Abstract
-
Objective
The aim is to study the diagnostic positive rates of metagenomic next-generation sequencing (mNGS), microbial culture, and serologic testing in suspected native spinal brucellosis, and to evaluate the clinical value of their combined application.
-
Methods
In this multicenter, retrospective observational study, 128 patients with suspected native spinal brucellosis from 6 medical centers (February 2020 to February 2025) were enrolled. Specimens from infection sites were subjected to microbial culture, mNGS, and serological testing (agglutination test).
-
Results
Of the 128 patients with suspected native spinal Brucella infections, 118 patients were diagnosed with Brucella spondylitis. Among the 118 confirmed Brucella spondylitis cases, mNGS demonstrated a positivity rate of 92.37% (109 of 118), significantly higher than that of culture (26.27%, 31 of 118) and agglutination test (83.05%, 98 of 118). In the 87 culture-negative samples, mNGS detected Brucella in 91.95% (80 of 87), compared to 82.76% (72 of 87) by agglutination test. mNGS confirmed Brucella infection in all 16 cases that were agglutination test negative. mNGS combined with agglutination tests can effectively complement each other, improving the sensitivity of diagnosis and thereby minimizing missed diagnoses to the greatest extent. Among the 10 nonbrucellar spinal pathologies, agglutination test showed a high false-positive rate of 90% (9 of 10), whereas mNGS had a 10% (1 of 10) false-positive rate. Therefore, the agglutination test has a relatively high rate of false positives.
-
Conclusion
mNGS detection represents an effective adjunct to microbial culture and the agglutination test. The concurrent use of all 3 methods enhances diagnostic accuracy and reduces the likelihood of missed and incorrect diagnoses, significantly improving patient prognosis and guiding personalized clinical treatment.
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Keywords: Metagenomic next-generation sequencing, Spinal Brucella infections, Microbial culture, Agglutination test, Combined detection, Etiological diagnosis
INTRODUCTION
Brucellosis is a zoonotic infectious disease that affects both humans and animals worldwide, with more than 500,000 cases reported annually. The disease primarily affects organs rich in mononuclear phagocytes, such as the liver, spleen, lymph nodes, and bone marrow [
1,
2]. The incidence of brucellosis increased markedly during the coronavirus disease 2019 pandemic [
3]. Studies have shown that 10%–85% of brucellosis cases present with osteoarticular complications, with the spine being the most common and severe site of musculoskeletal
Brucella infection [
4,
5]. However, the clinical manifestations, laboratory tests, imaging findings, and histopathological examination of native spinal Brucella infections (SBIs) are not always specific and the diagnosis is often delayed, challenging the acquisition of timely and accurate etiological information [
6]. Early and accurate diagnosis is crucial for the treatment of native SBI.
Currently, the diagnosis of native SBI relies primarily on laboratory tests, with microbial culture remaining the gold standard for diagnosis [
7]. Literature reports indicate that the rate of positive blood cultures ranges from 10% to 90%, with the culture requiring between 1 and 4 weeks. Additionally, as the infection progresses,
Brucella enters macrophages, making bacterial isolation more difficult [
8,
9]. Serological tests such as agglutination tests and enzyme-linked immunosorbent assays (ELISAs) have shown limitations in both specificity and sensitivity, especially in the early stages of infection or during chronic infection phases [
9]. Since native SBI requires long-term treatment with a combination of antibacterial drugs, and this antibacterial treatment regimen is rarely used for other types of bacterial infections [
7], there is an urgent clinical need for a new less time-consuming diagnostic method with high specificity and sensitivity to guide the early and precise implementation of antibacterial strategies.
Metagenomic next-generation sequencing (mNGS), an emerging molecular diagnostic method, has shown significant potential in the diagnosis of infectious diseases in recent years [
10]. Previous studies have reported the use of mNGS in the identification of bone and joint spinal infections, showing that mNGS markedly improves the diagnosis of pathogenic microorganisms in orthopedic infections [
11-
13]. However, there are few studies on the application of mNGS in diagnosing suspected native SBI, and no studies using spinal tissue samples to evaluate the efficacy of mNGS in diagnosing native SBI. Therefore, we conducted a multicenter retrospective study of native SBI. Aiming to systematically evaluate and compare the positive detection rate and diagnostic accuracy of mNGS, microbial culture, and serologic testing in the etiological diagnosis of native SBI, and to explore the clinical significance of its application in combination with serologic testing (Rose Bengal plate agglutination test) and microbial culture, in order to improve the diagnostic and therapeutic levels of brucellosis.
MATERIALS AND METHODS
1. Study Design
A retrospective analysis was conducted on patients with suspected native spinal brucellosis who had visited one of the 6 academic tertiary referral centers between February 2020 and February 2025. Routine infection marker measurements and immunological tests were conducted on admission, and blood cultures were performed for febrile patients. Cultures of infected spinal lesion tissues and pathological examinations were undertaken to identify the responsible pathogens, along with mNGS testing for specific pathogen identification. Samples of the infected lesions were obtained via puncture under C-arm or computed tomography (CT) guidance, or during surgery. Traditional cultures were separated and identified using a fully automated bacterial culture analyzer operated by professional microbiology personnel. For mNGS, specimens were sent under strict aseptic conditions to testing centers. After diagnosis, all patients received a combination therapy of doxycycline and aminoglycoside antibiotics [
14].
1) Inclusion criteria
(1) An initial diagnosis of brucellosis spondylitis based on clinical symptoms, signs, laboratory tests (especially positive agglutination tests), microbial culture, imaging examinations, and mNGS detection
(2) Procurement of lesion specimens using C-arm fluoroscopy or CT-guided puncture, or during surgery
(3) Comprehensive and detailed information on examinations of the specimens, including microbiological culture, histopathological examination, and mNGS testing
(4) Complete and reliable clinical data
2) Exclusion criteria
(1) Incomplete clinical data or failure to complete all routine examinations and mNGS testing
(2) Obvious exogenous contamination of samples during submission
(3) Postoperative spinal infection or infection of adjacent tissues involving the spine
(4) Histopathological examination indicating the presence of spinal tumor or aseptic inflammation
(5) Microbiological culture indicative of pyogenic bacteria
(6) Mycobacterium tuberculosis culture, acid-fast staining, histopathology, molecular pathology, Xpert MTB/RIF test, any of the above test results indicating Mycobacterium tuberculosis infection
(7) Laboratory tests indicating the presence of fungi or parasites
Based on these criteria, a total of 128 patients with suspected native SBI based on the spinal infection data from the 6 medical centers were enrolled. The included cases are all over 14 years old and have no pregnant patients. The study protocol was reviewed and approved by the Ethics Committee of the Affiliated Hospital of Qingdao University (approval No. QYFY WZLL 30496). The study uses the clinical multidimensional comprehensive diagnostic criteria as the “gold standard.” It also establishes a spinal infection expert group consisting of spinal surgeons, infectious disease specialists, radiologists, and microbiology experts. The expert group performs a multidimensional clinical diagnosis by integrating the patient’s clinical history, laboratory tests, imaging studies, mNGS testing, and clinical responses to targeted antibacterial therapy (
Fig. 1).
1) Clinical diagnostic criteria
The early symptoms of native spinal brucellosis infection are relatively subtle and are nonspecific. Clinical diagnosis should be based on the patient’s medical history and the following relevant examinations for a comprehensive, multidimensional assessment: (1) Local spinal region pain, especially axial back pain, often accompanied by systemic symptoms such as fever, chills, and fatigue. Some patients may experience neurological dysfunction, and the course of the disease often follows a chronic or subacute course; (2) Abnormal elevation of infection indicators such as C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), and procalcitonin (PCT); (3) CT and/or magnetic resonance imaging examinations conducted during the lesion stage show local erosion of the upper and lower vertebral body angles, anterior or diffuse intervertebral disc collapse, possibly accompanied by the formation of paravertebral or epidural abscesses; (4) Isolation of Brucella spp. from lesion tissue, pus, or blood samples; (5) Serological tests (agglutination test for total
Brucella antibody titer>1:160, or positive ELISA, polymerase chain reaction [PCR] tests) suggest Brucella; (6) Epidemiological history: history of residence in endemic areas, contact with cattle or sheep, or consumption of unpasteurized dairy products; (7) Effective antibiotic treatment against Brucella [
1,
8,
15-
19].
Ultimately, a multidisciplinary expert team specializing in spinal infection, including spinal surgeons, infectious disease physicians, radiologists, and microbiology specialists, should conduct a comprehensive analysis to determine the definitive diagnosis of native SBI.
2) mNGS diagnostic criteria
There is currently no consensus on the diagnostic criteria for mNGS examinations in infectious diseases of the spine, and differences exist among different platforms [
20,
21]. This study references studies that used the same sequencing platform and similar tissue samples [
22,
23], and by making appropriate modifications based on the sequencing results of this dataset, the following criteria were formulated: Set the relative abundance of
Brucella ≥5% and the number of sequences ≥5 as the diagnostic threshold. In the case of specific sequencing results that do not meet the established criteria, comprehensive judgment by a multidisciplinary team consisting of clinicians, medical microbiologists, and bioinformatics specialists is required, and if necessary, review the raw data of mNGS detection for further analysis and determination.
Samples, such as spinal tissue, vertebral bone, or pus from the infected site, should be placed immediately in sterile containers after collection and stored at low temperatures (typically -80°C). Total DNA is then extracted from the samples using appropriate kits or extraction methods (e.g., enzymatic digestion, physical disruption). The quality of the DNA is assessed using quantitative fluorescence methods and quality control procedures, followed by fragmentation of the nucleic acids, addition of adapters, and PCR amplification for the construction of libraries suitable for sequencing. The libraries are then loaded onto an NGS platform for high-throughput sequencing, generating large amounts of short-read sequencing data. After quality control and assembly, the reads are aligned with database sequences, enabling annotation and identification of the pathogen. Further analyses include assessments of abundance, functional annotation, and detection of resistance and virulence-associated genes [
21].
Data were analyzed using IBM SPSS Statistics ver. 27.0 (IBM Co., USA). The study uses clinical multidimensional comprehensive diagnostic criteria as the gold standard to evaluate the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of mNGS detection, microbial culture, and agglutination tests. Measurement data were first subjected to normality tests. Data that conformed to normal distribution were expressed as mean±standard deviation, while data that did not conform to normal distribution were expressed as median and interquartile range. Categorical variables were expressed as ratios and proportions. According to the normality test, the difference in white blood cell before and after treatment conformed to normal distribution, thus a paired sample mean t-test was used. The differences in CRP, PCT, and ESR did not conform to normal distribution, thus the sign rank sum test for paired design data was used. The study employed t-test for paired sample means, sign rank sum tests for paired design data, and chi-square tests to analyze the data, with p<0.05 indicating statistical significance.
RESULTS
1. Characteristics of Study Subjects
A total of 128 patients with suspected
Brucella spondylitis underwent mNGS,
Brucella culture, and serological testing (agglutination test). Of these, samples from 73 patients were obtained via CT/C-arm guided puncture, while 55 patients had samples collected during surgical procedures. In this study, all surgical procedures followed standard aseptic principles. Moreover, the instruments or implants used during the operation did not introduce additional complexity to the subsequent antiinfection treatment options. The spinal infection expert group comprehensively evaluated the patients’ clinical history, laboratory findings, imaging studies, mNGS detection, and the patients’ favorable clinical response to targeted antibacterial treatment, and ultimately diagnosed 118 patients with native spinal brucellosis, 9 patients with non-
Brucella spinal infection, and 1 patient with nonspinal infection (Kummell disease). The 118 confirmed cases were predominantly middle-aged or older, with ages ranging from 23 to 81 years and an average age of 59.76±9.57 years. The cohort included 82 males and 36 females. Clinically,
Brucella spondylitis presented with a heterogeneous array of symptoms, including localized manifestations, such as low back pain, and systemic symptoms, including fever and fatigue. Epidemiologically, 24 patients had a clear history of exposure, such as contact with livestock or residing in
Brucella-endemic regions. The lumbar spine was the site most frequently affected, accounting for 85.59% (101 of 118) of cases, followed by the thoracic spine at 10.17% (12 of 118), indicating a predilection for these spinal regions (
Table 1). The histopathological main manifestations of 118 cases of native SBI are inflammatory cell infiltration (mainly neutrophils and lymphocytes), necrosis, and bone tissue destruction, etc. (
Table 2).
Following targeted antimicrobial therapy, all enrolled patients demonstrated marked clinical improvement with sustained stabilization, fulfilling the predefined criteria for discharge, namely, significant relief of clinical symptoms, improvement in infection indicators. In this study, all confirmed patients underwent standardized antibiotic treatment for 3 months. After this period, the decision to extend treatment duration was based on imaging results, blood infection markers, and clinical manifestations. A total of 118 patients with native SBIs were treated with a targeted antibiotic regimen, and the infection indicators were significantly lower than those before treatment, with statistically significant differences (p<0.01) (
Table 3).
In the cohort of 118 patients with native SBI, mNGS yielded positive results in 109 cases, all of which were monomicrobial. The mNGS detection sensitivity was 92.37% (109 of 118).
Brucella cultures (both blood and tissue) were positive in 31 cases, all of which were monomicrobial, resulting in a culture sensitivity of 26.27% (31 of 118). The Rose Bengal plate agglutination test showed positivity in 98 cases, with a sensitivity of 83.05% (98 of 118). mNGS detection clearly indicates that the species level of
Brucella in the spine is mainly
Brucella melitensis, and the culture is primarily
Brucella ovis (
Fig. 2A and
B).
The 9 cases of non-
Brucella spinal infection all showed positive results on the Rose Bengal plate agglutination test, while microbial culture was negative in all cases, and mNGS did not detect the presence of
Brucella in any case. This indicates that the Rose Bengal plate agglutination test has a marked false positivity rate. Of the 9 patients with non-
Brucella spinal infections, mNGS identified pathogens other than Brucella in 8 cases, while the remaining case was found by real-time fluorescent quantitative PCR for tuberculosis to have been caused by
Mycobacterium tuberculosis. Three cases in which mNGS detected
Mycobacterium tuberculosis infection underwent further real-time fluorescent quantitative PCR for tuberculosis, and the results were all positive (
Table 4).
Among 118 cases of native
Brucella spinal infection, positive mNGS results were found in 92.37% (109 of 118) of cases, which was significantly higher than the positivity rate of the microbial culture at 26.27% (31 of 118) (χ
2=74.20, p<0.05). The positive rate of mNGS detection is higher than that of the Rose Bengal plate agglutination test at 83.05% (98 of 118). However, the difference is not statistically significant (χ
2=3.45, p>0.05). The positivity rate of the Rose Bengal plate agglutination test was significantly higher than that of the microbial culture at 26.27% (31 of 118) (χ
2=58.30, p<0.05) (
Fig. 3B).
The data showed that mNGS detected Brucella infection in 91.95% (80 of 87) of the culture-negative samples. The Rose Bengal plate agglutination test indicated Brucella infection in 82.76% (72 of 87) of the culture-negative samples. In 20 cases where the Rose Bengal plate agglutination test was negative, mNGS confirmed Brucella infection in all cases. In 9 cases where the mNGS results were negative, the Rose Bengal plate agglutination test was positive.
4. Diagnostic Value of Combined mNGS, Culture, and Agglutination Tests
In 118 cases of native spinal brucellosis, the positivity rate of microbial culture together with mNGS was 94.06% (111 of 118), higher than that of mNGS detection alone. The positivity rate of microbial culture combined with the Rose Bengal plate agglutination test was 88.14% (104 of 118), higher than that of the Rose Bengal plate agglutination test alone but lower than that found by mNGS detection. Among the 9 subjects with negative mNGS results, the agglutination tests were all positive, and positive microbial culture results were also obtained in 2 cases, representing traditional etiological diagnostic evidence. In these 20 subjects with negative agglutination tests, mNGS indicated
Brucella infection, and 5 of these cases had positive microbial culture results (
Table 5A). Therefore, the combination of mNGS testing and agglutination tests helps to minimize missed diagnoses to the greatest extent.
Among the 118 cases of native SBI, 29 were positive in both mNGS and microbial culture; 89 were positive in both mNGS and the Rose Bengal plate agglutination test, and these included 3 cases of partial consistency, where mNGS detected multiple pathogens including
Brucella. 26 cases were positive in both the Rose Bengal plate agglutination test and microbial culture. There were 7 cases in which both mNGS and microbial culture were negative, no cases where both mNGS and the Rose Bengal plate agglutination test were negative, and 15 cases where both the Rose Bengal plate agglutination test and microbial culture were negative. 82 cases showed inconsistent results between mNGS detection and microbial culture, while 29 cases had inconsistent results between mNGS and the Rose Bengal plate agglutination test, and 77 cases with inconsistent results between the Rose Bengal plate agglutination test and microbial culture (
Fig. 4).
Among the 128 suspected cases of native SBIs included in this study, the false-positive rate of the Rose Bengal plate agglutination test was 90% (9 of 10), while that of mNGS detection was 10% (1 of 10). The sensitivity, specificity, PPV, NPV, Kappa value, among mNGS detection, microbial culture, and the Rose Bengal plate agglutination test are detailed in
Tables 5B,
C, and
6. The Kappa value of mNGS detection is 0.60 (0.4–0.75), which is highly consistent with clinical diagnosis, while the consistency of culture and agglutination tests with clinical diagnosis is poor (≤0.4) (
Fig. 3A and
B).
DISCUSSION
Brucella spondylitis, a specific osteoarticular infection, is characterized by nonspecific clinical manifestations, chronicity, propensity for recurrence, and the likelihood of severe complications, making its diagnosis and treatment a persistent challenge in clinical practice. The treatment of brucellosis requires combination antibiotics (such as dual therapy of doxycycline+ aminoglycoside antibiotics) [
14,
24-
26]. Therefore, in cases where the pathogenic microorganism is unknown, the empirical use of antibiotics rarely includes
Brucella, often leading to delayed treatment and increased risk of complications. In recent years, the rapid development of mNGS has provided a new solution for the etiological diagnosis of infectious diseases [
27]. This study retrospectively analyzed the clinical data of 118 patients with
Brucella spondylitis from different medical centers and explored the significance of the combined application of mNGS, serological testing (the Rose Bengal plate agglutination test), and microbial culture to evaluate the diagnostic value of mNGS in
Brucella spondylitis, to provide a more reliable detection technique for the diagnosis of
Brucella spondylitis.
Microbial culture has always been the gold standard for the diagnosis of brucellosis. However, the present study found its positivity rate to be only 26.27%, significantly lower than the positivity rate of mNGS (92.37%).
Brucella is a facultative intracellular parasite. In the later stages of infection, the bacteria often hide within macrophages or form biofilms, significantly reducing the success rate of culture [
28,
29]. In contrast, mNGS detects pathogen nucleic acid fragments in samples directly, obviating the need for viable bacteria required by traditional cultured [
30].
In this study, 9 cases showed negative results in mNGS testing but were ultimately diagnosed as native spinal brucellosis by the spinal infection expert group. The diagnostic basis includes the following points: First, all 9 patients had positive agglutination tests, providing strong serological evidence. Second, 2 of these cases yielded positive microbial cultures, which is traditional etiological diagnostic evidence. Finally, all cases exhibited a typical clinical presentation and imaging features consistent with native spinal brucellosis, and showed a good clinical response to standardized anti-
Brucella treatment. Therefore, the study concludes that these 9 patients are not “false positives of the agglutination test.” In clinical practice, when mNGS shows false negatives, possible reasons include: (1) low pathogen load in local lesions; (2)
Brucella, being an intracellular parasite with a thick cell wall, may resist conventional extraction methods, resulting in insufficient lysis and inadequate nucleic acid release; and (3) a large amount of host cell nucleic acid in the infected tissue may dilute the pathogen signal, affecting the detection efficiency of microbial sequences. Therefore, it is essential to rely on other strong evidence (such as serology, culture, and treatment response) for comprehensive diagnosis, highlighting the necessity and importance of multidimensional comprehensive diagnosis to overcome the limitations of single testing. In this study, mNGS was observed to detect
Brucella in 91.95% (80 of 87) of culture-negative cases, fully demonstrating its high sensitivity. This advantage is particularly relevant in chronic infections or cases where bacterial activity is reduced after antibiotic exposure. Additionally, the time required for mNGS is 24–48 hours, significantly shorter than the 7–14 days required for culture, a crucial difference in the treatment of acute spinal infections. The positivity rate of the Rose Bengal plate agglutination test was 83.05%, not significantly different from that shown by mNGS detection. However, the Rose Bengal plate agglutination test was found to have a false-positive rate. In this study, the Rose Bengal plate agglutination test suggested
Brucella infection in 9 cases; however, when taking clinical manifestations and multiple examinations into account, the cases were ultimately confirmed to be non-
Brucella spinal infections, with the most common pathogens were
Mycobacterium tuberculosis (4 cases). The causes of the false-positive results in the the Rose Bengal plate agglutination test in this study may include: (1) cross-reactivity due to similarity between antibodies against
Brucella lipopolysaccharides and other bacterial antibodies; (2) the presence of specific antibodies (such as IgG) resulting from previous
Brucella infections; (3) specific antibodies in livestock workers caused by exposure to animal vaccines (such as the Rev-1 vaccine), showing positive results [
9,
31-
33]. Nevertheless, the mNGS results are not absolutely reliable. In this study, mNGS identified streptococcal infection in case 118, while the Rose Bengal plate agglutination test and pathological results both suggested
Brucella infection. After discussion by the panel of spinal infection experts, the final diagnosis was
Brucella infection, which was further confirmed during subsequent clinical treatment. It is possible that the sample may have been contaminated. Despite the high sensitivity of mNGS, it can be affected by sample contamination or interference by commensal bacteria [
34]. It is worth noting that combining mNGS detection with agglutination tests can reduce the rate of missed diagnoses, but both have an inherent false-positive rate. Therefore, when there is inconsistency between mNGS detection and agglutination tests (i.e., mNGS positive but agglutination test negative, or mNGS negative but agglutination test positive), the spinal infection expert group will conduct a comprehensive analysis based on clinical history, laboratory testing, imaging examination, and the status of antiinfective treatment. If necessary, agglutination tests, microbial culture, or even a repeat puncture for mNGS detection will be performed.
In this study, 10 subjects with negative test results (including 9 with nonbrucellar spinal infections and 1 with nonspinal infections) were thoroughly reviewed by a spinal infection expert group to assess the possibility of sampling errors. More specifically, among these 10 patients, samples from 3 patients were obtained via CT/C-arm-guided puncture, while samples from the other 7 patients were collected during open surgery. It was determined that these negative results were not due to sampling errors for the following reasons: First, among these 10 patients, 9 had positive serological agglutination tests. However, the final comprehensive clinical assessment (including response to targeted antibiotic treatment, subsequent imaging examinations showing no progression of infection, and other laboratory test results) did not support a diagnosis of brucellosis, thus more likely indicating false-positive serological results. Second, in the 3 cases where
Mycobacterium tuberculosis infection was detected by mNGS, further confirmation was obtained through real-time fluorescence quantitative PCR testing for tuberculosis. Finally, all samples were collected ensuring they were taken from the core areas of lesions indicated by imaging (for puncture samples) or from infection foci under direct vision during surgery (for surgical samples), and the sample volume was sufficient. Therefore, we believe that these negative results are more likely related to disease status (serological cross-reactivity) or the inherent limitations of detection technology, rather than improper sampling. More importantly, spinal mixed infections mainly occur postoperatively and after trauma. The spine itself is a sterile environment, and polymicrobial infections of the spine are extremely rare under natural conditions, so caution should be exercised during diagnosis [
23]. In this study, the 9 nonbrucellar spinal infections and 3 cases with multiple pathogens including
Brucella detected by mNGS were traced back by the expert group regarding their medical history, clinical picture, imaging examinations, laboratory tests, and responses to targeted anti-infection treatments, with no evidence of mixed infections found. Therefore, the 9 nonbrucellar spinal infection subjects do not meet the criteria for spinal mixed infection. Among the 3 subjects with multiple pathogens indicated by mNGS, the sequence count and relative abundance of
Brucella ranked first in the report, and the agglutination test was positive. Although culture did not provide direct evidence, all 3 cases had other auxiliary evidence supporting
Brucella infection. For these 3 cases, the spinal infection expert group determined that the other microorganisms detected were more likely background flora, while
Brucella was the pathogenic pathogen. Therefore, this study suggests that when interpreting mNGS results, careful interpretation must be combined with clinical context to avoid overdiagnosis of mixed infections. A recent study involving 492 cases of human brucellosis reported that 187 patients consumed unpasteurized dairy products [
35]. Another study involving 71 cases of brucellosis indicated that 66% of patients had a clear history of
Brucella exposure [
36]. In this study, only 24 patients reported a clear exposure history (such as contact with livestock or living in areas with high
Brucella prevalence), indicating a low rate of reported exposure. We analyzed that this may be due to several factors: (1) As a multicenter retrospective study, the depth and focus of medical history collection may vary among clinical doctors from different centers, potentially leading to some patients’ exposure histories not being systematically recorded or thoroughly inquired. (2) Brucella infection has a long incubation period and diverse clinical manifestations, making it difficult for some patients to accurately recall or associate non-recent, atypical exposure histories. (3) With increasing social mobility and the complexity of food supply chains, infections caused by indirect, nonoccupational exposure through consumption of contaminated dairy products or meats are becoming more common, and such exposure histories are more easily overlooked or underreported by patients during routine inquiries.
In cases where Brucella was clearly identified, microbial culture confirmed the species level in 38.71% (12 of 31) of cases, while mNGS confirmed the species level in 77.06% (84 of 109) of cases. Compared to culture, mNGS testing can further clarify the species level of Brucella, which is of great significance for tracing the source of infection, conducting epidemiological investigations, and predicting disease severity.
The emergence of mNGS technology represents a significant innovation in the clinical diagnosis and treatment of infectious diseases. Although microbial culture remains the gold standard for the diagnosis of native SBIs, the positivity rate of culture (26.27%) is low and cannot meet clinical needs. While the positivity rate of the Rose Bengal plate agglutination test is relatively high (83.05%), this study found a high false-positive rate. The clinical manifestations of native SBIs are diverse and often lack specific imaging features. Relying solely on one testing method may lead to misdiagnosis or missed diagnosis. In this study, the agglutination test yielded a positive rate of 83.05% (98 of 118), while that of mNGS detection was 92.37% (109 of 118). The increase in mNGS detection for diagnostic improvement is limited in terms of additional detection rate. Nevertheless, this study shows that both mNGS detection (7.63%, 9 of 118) and agglutination tests (16.95%, 20 of 118) exhibit false negative rates, and the combined application of multiple detection methods can complement one another and reduce the missed diagnosis rate. Therefore, we believe that this investment in patient health is worthwhile. Therefore, based on the findings of this study, for clinically suspected cases of native Brucella spinal infection, it is recommended to use the triple-testing approach of culture (blood/tissue)+mNGS+agglutination test (Rose Bengal plate or tube) to maximize diagnostic performance and reduce the possibility of missed diagnoses, making the diagnosis closer to perfection. For mNGS test results, it is suggested to establish strict diagnostic criteria based on specific characteristics (e.g., relative abundance of Brucella >15%, and sequence count >5), and form a multidisciplinary team of experts (spine surgeons, medical microbiologists, radiology expert, and clinical infection specialists) for comprehensive diagnosis.
Despite the significant advantages of mNGS in diagnosing native SBIs, there remain specific limitations to its clinical application. These include: (1) a lack of unified diagnostic standards for sequencing results, as well as potential differences in interpretation across different sequencing platforms and databases; (2) interference from host nucleic acids and background bacteria; (3) a testing cost ($500) that is significantly higher than that of culture and serological testing ($120).
The limitations of this study include: (1) Clinical heterogeneity constraints, as the study covered 6 medical centers and specimen collection, preservation, and testing procedures may vary across centers; (2) Cases with inconsistent test results were not assessed further using PCR or other testing methods; (3) As a retrospective study, the final diagnosis was determined by a spinal infection expert group based on a comprehensive assessment of multidimensional data, including clinical, imaging, laboratory, and follow-up information, as well as mNGS results. Therefore, the reference diagnosis is not completely independent of the mNGS tests to be evaluated, which may theoretically introduce bias into the calculated sensitivity and specificity, which represents an inherent limitation of this study design; (4) The number of nonspinal brucellosis cases in this study is small (n=10), and this disproportionate sample size may lead to instability in the NPV and Kappa statistics, thereby affecting the clinical significance assessment of the exclusion performance of negative mNGS results. Therefore, the diagnostic performance indicators reported in this study should be considered preliminary results. Future validation is needed in larger and more balanced cohorts, especially by expanding the nonbrucellosis control group, to further confirm the stability of these indicators.
CONCLUSION
The introduction of mNGS represents a revolutionary advancement in medical science. The present study highlights the substantial benefits of mNGS testing for the etiological identification of native spinal brucellosis, demonstrating that combining mNGS with agglutination tests helps to minimize the possibility of missed or misdiagnosis. Clinically, a tripartite strategy involving “mNGS+culture+agglutination test,” interpreted by multidisciplinary experts, is recommended to optimize diagnostic accuracy. Despite the higher cost and lack of standardization associated with mNGS, it represents a rapid and highly sensitive molecular diagnostic tool, offering an innovative approach for the early and precise diagnosis and treatment of native spinal brucellosis.
NOTES
-
Conflict of Interest
The authors have nothing to disclose.
-
Funding/Support
This work was supported by grants from the National Key Research and Development Program (2023 YFC2812004); Qingdao Natural Science Foundation (24-4-4-zrjj-154-jch); Qingdao City Healthcare Key Discipline Construction Project; the Technological Innovation Capability Improvement Project for Small and Medium-Sized Technology-Based Enterprises in Shandong Province (2023TSGC051l); Shandong Provincial Natural Science Foundation General Project (ZR2024 MH251); Shandong Province Medical and Health Science Project (202404070869).
-
Acknowledgments
The author thanks the research volunteers, the clinical staff conducting the trials, and the members of the clinical research team.
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Author Contribution
Conceptualization: LL, YL, YW, CY, WW, FS, ZH, SH, PC, TT, QL, XC, HZ, TP, XY, XP, XL, TW; Data curation: QZ, HW, ZZ, JL, YL, XL; Formal analysis: ZL, QZ, HW, ZZ, JL, XL; Funding acquisition: TW; Methodology: LL, YL, YW, CY, WW, FS, ZH, SH, PC, TT, QL, XC, HZ, TP, XY, XP; Writing – original draft: ZL; Writing – review & editing: ZL.
Fig. 1.Flowchart of the enrolled patient selection. mNGS, metagenomic next-generation sequencing.
Fig. 2.Distribution map of Brucella species in the spine. (A) mNGS detected the distribution of Brucella species in the spine (N=109). (B) Microbial culture detected the distribution of Brucella species in the spine (N=31).
Fig. 3.(A and B) Comparison of the diagnostic performance of culture, agglutination test, and mNGS detection for spinal Brucella infections. mNGS, metagenomic next-generation sequencing; PPV, positive predictive value; NPV, negative predictive value. ns, not significant. *p<0.05, statistically significant differences.
Fig. 4.Analysis of the consistency and inconsistency of 3 methods: cultivation, agglutination test, and metagenomic next-generation sequencing detection.
Table 1.Basic characteristics of the research population (N=118)
Table 1.
|
Characteristic |
Value |
|
Age (yr) |
59.76 ± 9.57 |
|
Sex, male:female |
82:36 |
|
Infection site |
|
|
Cervical vertebra |
3 (2.54) |
|
Thoracic vertebra |
12 (10.17) |
|
Lumbar vertebra |
101 (85.59) |
|
Thoracolumbar spine |
2 (1.69) |
|
History of exposure to high-risk factors |
|
|
Yes |
24 (20.34) |
|
No |
94 (79.66) |
|
Antibiotic history |
|
|
Applied |
66 (55.93) |
|
Not applied |
52 (44.07) |
|
Underlying disease |
|
|
Diabetes |
12 (10.17) |
|
Hypertension |
28 (23.73) |
Table 2.Histopathological features of spinal Brucella infection (N=118)
Table 2.
|
Histopathological features |
No. (%) |
|
Basic inflammatory pattern |
|
|
Chronic suppurative inflammation |
66 (55.93) |
|
Acute inflammatory activity |
39 (33.05) |
|
Inflammatory cell infiltration (neutrophils, lymphocytes) |
70 (59.32) |
|
Bone tissue destruction, necrosis, and dead bone |
29 (24.58) |
|
Fibrous tissue or vascular proliferation |
18 (15.25) |
|
Granulation tissue hyperplasia |
16 (13.56) |
|
Rod-shaped or club-shaped bacteria |
2 (1.69) |
|
Acid-Fast Bacilli (-) |
118 (100) |
|
No obvious abnormalities were observed |
13 (11.02) |
Table 3.Changes in infection indicators before and after the targeted anti-infection treatment
Table 3.
|
Infection indicators |
Before treatment |
After treatment |
Mean difference (95% CI) |
t-value/z-value |
p-value |
|
WBC (× 10⁹/L) |
4.99 (4.34–6.49) |
4.86 ± 1.50 |
0.63 (0.27–0.99) |
3.46†
|
< 0.01 |
|
CRP (mg/L) |
28.59 (12.43–58.88) |
9.61 (4.45–17.79) |
- |
-6.03‡
|
< 0.01 |
|
PCT (μg/L) |
0.34 (0.10–0.70) |
0.10 (0.10–0.10) |
- |
-5.11‡
|
< 0.01 |
|
ESR (mm/hr) |
49.01 ± 27.89 |
19.00 (11.00–36.75) |
- |
-6.64‡
|
< 0.01 |
Table 4.Methods for detecting non-Brucella spinal infection cases (9 cases)
Table 4.
|
Case No. |
Culture |
Agglutination test |
mNGS |
QPCR |
Clear diagnosis |
|
1 |
− |
+ |
− |
+ |
Mycobacterium tuberculosis
|
|
2 |
− |
+ |
Mycobacterium tuberculosis
|
+ |
Mycobacterium tuberculosis
|
|
3 |
− |
+ |
Staphylococcus epidermidis
|
− |
Staphylococcus epidermidis
|
|
4 |
− |
+ |
Parvimonas micra
|
− |
Parvimonas micra
|
|
5 |
− |
+ |
Mycobacterium tuberculosis
|
+ |
Mycobacterium tuberculosis
|
|
6 |
− |
+ |
Mycobacterium tuberculosis
|
+ |
Mycobacterium tuberculosis
|
|
7 |
− |
+ |
Mycobacterium tuberculosis
|
+ |
Mycobacterium tuberculosis
|
|
8 |
− |
+ |
Escherichia coli
|
− |
Escherichia coli
|
|
9 |
− |
+ |
Staphylococcus aureus
|
− |
Staphylococcus aureus
|
Table 5.
2×2 Contingency tables
(A) Cross-analysis of mNGS and agglutination test diagnostic results
Table 5.
|
Agglutination test |
mNGS
|
Total |
|
Positive |
Negative |
|
Positive |
89 |
9 |
98 |
|
Negative |
20 |
0 |
20 |
|
Total |
109 |
9 |
118 |
Table 5.(B) The agglutination test and the 2×2 contingency table of clinical multidimensional comprehensive diagnostic criteria
Table 5.
|
Agglutination test |
Clinical multidimensional comprehensive diagnostic criteria
|
Total |
|
Positive |
Negative |
|
Positive |
98 |
9 |
107 |
|
Negative |
20 |
1 |
21 |
|
Total |
118 |
10 |
128 |
Table 5.(C) The agglutination test and the 2×2 contingency table of clinical multidimensional comprehensive diagnostic criteria
Table 5.
|
mNGS |
Clinical multidimensional comprehensive diagnostic criteria
|
Total |
|
Positive |
Negative |
|
Positive |
109 |
1 |
110 |
|
Negative |
9 |
9 |
18 |
|
Total |
118 |
10 |
128 |
Table 6.The diagnostic performance of 3 detection methods: microbial culture, agglutination test, and mNGS
Table 6.
|
Testing method |
Accuracy (%) |
Sensitivity (%) |
Specificity (%) |
PPV (%) |
NPV (%) |
Kappa value |
|
Culture |
32.03 |
26.27 |
100.00 |
100.00 |
10.31 |
0.053 |
|
Agglutination test |
77.34 |
83.05 |
10.00 |
91.59 |
4.76 |
-0.046 |
|
mNGS |
92.19 |
92.37 |
90.00 |
99.09 |
50.00 |
0.60 |
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, Qiang Zhang2
