Clinical features and outcomes of brucellosis complicated with Epstein-Barr virus infection

Objective To analyze the clinical, laboratory characteristics and prognosis of brucellosis patients with co-existing Epstein-Barr virus (EBV) infection at a major hospital in Shandong, a brucellosis epidemic region of China. Methods A total of 576 inpatients diagnosed with brucellosis at Yidu Central Hospital, between July 2013 and July 2018, were selected and tested for EBV DNA. 22 patients were found to be positive for co-infection with EBV. The clinical data of these 22 patients (observation group) and 100 patients (control group) with only brucellosis were retrospectively compared. Results The observation group (the group with the EBV co-infection) had more severe clinical manifestations in the form of fever, headache, and hepatosplenomegaly. Further, the observation group also had a significantly higher number of patients with elevated alanine transaminase(ALT) and aspartate-aminotransferase (AST), reduced WBC, and elevated PLT count. The incidence of abnormal levels of cardiac enzyme was also significantly higher in the observation group, as was the recovery time and average hospitalization period. Conclusions It is important to consider EBV infection and other potentially latent viral infections in patients with brucellosis, as these infections can further complicate the disease course. Further, patients diagnosed with a co-infection should be administered combined antibacterial and antiviral treatment and kept under observation and followed up for a longer period.


Introduction
Brucellosis, which is also known as Malta fever, and Mediterranean fever, is an neglected zoonotic disease 1 . Human brucellosis is more than 500,000 new cases in the world annually 1,2 . Brucellosis remains an important zoonoses,which is officially classified as B infectious disease in China. Human brucellosis is endemic in several provinces, including Inner Mongolia, Shanxi, Heilongjiang, Hebei and Xinjiang 3 .
Further, there is evidence that the disease has spread from endemic to non-endemic areas, and has taken on the changing epidemiology. The incidence of brucellosis has been increasing annually since 2001, partly due to the well-developed animal (small ruminants and cattle) husbandry production in Shandong Province 4,5 .
Brucellosis, which commonly occurs in sheep, cattle, and pigs, is transmitted to humans through many routes, including the digestive and respiratory tract, but it is mainly spread through direct contact with the skin and mucous membrane 6 . It is a complex condition that has diverse clinical manifestations, an atypical onset, and its treatment period is also typically prolonged. Some of the common symptoms are irregular fever, fatigue, excessive sweating, anorexia, chills, muscle pain, joint pain, hepatosplenomegaly, and poor appetite 7 . Early diagnosis is difficult, and misdiagnosis or missed diagnosis is common in non-endemic areas 1 .If co-infection with other viruses occurs at the same time, the disease gets further complicated and is likely to increase in severity. This makes diagnosis and treatment more difficult. A likely agent of co-infection is Epstein-Barr virus (EBV), which is a common γ herpesvirus subfamily that has a high infection rate in population 8,9 . Often, this virus remains in a latent state and gets activated in a state of immune dysfunction. Further, the immune mechanism of EBV-latent-activated infection is very complicated 10,11 .
It is worthy to note that brucellosis is also re-emergering in Shandong Province 12,13 . However, studies report the detail clinical characteristics of human brucellosis from Shandong are lacking. Especially, previous information about brucellosis coinfection with EBV was unclear. In this study, cases of brucellosis with concomitant EBV infection were retrospectively analyzed. The clinical and laboratory features, as well as the treatment and prognosis of the co-infection were discussed for the first time..

Hospital and participants
This retrospective study was conducted at the Infectious Diseases Department of   B, and C), cytomegalovirus, were excluded. HIV was not detected in this study.

Methods
The 22 cases of brucellosis with concomitant EBV infection were analyzed using recorded hospital data, including epidemiology, clinical manifestations and complications. The laboratory examinations and clinical data included blood routine, biochemistry, urine routine, treatment plan, and prognosis. Patients with brucellosis alone (n = 100) were randomly selected as the control group. The age and sex distribution of the two groups was comparable, and the differences in clinical features were compared. The blood routine exam was conducted with the SYSMEX-XE-2100D automatic blood cell analyzer. The Roche COBAS-C-701 automatic biochemical analyzer was used to detect liver and kidney function. The AMPLLY9800 fully automatic PCR analyzer was used to determine the EBV DNA load by Taqman Real-time PCR The EBV DNA load of >1.0 ´ 10 2 IU/ml was considered to indicate EBV infection. Anti-IgM was also detected by ELISA.

Treatment
For brucellosis, doxycycline was administered two times a day at a dose of 100 mg each time, and rifampicin was administered once a day at a dose of 600 mg according to the guidelines of the Ministry of Health. Based on the temperature and complications of the patient, an alternate regimen recommended by the guidelines is a combination of levofloxacin (0.5 mg once a day), cotrimoxazole (two times a day, 1.0 mg each time), and amikacin (two times a day, 0.4 mg each time). The total duration of treatment was 18 weeks. Patients with concomitant EBV infection were first administered intravenously with ganciclovir (two times a day, at a dose of 7 with a total course of 21 days.

Statistical analysis
All statistical analyses were performed using the SPSS software (version 22.0; SPSS, Chicago, IL, USA). The distribution of variables in the groups was compared with the Chi-squared test or Fisher's exact test for categorical variables. P-values <0.05 were considered significant.

Ethics issues
The ethics committee of WYCH approved the study. Written informed consent has been obtained from all patients in accordance with the Declaration of Helsinki.

General information
The 22 cases of concomitant infection were considered as the observation group, and this group comprised 10 males and 12 females and had a median age of

Clinical manifestations
As shown in Table 1, the incidence of headache, hepatosplenomegaly and lymphnode enlargement was significantly higher in the observation group.

Complications
As shown in Table 3, a significantly higher number of patients in the observation group had abnormal hepatic enzymes and cardiac enzyme.

Follow-up and outcome
Satisfactory results were obtained in both groups. The therapeutically failure and relapses were not reported in our study after 18-weeks treatment. However, as evident in the Table 4 below, the recovery period and hospital stay were significantly longer in the observation group. The fever in the observation group lasted for a long time, and the abnormal hepatic enzymes and cardiac enzyme, were higher than those in the control group, and the average blood routine recovery and hospital stay were prolonged. EBV DNA load was all less than 1.0 ⋅ 10 2 IU/ml and anti-IgM was all negative after 1 months follow-up.

Discussion
Shandong province is divided into 17 prefectures, 137 counties, and 1,941 townships. In 2016, the annual incidence rates of human brucellosis rose to 4.1 cases per 100,000 population 15  Accordingly, the pathogenesis of brucellosis has also been found to involve the activation of microglia and cytotoxic T lymphocytes 19,20 .
In Importantly, it was found that the probability of abnormal hepatic enzymes and cardiac enzyme was significantly elevated in the concomitant infection group.
Rifampicin, a first-line drug for brucellosis, could also lead to abnormal hepatic enzymes. It is necessary to avoid rifampicin in the concomitant cohort. Therefore, second-line drugs and hepatoprotective drugs needed to be used at the same time, which led to the prolongation of treatment cycle and the increase of treatment cost.

Conclusions
This study describes the clinical, laboratory characteristics and prognosis of brucellosis patients with co-existing Epstein-Barr virus infection in Shandong, China.
Given that brucellosis is still endemic in this region, it is important to consider EBV infection and other potentially latent viral infections in patients with brucellosis.
Limitation of the study: The present study is retrospective in nature. Additionally, since most patients were not followed up regularly, the changes in EBV Cuiping Wu, Dongri Piao, and Wentao Yang conceived the study and contributed designing the study. Hai Jiang contributed designing the study, analysis and interpretation of data and reviewed the initial draft manuscript. All authors read and approved the final manuscript.