A 55-year-old male shepherd without preexisting diseases began to experience an intermittent fever up to 38–39 °C and back pain on July 1, 2018. The patient was started on oral cephalosporin for 3 months (self-administered), but his symptoms persisted. The patient was sent to the First Affiliated Hospital of Shihezi University with a normal body temperature at 36.6 °C, where he underwent transthoracic echocardiography (TTE) on October 11, 2018. Vegetation (0.7 × 1.5 cm) was discovered on the posterior leaflet of the mitral valve by a TTE examination (Fig. 1a), which caused a mild regurgitation. The serum tube agglutination test (SAT) for Brucella was positive with a titer of 1:800, but the culture result was negative. The blood test results showed that the hemoglobin levels were reduced to 120 g/L (see Additional file 1). The levels of platelets, RBC, and albumin were lower than the normal range. However, the levels of globulin and erythrocyte sedimentation rate (ESR) were higher than the normal range (see Additional file 1). A combination of treatment with doxycycline (200 mg/day) and rifampicin (900 mg/day) was administered to the patient. However, the clinical symptoms were not relieved, and the patient was experiencing a serious backache. The MRI examination revealed that a cold abscess had formed on both sides of the psoas major muscles and the left side of the erector spinae muscle (Fig. 2a), and the CT results indicated hyperplasia of the second and third vertebra (Fig. 2c) on October 15, 2018. The MRI showed a low signal intensity on the T1-weighted images and a high signal intensity on the T2-weighted images of the second and third vertebra. Because of financial constraints, the patient refused to undergo surgical treatment. Hence, he was prescribed oral doxycycline (100 mg/dose, twice a day) and rifampicin (600 mg/dose, once daily) as recommended by the World Health Organization.
Because of a lack of timely medical follow-up, on November 6, 2018, the patient’s condition deteriorated, the size of the vegetation increased to 0.8 × 2.5 cm (Fig. 1b) along with a hemorrhage that occurred on the gingiva, and a hemorrhagic spot formed on both lower limbs. An echocardiogram confirmed the presence of severe regurgitation. Additionally, the left atrium and ventricle were enlarged, and the left ventricular ejection fraction was 64%. The blood test results showed that the platelet level dropped rapidly over time, reaching its lowest level at 29 × 109/L on November 6, 2018 (see Additional file 1). The MRI examination showed that both the second and third vertebra hyperplasia and abscess in the muscle increased significantly on November 19, 2018 (Fig. 2b, d). The antibiotics for brucellosis were changed to levofloxacin (400 mg/dose, once daily), doxycycline (100 mg/dose, twice a day), and sulfamethoxazole tablets (600 mg/dose, once daily), and the patient was treated with medicines to enhance the platelet levels at the same time.
However, the patient’s situation deteriorated again, and heart failure was discovered on October 22, 2019. The size of the vegetation was 1.3 × 1.5 cm (Fig. 1c). The left atrium and ventricle were further enlarged, and the left ventricular ejection fraction was 58%. The end-diastolic volume (EDV) and end-systolic volume (ESV) of the left ventricular were 304 ml and 105 ml, respectively. A strong regurgitation signal presented on the mitral valve, with a pulse rate of 67 beats/min. The fraction shortening (FS) value was 36%. Although the platelet level had recovered to 52 × 109/L, the hemoglobin level sharply decreased to 85 g/L (see Additional file 1). The NT-proBNP levels reached 4152.0 pg/mL, 33 times higher than the normal level. It is worth noting that the level of creatinine was normal, except on October 22, 2019, when it reached its peak level at 252.6 umol/L (see Additional file 1). Additionally, the bone marrow aspiration result showed that the proliferation of bone marrow was considerably reduced, the granulocyte and erythrocytes were multiplicative, and the proportion of neutrophilic segmented granulocytes increased with the development of granulocyte lineage hyperplasia in the bone marrow (data not shown). At the moment of submission of this manuscript, the patient remains in bed at home because of severe debility caused by brucellosis. Now, the patient must undergo regular renal dialysis as a follow-up, and on June 26, 2020, the patient had renal dialysis. The clinical symptoms and diagnostic results at different times are listed in Additional file 1. The onset, diagnosis, and treatment of the disease in this patient are shown in Fig. 3.
Serological tests
The diagnosis of brucellosis was based on the rose bengal plate test (RBPT) and the SAT. The RBPT and SAT Brucella antigen were purchased from the Institute of Infectious Disease of the China Centers for Disease Control and Prevention. The SAT result was 1:800.
Pathogen isolation and identification
Five milliliters of venous blood was collected from the patient on October 11, 2018 and October 22, 2019, and the blood samples were injected into a biphase blood culture and incubated at 37 °C for 5 days. Conventional biological methods were used for the isolation and identification of the bacteria [4]. The minor phenotypic differences were used to distinguish the biovars of Brucella, including serotyping, phage typing, fuchsin and thionin dye sensitivity, CO2 requirement, H2S production, and metabolic properties, and Brucella melitensis 16 M was used as the reference strain. This process was completed at the Brucellosis Laboratory, the National Institute for Communicable Disease Control, and the Center for Disease Prevention and Control (CDC) of China in Beijing.
The specific sequences of the IS711 primers have been described in previous work [5]. The reaction system for the gene sequencing included 13 μL ddH2O, 15 μL master mix, 0.5 μL of each primer, and 1.5 μL of the DNA template. The amplification conditions were as follows: 95 °C for 5 min, 30 cycles at 95 °C for 2 min, 55 °C for 2 min, 72 °C for 2 min, and a final incubation at 72 °C for 4 min. The positive PCR products were purified using the TIAN-gel Mini Purification Kit (TIANGEN, Beijing, China) and sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). R version 3.6.1 was used to construct the tree according to the packages “ggplot2,” “ggtree” [6], and “colorspace.” The DNA extractions were performed using a whole bacterial genome nucleic acid extraction kit [Tiangen Biotech (Beijing) Co., Ltd., Beijing, China]. The IS711 primers were synthesized by the Sangon Biotech Co., Ltd. (Beijing, China).
