Abstract
Metagenomic next-generation sequencing (mNGS) in diagnosis of human brucellosis is comparatively unexplored. This report details five human brucellosis cases diagnosed using mNGS based on Illumina sequencing platform, comprising three females and two males, four with epidemiological exposure. In cases 1 and 2, plasma mNGS results showed one positive and one negative for Brucella melitensis, and subsequent blood cultures were both positive. Cases 3, 4 and 5 involved spinal brucellosis, some with paravertebral abscesses. mNGS from infectious tissue samples successfully detected Brucella, with read counts ranging between 30 and 1314, yet cultures were negative in cases 4 and 5. Following antibiotic and surgical treatments, all patients showed clinical improvement. This report shows mNGS testing enhances the detection sensitivity of brucellosis diagnosis.
Keywords: : Brucella melitensis, brucellosis, diagnosis, meta-genomic next-generation sequencing (mNGS), spinal brucellosis
Plain Language Summary
What is this summary about?
Brucella is a type of bacteria that can infect humans and animals. It causes a disease called brucellosis. Symptoms of brucellosis include fever and fatigue, among others. Meta-genomic next-generation sequencing (mNGS) is a tool for sequencing the DNA of bacteria. In this report, we use mNGS to diagnose human brucellosis in five cases.
What were the results?
Brucella was found in the blood of two infected people, but mNGS found Brucella in only one. Of three people with Brucella infection of the spine, mNGS found Brucella in the infected tissue but Brucella was only cultured in one case. Following antibiotic and surgical treatments, all five patients showed improvement of their symptoms.
What do the results of the study mean?
mNGS is a relatively rapid and effective diagnostic method that can improve the detection of Brucella in brucellosis.
Plain language summary
Article highlights.
Background
Human brucellosis is one of the most common bacterial zoonosis globally.Bacterial culture is considered the gold standard in diagnosis of human brucellosis. Metagenomic next-generation sequencing (mNGS) facilitates the detection of a wide array of pathogens, including rare, novel and atypical agents in complex infectious diseases.
Presentation of cases
This report details five human brucellosis cases. Blood cultures were positive in two patients with acute Brucella infection, but mNGS was negative in one patient. Of the three patients with Brucella spondylitis, infectious tissue mNGS testing results were all positive, with reads of 30–1314, whereas culture was positive in only one patient.
Conclusion
mNGS testing enhances the detection sensitivity of human brucellosis diagnosis.
1. Background
Brucella is a significant pathogen, affecting both humans and animals. It is responsible for brucellosis, one of the most common bacterial zoonosis globally, with an estimated 2.1 million new human cases annually [1]. The non-specific symptoms of human brucellosis often led to misdiagnoses. Additionally, its treatment poses challenges due to the need for prolonged administration of unique combinations of antimicrobial drugs, distinct from those typically used for other infectious diseases [2]. Consequently, accurate and early diagnosis of human brucellosis is critical.
The conventional microbiological diagnosis of human brucellosis includes culture, serology and nucleic acid amplification tests, each with its limitations. Bacterial isolation, while considered the gold standard, has variable sensitivity based on the organism's presence in the blood, ranging from 10 to 90% [3,4]. However, it typically require one to four weeks for detection, which can delay diagnosis and treatment. Serological tests, lacking objectivity and clarity, often yield results that are difficult to interpret and sometimes inconclusive [5]. However, the cost-effectiveness and simplicity make them preferable to bacterial isolation or nucleic acid amplification tests, particularly in resource-limited settings. Nucleic acid amplification tests, although more sensitive, safe and rapid than bacterial isolation and more specific than existing serological tests for diagnosing human brucellosis [6], cannot reliably indicate active infection [4].
Metagenomic next-generation sequencing (mNGS) has emerged as a valuable tool for diagnosing a range of infectious diseases [7–11]. However, its application in diagnosing human brucellosis remains relatively unexplored. At clinical laboratory of our hospital (the First Affiliated Hospital, Zhejiang University School of Medicine), mNGS performing on plasma cell-free nucleic acids (plasma mNGS assay) and other samples has been developed based on illumina NextSeq 500 platform since 2021, which greatly improves the diagnostic effectiveness of difficult critical infectious diseases [8,12,13]. The diagnostic sensitivity of the method could reach 95.2% [12]. Detailed experimental parameters about sample collection, nucleic acid extraction, library construction, sequencing and bioinformatic analysis are extensively outlined in our prior researches [7–9,12,13]. Library pools were loaded onto Illumina Nextseq 500 sequencer for a 50-cycle single-end sequencing (SE-50) run, producing around 20 million reads per sample. As negative controls, peripheral blood mononuclear cell samples were simultaneously prepared from healthy donors at a concentration of 105 cells/ml for each batch, following the identical protocol. Additionally, sterile deionized water was processed along with the samples as non-template controls. The pathogens (include Brucella) identified and their associated sequencing read counts (stringent mapped read number, defined as the read count per pathogen at a sequencing depth of 20 million reads) are communicated to the clinical treatment team by the clinical laboratory. In the past 2 years, several brucellosis cases have been identified in our hospital, with five patients eventually diagnosed with brucellosis following simultaneous blood or tissue culture and mNGS testing.
2. Presentation of cases
2.1. Case 1
On 20 April 2023, a 60-year-old man was admitted to our hospital with a ten-day history of fever (peaking at 40°C), chills and cough. Previously treated for community-acquired pneumonia at a regional hospital from 13 to 19 April, his condition had not improved despite intravenous antibiotics, including cefoperazone-sulbactam, imipenem-cilastatin and doxycycline (the dose and duration were unclear). His initial treatment was prompted by elevated C-reactive protein (CRP) levels of 229 mg/l and a computed tomography scan indicating inflammation in the right lung's middle lobe. The patient had a long-standing history of hypertension, controlled with daily amlodipine.
Upon admission, he presented with a high fever of 38.6°C. Blood tests showed a white blood cell (WBC) count of 6.01 × 109/l, neutrophil count of 5.28 × 109/l, platelet (PLT) count of 48 × 109/l, CRP of 192.59 mg/l, procalcitonin (PCT) of 1.47 ng/ml, alanine aminotransferase (ALT) of 116 u/l, total bilirubin of 65.0 μmol/l and a significantly elevated D-dimer level of 59,241 ug/l·FEU. A pulmonary artery CT scan revealed partial branch embolisms in the lower pulmonary arteries. Anticoagulation therapy with low molecular weight heparin commenced once platelet counts increased. Blood cultures and mNGS testing were conducted on 21 April. Two days later, plasma mNGS detected Brucella melitensis with a read count of 4. Following patient history review, it was revealed he had been raising sheep at home, leading to a suspicion of brucellosis. Consequently, treatment with omadacycline (initially 100 mg twice daily intravenously, then 100 mg daily intravenously) and levofloxacin (500 mg daily orally) was initiated alongside the ongoing intravenous anti-infective therapy. The diagnosis of brucellosis was confirmed on 27 April when blood cultures reported Brucella melitensis. By this time, the patient had been afebrile for 3 days. Post-discharge, he continued oral administration of omadacycline (300 mg daily orally) and levofloxacin (500 mg daily orally) for 6 weeks, with no further symptoms of discomfort.
2.2. Case 2
On 26 June 2023, a 67-year-old woman was admitted to our hospital, presenting with a 2-month history of fever and fatigue. Initially treated for community-acquired pneumonia at a regional hospital, she experienced temporary improvement with intravenous antibiotics. However, her symptoms persisted post-discharge, characterized by a continual sense of weakness despite no further temperature monitoring. On 21 June, she experienced a resurgence of symptoms, including fever (peaking at 38.3°C), fatigue and anorexia. Laboratory tests at a regional hospital's outpatient clinic showed normal WBC count and CRP levels, but her condition did not improve, leading to her hospitalization at our facility.
The patient had a 15-year history of hypertension, with the highest recorded at 180/100 mmHg, managed with amlodipine (5 mg daily orally), and her blood pressure was well-controlled after treatment.
Upon admission, her blood test results were as follows: WBC count of 3.46 × 109/l, hemoglobin (Hgb) 119 g/l, PLT count 154 × 109/l, atypical lymphocyte ratio 5%, CRP 4.5 mg/l, and PCT 0.07 ng/ml. Interestingly, plasma mNGS revealed no pathogens. Given the unclear etiology of her prolonged fever, a positron emission tomography-computed tomography scan was conducted three days post-admission. It showed multiple lymphadenectasis with the highest standard uptake value reaching 10.2, raising the possibility of lymphoma (Figure 1A). Subsequent bone marrow aspiration and lymph node biopsy ruled out neoplastic processes. However, two blood cultures taken upon admission later identified Brucella melitensis. The patient was then prescribed oral anti-infective therapy with doxycycline (100 mg twice daily orally) and rifampicin (600 mg daily orally). Her condition improved significantly, leading to her discharge. The medications were continued for 7 weeks before being discontinued.
Figure 1.
Imaging abnormalities in brucellosis patients. (A) In case 1, positron emission tomography-computed tomography revealed enlarged lymph nodes and abnormal metabolic activity. (B) In case 3, magnetic resonance imaging (MRI) depicted an infection in the third and fourth lumbar vertebrae, accompanied by surrounding abscess formation. (C) MRI in case 4 showed vertebral infection with peripheral abscess. (D) Abnormal signal patterns in the fourth and fifth lumbar vertebrae in case 5 as revealed by MRI.
2.3. Case 3
A 60-year-old woman, who presented with a two-month history of fever(maximum temperature 39°C) and lumbago, was admitted at our hospital on 5 October 2023. Initially, she was admitted to a primary hospital and was diagnosed with lumbar degeneration. After the application of glucocorticoids and other symptomatic treatments, her temperature returned to normal and lumbago improved. On 29 September, her lumbago worsened, and the fever recurred. The patient was admitted to a primary hospital, but the symptoms were not relieved, and the Rose-Bengal plate test was positive. The patient came to our hospital for further treatment.
The patient had a history of autoimmune disease, which had been treated with hydroxychloroquine for a long time. Brucellosis was diagnosed half a year ago and was cured.
On admission, her temperature was 38.3°C. Blood tests revealed the following: WBC 7.11 × 109/l, Hgb 84 g/l, PLT 352 × 109/l; CRP 26.54 mg/l; PCT 0.02 ng/ml. Magnetic resonance imaging (MRI) showed third and fourth lumbar vertebrae infection with surrounding abscess formation (Figure 1B). Surgery was performed on 7 October. Brucella melitensis was detected by tissue mNGS, with a reads count of 1314. Subsequently, tissue culture results showed Brucella spp. Doxycycline (100 mg twice daily orally) and rifampicin (450 mg daily orally) were used as anti-infective drugs. At the time of completing this report, the patient is still receiving anti-infective therapy.
2.4. Case 4
A 71-year-old woman was admitted to our hospital on 24 March 2022, with a history of a painless, encapsulated mass on her back for the past month. Initially, she sought treatment at a primary hospital, where a CT scan revealed an inflammatory lesion in the dorsal soft tissue, extending from the tenth thoracic vertebrae to the second lumbar vertebrae, consistent with an abscess. On 8 March, an abscess puncture and drainage procedure was conducted, with pus culture indicating Brucella spp. She received treatment with doxycycline (100 mg twice daily orally) and rifampicin (450 mg daily orally). A follow-up CT scan uncovered an infection involving the right 11th rib and the 9th and 10th thoracic vertebrae, leading to her referral to our hospital for further management.
Upon admission, the patient was afebrile with stable vital signs. Blood tests showed a WBC count of 5.61 × 109/l, Hgb of 107 g/l, PLT count of 259 × 109/l, CRP of 9.25 mg/l and PCT of 0.10 ng/ml. MRI indicated an infection from the 7th to the 11th thoracic vertebrae, along with a paravertebral abscess (Figure 1C). The patient subsequently underwent thoracic fusion surgery. Tissue samples from the lesion were sent for culture and mNGS. While no pathogens were identified in the culture, mNGS detected Brucella melitensis with a read count of 30. The anti-infective regimen was adjusted to include doxycycline (100 mg twice daily orally), rifampicin (450 mg daily orally) and moxifloxacin (400 mg daily orally). The patient was discharged and continued treatment for 6 months, after which her condition was deemed stable and medication was discontinued.
2.5. Case 5
A 55-year-old man, with a 5-month history of persistent lower back pain, was admitted to our hospital on 18 October 2022. Previously, he had been treated at a regional hospital for back pain, numbness in the lower limbs and fever, where he was diagnosed with brucellosis based on a positive agglutination test. His condition initially improved following a 6-week course of antibiotic therapy with doxycycline (100 mg twice daily orally) and rifampicin (600 mg daily orally). However, ten days prior to his current admission, his lower back pain exacerbated, accompanied by numbness in the lower limbs, fatigue and poor appetite.
Upon admission, his temperature was normal and vital signs were stable. Blood tests indicated a WBC count of 6.12 × 109/l, Hgb of 137 g/l, PLT count of 211 × 109/l, CRP of 12.80 mg/l and PCT of 0.02 ng/ml. Blood cultures were negative, but the Rose-Bengal plate test was positive. MRI scans revealed abnormal signals in the fourth and fifth lumbar vertebrae and disc, as well as spinal stenosis (Figure 1D). Surgical resection of the lumbar lesion was performed. Tissue cultures from the lesion were negative, but mNGS identified Brucella melitensis, with a read count of 58. The patient was subsequently treated with doxycycline (100 mg twice daily orally) and rifampicin (600 mg daily orally). His condition improved, and he was discharged from the hospital. The medications were continued for 7 months before being discontinued.
3. Discussion & conclusion
Brucellosis is acknowledged as one of the most prevalent zoonotic infections worldwide [14,15]. At least 170 countries have reported human brucellosis cases [16]. Asia and Africa account for most of the risk and incidence of human brucellosis [1]. Persistent traditional agricultural practices and lifestyles, and consumption of fresh dairy products, such as raw milk, contribute to this high prevalence [16]. In China, brucellosis continues to be a significant public health concern [17], predominantly affecting pastoral areas [18]. In Zhejiang Province, where our study was conducted, brucellosis is not widespread, and serological testing is typically handled by the Centers for Disease Control and Prevention. This necessitates clinicians' suspicion of brucellosis before testing, which can lead to diagnostic challenges.
The isolation of Brucella establishes a definitive diagnosis, particularly when the infection is not clinically anticipated [19,20]. However, the culture detection of Brucella is impeded by the slow growth of the genus, laboratory safety concerns and diminished sensitivity in prolonged and focal infections [6].
Our experience indicates that mNGS facilitates the detection of a wide array of pathogens, including rare, novel and atypical agents in complex infectious diseases [10,21]. Its advantages include high sensitivity and rapid detection.However, due to the limitations of cost and technical requirements, mNGS is currently difficult to be used for large-scale detection of Brucella. Moreover, mNGS does have some potential limitations, such as the difficulty of DNA extraction, the possibility of contamination and no uniform standard for assaying positive results of pathogens. Prior studies on mNGS for diagnosing human brucellosis have primarily been limited to case reports. Jun et al. [22] documented a case of central nervous system infection with Brucella melitensis confirmed by mNGS. Similarly, Xi Huiyu et al. [23] reported on a patient with infectious endophthalmitis caused by Brucella spp., diagnosed through mNGS. So far only one retrospective study revealed among the 48 patients with osteoarticular brucellosis, the sensitivity, specificity, positive predictive value and negative predictive value of mNGS were 83.33, 100.00, 100.00 and 55.56%, respectively [24].
In our study, the clinical characteristics of the five patients are shown in Table 1, and the patients were diagnosed with brucellosis via culture, mNGS, or serological tests (Table 2). Cases 1 and 2 involved acute human Brucella infection without abscess formation. In case 1, Brucella was detected in both blood culture and plasma mNGS. Case 2 was eventually confirmed through blood culture, after plasma mNGS, positron emission tomography-computed tomography, bone marrow puncture and lymph node biopsy yielded no definitive results. Due to Brucella‘s slow-growing, intracellular nature [6], and the challenges in DNA extraction and contamination control in mNGS, similar to Mycobacterium tuberculosis [10], the detectable nucleic acid load is typically low. Efficient cell wall disruption is essential for effective organism lysis and nucleic acid release when using mNGS for Brucella detection.
Table 1.
Clinical characteristics of five patients with brucellosis.
| Patient | 1 | 2 | 3 | 4 | 5 |
|---|---|---|---|---|---|
| Age (years) | 60 | 67 | 60 | 71 | 55 |
| Gender | Male | Female | Female | Female | Male |
| Clinical manifestations | Fever, chills and cough | Fever, fatigue | Fever, lumbago | A painless and enclosed mass at back | Lumbago, numbness in the lower limbs and fever |
| Epidemiological history | Yes | Yes | Yes | No | Yes |
| Laboratory tests | |||||
| white blood cell (10*9/l) | 6.01 | 3.46 | 7.11 | 5.61 | 6.12 |
| C-reactive protein(mg/l) | 192.59 | 4.5 | 26.54 | 9.25 | 12.80 |
| Procalcitonin(ng/ml) | 1.47 | 0.07 | 0.02 | 0.10 | 0.02 |
| Anti-infective regimens | Omadacycline (300 mg daily orally) and levofloxacin (500 mg daily orally) | Doxycycline (100 mg twice daily orally) and rifampicin (600 mg daily orally) | Doxycycline(100 mg twice daily orally) and rifampicin(450 mg daily orally) | Doxycycline (100 mg twice daily orally), rifampicin (450 mg daily orally) and moxifloxacin (400 mg daily orally) | Doxycycline (100 mg twice daily orally) and rifampicin (600 mg daily orally) |
Table 2.
Etiological examination results of five patients with brucellosis.
| Patient | Specimens for culture | Culture | Serology | Specimens for mNGS | Reads for mNGS |
|---|---|---|---|---|---|
| 1 | Whole blood | Positve | – | Plasma | 4 |
| 2 | Whole blood | Positive | – | Plasma | – |
| 3 | Tissue | Positive | Positive | Tissue | 1314 |
| 4 | Pus/tissue | Positive/negative | – | Tissue | 30 |
| 5 | Whole blood/tissue | Negative/negative | Positive | Tissue | 58 |
Osteoarticular involvement is the most common complication of brucellosis, with a prevalence of approximately 2–77% [25]. Brucella spondylitis is the most common (2–60%) and severe manifestation of the osteoarthritis system, prevalent in elderly patients [26]. However, its focal complications have similarities to certain forms of pinal tuberculosis [27], making traditional bacterial isolation challenging for differentiating the two diseases. In this context, mNGS may offer a superior detection method. Cases 3, 4 and 5, which involved spinal brucellosis with or without paravertebral abscess, were diagnosed through local tissue or pus culture, mNGS, and serological tests. Notably, cultures from infectious tissue samples were negative in case 4 and 5, but mNGS successfully detected Brucella in all tissue samples, with read counts ranging from 30 to 1314. We believe that higher read counts indicate a greater Brucella presence. Therefore, mNGS may have a higher positive rate for local tissue samples in Brucella detection.
There are some limitations to our reporting. The whole process of diagnosis and treatment of the five brucellosis patients were completed by different medical teams in our hospital, hence the variability in patient management. While all five patients showed clinical improvement following treatment, the durability of this improvement and the possibility of relapse are not yet known due to the absence of long-term follow-up data.
4. Conclusion
In conclusion, our cases demonstrate that mNGS is a relatively rapid and effective diagnostic method, enhancing the efficiency of diagnosing human brucellosis, especially in non-endemic areas. When clinicians suspect Brucella infection in patients with atypical symptoms, plasma mNGS should be considered. In cases of localized lesions, priority should be given to mNGS testing at the affected site. We hope to explore the diagnosis of brucellosis by mNGS in a larger population and complete drug resistance analysis by mNGS. We believe it will be helpful for the clinical application of mNGS in the diagnosis of brucellosis.
Funding Statement
This work was supported by National Key R&D Program of China (2022YFC2304500, 2022 YFC2304505) and Zhejiang Province Natural Science Foundation (LY23H200001).
Author contributions
Conceptualization, X Zhang and M Yang; data curation, X Zhang, X Yao and H Chen; writing—original draft preparation, X Zhang and D Han; writing—review and editing, X Zhang and M Yang. All authors have read and agreed to the published version of the manuscript.
Financial disclosure
This work was supported by National Key R&D Program of China (2022YFC2304500, 2022 YFC2304505) and Zhejiang Province Natural Science Foundation (LY23H200001). The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
Competing interests disclosure
The authors have no competing interests or relevant affiliations with any organization or entity with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Writing disclosure
No writing assistance was utilized in the production of this manuscript.
Ethical conduct of research
This report was conducted according to the guidelines of the Declaration of Helsinki, and approved by the the Ethical Committee of the First Affiliated Hospital, School of Medicine, Zhejiang University (reference number: IIT20240380A). Written informed consent was waived with the approval of the Ethics Committee.
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