases with abnormal values to cases with normal values, median values, and ranges for each measure.
      Laboratory testEarly stage admission (<5 days after onset)Intermediate stage admission (5–8 days after onset)Later stage admission (9–15 days after onset)Normal ranges
      AST (U/l)2/5, 45 (32–86)27/28, 312 (38–1856)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      12/15, 212 (21–930)8–40
      ALT (U/l)2/5, 27 (21–57)27/28, 157 (34–955)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      14/15, 127 (36–406)5–40
      Leukocytes (× 109/l)2/5, 4.42 (2.92–7.22)26/28, 1.90 (0.59–6.44)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      11/15, 1.84 (0.56–5.95)4–10
      Platelets (× 109/l)5/5, 91 (75–98)27/28, 26 (4–102)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      15/15, 23 (5–84)100–300
      CK (U/l)1/5, 102 (77–180)27/28, 755 (105–6423)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      14/15, 735 (158–9805)38–174
      LDH (U/l)0/5, 73 (47–105)27/28, 847 (51–4598)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      28/28, 605 (302–11619)109–225
      APTT (s)1/5, 42.2 (33.7–47.2)27/28, 55.4 (35.5–171.8)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      10/15, 51.8 (31.9–109)23.5–43.5
      TT (s)0/5, 14.5 (11.7–17.6)27/28, 28.0 (15.4–118.4)
      p<0.05 vs. the early stage, as determined by the Mann–Whitney test.
      10/15, 33.1 (15.7–118.4)16–20
      ALT, alanine aminotransferase; AST, aspartate aminotransferase; CK, creatine kinase; LDH, lactate dehydrogenase; APTT, activated partial thromboplastin time; TT, thrombin time.
      a Data shown are the ratios of cases with abnormal values to cases with normal values, median values, and ranges for each measure.
      b p < 0.05 vs. the early stage, as determined by the Mann–Whitney test.

      3.4 Therapeutic responses

      Following treatment, we found that body temperature decreased to the normal level within 1–5 days post treatment in 32 out of 48 patients. Furthermore, of the 26 patients with severe neutropenia treated with G-CSF, 15 experienced a dramatic increase in white blood cell count. Some patients with heavy bleeding were treated with positive transfusion of platelets and clotting factors, as well as vasopressin, somatostatin, oxytocin, and others. Their bleeding was controlled and their blood pressure returned to the normal range. Following treatment with positive pressure ventilation through a breathing machine, particularly with ECMO, patients had obvious improvements in their shortness of breath, oxygen saturation, pulmonary edema, and ARDS. In addition, treatment with oxygen, nutrition, cardiac drugs, and digitalis significantly improved the cardiac function in a few patients with heart failure. Moreover, treatment with diuretic drugs and/or kidney dialysis dramatically ameliorated the renal function in 11 patients with severe kidney failure. Finally, treatment with either liver-protecting drugs or artificial liver support system rapidly improved the liver function in a few patients with severe liver dysfunction.
      During the convalescent period, the body temperature of most patients gradually returned to a normal level. Their body functions gradually recovered. Overall, 35 out of 48 patients recovered completely and 13 patients died due to the disease severity or severe complications, including deep coma and pulmonary fungal infection. Stratification of patients with or without complications and further bivariate analysis indicated that leukocytes <2 × 109/l, platelets <30 × 109/l, ALT >200 U/l, LDH >500 U/l, CK >400 U/l, and creatinine >150 mmol/l were risk factors for mortality (Table 3). Similarly, the percentage death in patients with a complication of pulmonary infection or central nervous system (CNS) manifestations was significantly higher than that of those without these complications. The severity was an independent factor for a poor prognosis in patients with HYSHF in this population. Multiple logistic regression analysis of eight risk factors indicated that ‘CNS manifestations’ was an independent factor for a poor prognosis in patients with HYSHF in this population (p < 0.05). However, the age and gender of the patients and their administration time were not significantly associated with a poor prognosis in this population (p > 0.05).
      Table 3Stratification analysis of mortality
      ComplicationPatients with the complicationPatients without the complicationp-Value
      TotalDied%TotalDied%
      Bleeding manifestations35925.713430.8>0.05
      CNS manifestations23939.125416.0<0.05
      Creatinine >150 mmol/l11763.637616.2<0.05
      ALT >200 U/l14642.934720.6<0.05
      AST >200 U/l311032.317317.6>0.05
      Platelets <30 × 109/l411331.7700<0.05
      LDH >500 U/l251040.023313.0<0.05
      CK >400 U/l321134.416212.5<0.05
      Leukocytes <2 × 109/l261034.522313.6<0.05
      Bilirubin >19 mmol/l391128.29222.2>0.05
      Secondary pulmonary infection15746.733618.2<0.05
      ALT, alanine aminotransferase; AST, aspartate aminotransferase; CNS, central nervous system; CK, creatine kinase; LDH, lactate dehydrogenase.

      4. Discussion

      VHFs are characterized by malaise, myalgia, and prostration due to systemic abnormalities of vascular permeability and regulation. Patients with a VHF often develop bleeding manifestations, particularly in severe cases, which are usually diffuse, reflecting widespread vascular damage. While hemorrhagic fever with renal syndrome (HFRS), CCHF, and dengue fever are common diseases in China,
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      HYSHF was also considered as SFTS.
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      We have reported the cases of 48 patients who had a clinical history compatible with HYSHF in a local region of Hubei and Henan provinces, Central China; these patients were confirmed to have the HYSV infection by IFA and RT-PCR.
      An epidemiological study indicated that these patients came from rural areas of Huaiyangshan and that the virus has spread since 2009, suggesting a sporadic nature. Most patients were older than 40 years of age. This is different to HFRS, CCHF, and dengue fever, which predominantly affect younger adults.
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      It is possible that fewer young men are now living in this region and that they had moved into the cities for work. Alternatively, many young men might become infected with HYSV, but have mild symptoms and not seek medical help. No child was found to be affected, which might be because pediatricians are unaware of the disease. In addition, older patients with HYSV infection displayed more severe clinical symptoms and were referred to our hospital, contributing to the higher frequency of patients.
      Given that the patients had a history of tick bite and that HYSV has been isolated from Haemaphysalis ticks,
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      this disease might be mediated mainly by Haemaphysalis ticks. It is possible that farmers have a greater chance of being bitten by a Haemaphysalis tick and becoming infected. Although the person-to-person transmission of HYSV (or SFTSV) through blood contact or personal contact has recently been reported,
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      these 48 cases occurred sporadically in the endemic region. We did not observe any case in a close family member of a patient with HYSHF and not a single case occurred in the healthcare providers at the hospital who cared for these patients. Accordingly, it is possible that these 48 cases were infected with HYSV through a tick bite rather than by person-to-person transmission. We are interested in further systematically investigating how many residents are infected with HYSV in this area and what immune responses have developed in this population. Interestingly, recent studies have suggested that new cases have also occurred outside the Huaiyangshan area during the past 2 years,
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      Severe fever with thrombocytopenia syndrome virus, Shandong Province.
      indicating that HYSHF has already spread outside the Huaiyangshan area; however no case of HYSHF has been reported in Zaoyang City, which is adjacent to Suizhou City. We are interested in further investigating the real endemic areas and how the HYSV spreads in these areas.
      The final diagnosis of HYSHF depends on a compatible clinical history with laboratory evidence of HYSV infection. Because these patients initially present nonspecific clinical symptoms and laboratory findings, physicians may diagnose the illness as a common flu or urinary infection at the early stage of the disease process. Although the RT-PCR-based assay is highly sensitive, we found that nine patients with similar clinical manifestations were negative for HYSV RNA detection, and they were diagnosed with lymphoma, Gram-negative bacterial septicemia, and coxsackie virus infection. However, we cannot completely exclude the possibility of co-infection with HYSV in those patients. The false-negative detection of HYSV may be caused by low levels of viral RNA or a sub-optimal time for testing. Therefore, the specificity and sensitivity of this assay for the detection of HYSV need to be improved. Given that this was a new virus-mediated disease, clinicians were generally unaware of the emergence of the disease in China. Furthermore, many laboratories in small hospitals lack the equipment and skilled personnel to test for HYSV. Hence, the development of a new detection kit with an easier method for clinical practice is urgently required. Although the characterization of antibody responses, particularly a 4-fold increase in antibody titers, is valuable,
      • Jiao Y.
      • Zeng X.
      • Guo X.
      • Qi X.
      • Zhang X.
      • Shi Z.
      • et al.
      Preparation and evaluation of recombinant severe fever with thrombocytopenia syndrome virus nucleocapsid protein for detection of total antibodies in human and animal sera by double-antigen sandwich enzyme-linked immunosorbent assay.
      serological analysis is mainly used for retrospective and epidemiological studies, and its value in the early diagnosis has not been confirmed. The in vitro isolation of HYSV from blood or other body fluids requires specialized laboratories. This, together with the 2–6 weeks required to obtain results,
      • Zhang Y.Z.
      • Zhou D.J.
      • Xiong Y.
      • Chen X.P.
      • He Y.W.
      • Sun Q.
      • et al.
      Hemorrhagic fever caused by a novel tick-borne bunyavirus in Huaiyangshan, China.
      make this protocol unfeasible for the early diagnosis. As we found that leukopenia, thrombocytopenia, and multiple organ dysfunction appeared several days after the onset of the febrile illness (Table 1), we recommend that clinicians should suspect HYSHF and monitor the clinical laboratory tests regularly if individuals have a history of tick bite, exposure to animals, and the presence of relevant symptoms, particularly if the patient is an animal handler or farmer from an endemic area.
      Currently no specific treatment for HYSHF is available. We treated all of the patients according to their clinical symptoms and laboratory test results. We found that the surveillance of vital signs, patient consciousness, blood cell counts, and blood biochemistry results were the cornerstone of treatment. Because these patients were suspected of having toxic shock syndrome, urinary infections, meningitis, or hematological diseases at the early stage of the disease in the local clinic, they were treated with cephalosporin, levofloxacin, and corticosteroids. Although the effect of these antibiotics on disease progression is unclear, we did find that five patients who were treated with high doses of corticosteroids developed fungal infections with severe outcomes. Thus, we do not recommend treatment with broad-spectrum antibiotics or corticosteroids for those with undiagnosed disease, particularly those suspected of having HYSHF, even if the patients have encephalopathy and arrhythmias, because many older patients with HYSHF were immunocompromised with progressive leukopenia. More importantly, high virus loads are correlated with systemic cytokine storm and a poor prognosis.
      • Gai Z.
      • Liang M.
      • Zhang Y.
      • Zhang S.
      • Jin C.
      • Wang S.W.
      • et al.
      Person-to-person transmission of severe fever with thrombocytopenia syndrome bunyavirus through blood contact.
      • Gai Z.T.
      • Zhang Y.
      • Liang M.F.
      • Jin C.
      • Zhang S.
      • Zhu C.B.
      • et al.
      Clinical progress and risk factors for death in severe fever with thrombocytopenia syndrome patients.
      We suspect that the use of corticosteroids may impede the clearance of HYSV in patients. Finally, treatment with broad-spectrum antibiotics and corticosteroids did not help to relieve the encephalopathy or to shorten the course of disease in those patients.
      Patients were diagnosed with ehrlichiosis in April 2009 at our hospital.
      • Li W.
      • He Y.W.
      Clinical analysis of 36 patients with human ehrlichioses.
      Because of the great similarity in the clinical symptoms and laboratory examination results of patients with HYSHF and those with ehrlichiosis,
      • Li W.
      • He Y.W.
      Clinical analysis of 36 patients with human ehrlichioses.
      • Walker D.H.
      Task Force on Consensus Approach for Ehrlichiosis
      Diagnosing human ehrlichioses: current status and recommendations. Despite shortcomings, immunofluorescence testing remains the best choice, with PCR and culture methods being valuable.
      we had considered some patients as having ehrlichiosis in 2009 before HYSV was identified. Interestingly, we found that treatment with doxycycline reduced body temperature within a couple days post medication and improved clinical symptoms and laboratory values as well, consistent with a recent report.
      • Zhao C.H.
      • Chen W.H.
      • Zhou W.F.
      • Zhang C.M.
      • Xi Z.
      Clinical analysis of four cases of severe fever with thrombocytopenia syndrome in Hubei Province.
      One possible reason for this is that treatment with doxycycline controlled a secondary bacterial infection in those patients who had leukopenia and poor immunity. Because antibiotics should have no effect on the control of a viral infection, further studies are needed to clarify the efficacy of treatment with doxycycline in patients with HYSHF.
      In summary, we have described the characteristics of clinical symptoms, laboratory examinations, primary epidemiological factors, and therapeutic responses of 48 patients with HYSHF. Because patients with HYSHF displayed clinical symptoms similar to those of other diseases, it is crucial for clinicians to recognize and be aware of the recent emergence of this new disease in China. Individuals with a history of tick bite, exposure to animals, and the presence of a symptom complex compatible with HYSV infection should be suspected of HYSHF. We found that, once a patient was suspected with HYSHF, she/he should be treated immediately and closely monitored because of rapid progression of this disease. We do not recommend treatment of these patients with broad-spectrum antibiotics and corticosteroids, which may cause severe fungus infections. We are interested in further investigating the pathogenic process of the disease and the therapeutic values of available treatments to reduce the mortality. Epidemiologically, we need to identify the infectious sources, the transmission routes of the disease, the sub-clinical infection rate in the population in the endemic area, and the potential strategies for the prevention of HYSV infection.

      Acknowledgement

      We thank Medjaden Bioscience for their help in preparing this manuscript.
      Ethical approval: Written informed consent was obtained from conscious patients or relatives of unconscious patients. The experimental protocols were established in accordance with the medical research regulations of the Ministry of Health, China, and were approved by the Ethics Committee of the Affiliated Union Hospital of Tongji Medical Colleges.
      Conflict of interest: No conflict of interest to declare.

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