A multi-center cross-sectional study was conducted in five cities in China, namely Bayannur City in Inner Mongolia Autonomous Region, Ili and Changji City in Xinjiang Uygur Autonomous Region, and Jinan and Dongying City in Shandong Province, from December 2019 to August 2021. These cities were selected based on their high incidence rates of brucellosis, being among the top 10% in their respective provincial-level administrative divisions (PLADs). All project sites were designated medical institutions for brucellosis treatment and had the necessary capacity to conduct the required examinations and laboratory tests. To maintain biosafety, Brucella blood cultures were performed at CDC laboratory, while other tests were carried out at the project sites. The laboratory test results were consistent across all project sites. The study protocol was approved by the Research Ethics Review Committee of the Chinese Center for Disease Control and Prevention (Approval number: 201942), and signed informed consent was obtained from all participants prior to the investigation.

A cross-sectional study was conducted to estimate the sample size for assessing the prevalence of focal complications in human brucellosis. The “Confidence Intervals for One Proportion” module from Power Analysis and Sample Size Software (Version 15, NCSS LLC., East Kaysville, Utah, United States) was used to perform the sample size calculation. Based on a previous study reporting a range of brucellosis complication rates from 27.7–89.7% [11, 14], we chose a conservative estimate of 50% to ensure the maximum sample size. Considering confidence levels: 95% confidence intervals (CIs) formula was Exact (Clopper-pearson), the width of the CIs (two sided) was 10%, the dropout rate was 20%, the minimum sample size was 503 individuals. Ultimately, a total of 880 brucellosis patients were included in the study.

A multi-stage random sampling method was utilized to select specific hospitals and patients with human brucellosis. In regions of Inner Mongolia, Xinjiang, and Shandong with high incidence rates, five county-level designated medical institutions were chosen. From December 2019 to August 2021, doctors from these designated medical institutions enrolled eligible brucellosis patients who met the criteria, following the principle of informed consent. Enrollment continued until the desired sample size was achieved.

The inclusion criteria for cases were as follows: (1) patients diagnosed with brucellosis; (2) aged 15 years and above; (3) provided informed consent and willingly participated in the study.

The criteria for case exclusion were: (1) infected with the human immunodeficiency virus (HIV), undergoing chemotherapy, having other immune system disorders, or suffering from other severe illnesses; (2) suffering from mental disorders, deafness and other disorders resulting in poor communication and inability to cooperate with the investigation; (3) pregnant woman.

The definition of confirmed cases of brucellosis includes patients who have both a history of exposure and clinical symptoms, and meet one of the following criteria: (1) Isolation and culture of Brucella bacteria from the patient's blood or other specimens; (2) A serum agglutination test (SAT) titer of ≥ 1:100 (++) or a duration of illness of over one year with a titer of ≥ 1:50 (++); (3) A Coombs test titer of ≥ 1:400 (++).

After obtaining informed consent, each patient diagnosed with brucellosis was interviewed by a qualified physician using a standardized questionnaire. The purpose of the questionnaire was to gather the following information: gender, age, nationality, occupation, region of residence, level of education, exposure to animals (specifically sheep & goats, cattle, etc.), methods of animal exposure (such as raising, grazing, slaughter, delivering lambs, etc.), consumption of unpasteurized food, family history of brucellosis, and number of days since symptoms onset. Additionally, relevant variables from the Hospital Information System were extracted, such as date of diagnosis, presence of underlying diseases, previous history of brucellosis, symptoms and signs experienced (such as fever, chills, fatigue, etc.), complications, and laboratory test results.

A standardized protocol was implemented to ensure consistency in interviewer training and quality control supervision throughout all survey instances. Cases were included based on specific inclusion and exclusion criteria. Face-to-face interviews were conducted with all eligible cases by physicians who received professional training. Each questionnaire underwent thorough review by qualified supervisory staff. Data management specialists checked the collected questionnaires for completeness and logical consistency. The laboratory tests were carried out by properly trained laboratory technicians following national standardized methods and procedures.

The clinical stages of brucellosis were classified as follows: the acute stage, which lasted less than 3 months from the onset of symptoms to admission; the subacute stage, which lasted from 3 to 6 months; and the chronic stage, which lasted longer than 6 months. Age was calculated by determining the time period between the date of study participation and the date of birth for each brucellosis case. Fever was defined as axillary temperature of > 37.3 °C. Anemia: hemoglobin (Hb) female and children < 110 g/L, male < 120 g/L. Leukopenia: white blood cells (WBC) < 4 × 109 /L; Leukocytosis: WBC > 10 × 109 /L; Thrombocytopenia: platelet < 100 × 109 /L. ESR elevation: female > 20 mm/h and male > 15 mm/h.

In this study, we defined “focal complication” as a patient with symptoms of brucellosis who has at least one affected organ and tests positive for brucellosis through serology or culture. We defined “osteoarticular involvement” as the presence of inflammatory signs (swelling, pain, functional disability, heat, or redness) in any peripheral osteoarticular location, along with radiographic evidence of abnormalities. Peripheral arthritis was diagnosed based on clinical findings of joint swelling, effusion, and limited motion, as well as X-ray imaging. Sacroiliitis, spondylitis, and paravertebral abscesses were diagnosed based on clinical findings, as well as bone scans or magnetic resonance imaging (MRI). Neurological complications were confirmed in cases where the patient had a positive Brucella culture and/or positive blood or spinal Brucella culture with abnormal cerebrospinal fluid, as well as symptoms and signs of encephalitis or meningitis, while excluding other neurological diseases. Cardiovascular complications were identified by the presence of signs and symptoms such as heart murmur, retrosternal pain, and abnormalities in electrocardiogram (ECG) or ultrasonic cardiogram (UCG), after ruling out other causes and/or with positive Brucella culture in cases of pericardial effusion. Hematological complications were diagnosed based on abnormal clinical manifestations (anemia or bleeding) and abnormal laboratory findings, while excluding other causes. Genitourinary complications such as Orchitis, epididymitis, and pelvic inflammation were diagnosed based on signs and symptoms of urogenital system inflammation (orchialgia, testicular enlargement, and lower back pain in men; lower abdominal pain in women), confirmed by ultrasound. Respiratory system complications presented as bronchitis, pneumonia, or pleural effusion, and could be confirmed through chest X-rays, computed tomography (CT) scans, or MRI scans once other possible causes were ruled out. Cutaneous complications were defined as clinical manifestations of skin rash, purpura, ecchymosis, erythema nodosum, ulceration, or abscess in confirmed cases of brucellosis, while excluding other causes.

Data storage was done using EpiData version 3.1 (EpiData Association, Odense, Denmark). The normality assumption for quantitative variables was assessed using the Kolmogorov–Smirnov (KS) test with Lilliefors correction for significance. Non-normally distributed variables were described using median and inter-quartile ranges (IQRs). Categorical variables were described using frequency and percentage. Differences in proportions were tested using Pearson’s χ2 test or Fisher’s exact test. Logistic regression models were used to investigate the association between brucellosis complications and demographic, clinical, and laboratory characteristics. Univariate logistic regression was used to screen variables, considering P < 0.1 as statistically significant. Correlation analyses were performed using Spearman’s or Pearson’s correlation, and variables with strong correlations were removed. Factors selected from the screening were included in a multivariate analysis using a backward stepwise procedure. Multiple categorical variables were included in the model as dummy variables. Odds ratio (OR) and 95% CIs were calculated for categorical variables using a two-tailed test. Model evaluation and refinement were conducted using covariance checking, overdispersion correction, and other methods. Receiver operating characteristic (ROC) curves were generated to determine optimal cutoff values for diagnosing complicated brucellosis. Sensitivity and specificity were computed for each parameter in distinguishing complicated from uncomplicated brucellosis, and the area under the curve (AUC) was calculated. Data analysis was performed using SAS version 9.4 (SAS Institute Inc., Cary, USA). All tests were two-sided with significance set at P ≤ 0.05.