Coxiella burnetii and Bartonella Endocarditis Diagnosed by Metagenomic Next-Generation Sequencing

(1) Background: Culture-negative endocarditis is challenging to diagnose. Here, we retrospectively identified 23 cases of Coxiella burnetii and Bartonella endocarditis by metagenomic next-generation sequencing. (2) Methods: Twenty-three patients with culture-negative endocarditis were retrospectively enrolled from Guangdong Provincial People’s Hospital (n = 23) between April 2019 and December 2021. Metagenomic next-generation sequencing was performed on blood (n = 22) and excised cardiac valvular tissue samples (n = 22) for etiological identification, and Sanger sequencing was performed for pathogenic diagnostic verification. The demographic and clinical data of the 23 patients were obtained from hospital electronic health records. (3) Results: A total of 23 male patients (median age, 56 years (interquartile range, 16)) with culture-negative endocarditis were diagnosed with Coxiella burnetii (n = 21) or Bartonella (n = 2) species infection by metagenomic next-generation sequencing. All patients underwent cardiac surgery. The resected tissue exhibited both a significantly higher number of unique suspected pathogen read-pairs and more unique pathogen read-pairs than the blood specimens. The results of Sanger sequencing tests on all remaining tissue and blood specimens were positive. Oral doxycycline was added to the antibiotic regimen for at least 1.5 years according to etiology. A total of 21 patients (91%) were discharged, and 20 patients were healthy at the 21-month (interquartile range, 15) follow-up visit. One patient exhibited endocarditis relapse with the same pathogen from inadequate antibiotic administration. The last 2 patients (9%) developed septic shock and multiple organ dysfunction syndrome postoperatively and died shortly after discharge. (4) Conclusions: CNE caused by C. burnetii and Bartonella species is challenging to diagnose and exhibits poor outcome due to delayed treatment. In response, mNGS, characterized by high sensitivity and rapid results, is an effective alternative for the etiological identification of C. burnetii and Bartonella endocarditis.


Introduction
Culture-negative endocarditis (CNE) is becoming more common in endocarditis patients, especially in tertiary hospitals. CNE has relatively high incidence rates of 15-40% in all cases of infective endocarditis and is an independent predictor of in-hospital mortality [1,2]. Coxiella burnetii is the most common causative pathogen of CNE (46.0-61.4%), followed by Bartonella species (15.1-20.7%) [3][4][5]. C. burnetii is a fastidious, Gram-negative and obligate intracellular bacterium, while Bartonella species are fastidious, Gram-negative, facultative intracellular bacteria with a unique intraerythrocytic lifestyle. This lifestyle 2 of 11 not only enables the escape of their detection by the host immune system but can also cause false negative results on conventional laboratory tests [5]. The recommended major criteria of the modified Duke criteria for the diagnosis of C. burnetii endocarditis rely on an immunofluorescence assay (IFA) titer of ≥1:800 for C. burnetii anti-phase I immunoglobulin G (IgG). Serological cross-reactions between C. burnetii, Bartonella and Chlamydia [6,7] and inconvenient sample transfers to reference laboratories limit its clinical application. Due to the limitations of conventional laboratory tests and the unidentifiable clinical manifestations of these conditions, the delayed diagnosis or misdiagnosis of C. burnetii and Bartonella endocarditis are common and frequently result in inadequate treatment. To reach an etiological diagnosis and explore unique clinical manifestations in these particular patients, an alternative pathogenic assessment should be performed to achieve a correct and timely diagnosis. Here, we describe 23 cases of CNE caused by C. burnetii and Bartonella identified by metagenomic next-generation sequencing (mNGS).

Materials and Methods Ethics statement:
The study was approved by the ethics committee of the faculty of medicine at the Guangdong Provincial People's Hospital (approve No. KY-Q-2022-173-01). Individual patients informed consents were all guaranteed except the death.

Patients
Between April 2019 and December 2021, twenty-three consecutive patients with C. burnetii or Bartonella endocarditis who underwent cardiac surgery at Guangdong Provincial People's Hospital for valvular disease were included into this study. All patients met the modified Duke criteria for the clinical diagnosis of infective endocarditis [8]. The excised valvular tissue and blood specimens obtained during cardiac surgery were used to identify the pathogen by mNGS. Sanger sequencing was performed on the remaining specimens for etiologic verification. In addition, 31 patients with infective endocarditis caused by Staphylococcus aureus and 30 patients with infective endocarditis caused by Streptococcus, who were randomly selected from 258 infective endocarditis patients treated in our hospital between April 2019 and December 2021, were retrospectively studied as disease controls. All patient underwent cardiac surgery, and blood and tissue samples were subjected to mNGS for etiological identification.

Data Collection
The demographic and clinical data of the 84 patients were obtained from the electronic health records.

Metagenomic Next-Generation Sequencing
Plasma was prepared from the blood specimens, and circulating cell-free DNA (cfDNA) was isolated from the plasma with a QIAamp Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. DNA from the valvular tissue was extracted using the QIAamp DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany). The quantity and quality of DNA were assessed using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), respectively. DNA libraries were prepared using the KAPA Hyper Prep kit (KAPA Biosystems, Oslo, Norway) according to the manufacturer's protocols. An Agilent 2100 bioanalyzer was used for quality control, and DNA libraries were single-end sequenced (75 bp) on an Illumina NextSeq 550Dx system (Illumina, San Diego, CA, USA).
An in-house bioinformatics pipeline was used for pathogen identification. Briefly, high-quality sequencing data were generated by removing the low-quality reads, adapter contamination and duplicated and short (length < 36 bp) reads. Human host sequences were identified by mapping to the human reference genome (hs37d5) using Bowtie2 software. Reads that could not be mapped to the human genome were retained and aligned with the microbial genome database for pathogen identification. Our microbial genome database contained bacterial, fungal, viral and parasite genomic sequences (downloaded from https://www.ncbi.nlm.nih.gov/genome/) accessed on 1 February 2022.
The following criteria were used to define positive mNGS results: For Mycobacterium, Nocardia and Legionella pneumophila, the result was considered positive if the data of a species detected by mNGS exhibited a species-specific read number ≥1.
For bacteria (excluding Mycobacterium, Nocardia and Legionella pneumophila), fungi, viruses and parasites, the result was considered positive if the data of a species detected by mNGS exhibited at least three nonoverlapping reads.
If the number of detected pathogens reads was ≥10-fold greater than that in the no template control (NTC), the pathogen detection was considered positive.

Organism-Specific Sanger Sequencing
Organism-specific Sanger sequencing was used for the verification of the mNGS results of the remaining specimens.

Statistical Analysis
All statistical analyses were performed with SPSS software, version 25.0 (IBM, Armonk, NY, USA). Continuous variables are summarized as the median with interquartile range (IQR). Categorical variables are summarized as frequencies and percentages. Comparisons were made between patients with infections caused by different pathogens. One-way ANOVA or the Welch test was used to compare continuous variables, and the chi-square test or Fisher's exact test was used for categorical variables.
The   The case series of the C. burnetii and Bartonella species endocarditis groups are described in detail in Supplementary Materials S1.

Discussion
We presented 84 diagnostically challenging cases of acute or subacute endocarditis in which the use of mNGS on blood and valve tissue samples identified the pathogens involved. These cases included one case caused by Bartonella quintana, one case caused by Bartonella henselae and 21 cases caused by C. burnetii, including one patient who was also infected with Streptococcus gordonii. Sanger sequencing of the blood and valvular tissues was performed in these 23 patients for etiological verification. In Figure 3, we summarize the diagnosis and treatment process of endocarditis in our institute based on a 10-year experience of surgical management in infective endocarditis. Timely diagnosis, multiple disciplinary teams and effective antibiotic treatment [9] combined with surgery were key points for treatment. The case series of the C. burnetii and Bartonella species endocarditis groups are described in detail in Supplementary Materials S1.

Discussion
We presented 84 diagnostically challenging cases of acute or subacute endocarditis in which the use of mNGS on blood and valve tissue samples identified the pathogens involved. These cases included one case caused by Bartonella quintana, one case caused by Bartonella henselae and 21 cases caused by C. burnetii, including one patient who was also infected with Streptococcus gordonii. Sanger sequencing of the blood and valvular tissues was performed in these 23 patients for etiological verification. In Figure 3, we summarize the diagnosis and treatment process of endocarditis in our institute based on a 10-year experience of surgical management in infective endocarditis. Timely diagnosis, multiple disciplinary teams and effective antibiotic treatment [9] combined with surgery were key points for treatment.
Mild clinical manifestations of infection and severe levels of bacterial invasion and damage in the cardiac valve and aorta were characterized in patients with C. burnetii and Bartonella encarditis; in contrast, endocarditis caused by Staphylococcus aureus or Streptococcus was accompanied by evident sepsis that was easily recognized early. The severity and mortality of a patient's condition can be predicted by clinicians using a variety of patient factors, including demographic, clinical, immunologic, hematologic, biochemical and radiographic findings, especially various laboratory test abnormalities and biomarkers of end-organ dysfunction [11]. Nonspecific symptoms of infection and a lack of pathogenic identification in C. Burnetii and Bartonella endocarditis may decrease the vigilance of clinical physicians or surgeons, leading to delayed diagnosis or misdiagnosis and poor outcome [1,2]. Patients with C. Burnetiid or Bartonella endocarditis may not undergo a cardiac examination or surgery until a new valvular regurgitation develops or the worsening of a preexisting valvular regurgitation occurs, which could indicate valve perfora- Mild clinical manifestations of infection and severe levels of bacterial invasion and damage in the cardiac valve and aorta were characterized in patients with C. burnetii and Bartonella encarditis; in contrast, endocarditis caused by Staphylococcus aureus or Streptococcus was accompanied by evident sepsis that was easily recognized early. The severity and mortality of a patient's condition can be predicted by clinicians using a variety of patient factors, including demographic, clinical, immunologic, hematologic, biochemical and radiographic findings, especially various laboratory test abnormalities and biomarkers of end-organ dysfunction [11]. Nonspecific symptoms of infection and a lack of pathogenic identification in C. burnetii and Bartonella endocarditis may decrease the vigilance of clinical physicians or surgeons, leading to delayed diagnosis or misdiagnosis and poor outcome [1,2]. Patients with C. burnetiid or Bartonella endocarditis may not undergo a cardiac examination or surgery until a new valvular regurgitation develops or the worsening of a preexisting valvular regurgitation occurs, which could indicate valve perforation, valve damage and/or, abscess [12]. Therefore, a rapid and effective diagnostic strategy is needed to reach the correct diagnosis of C. burnetii and Bartonella endocarditis and improve their outcome.
Unlike serological IFAs, molecular methods using the PCR-based amplification of pathogenic DNA fragments obtained from both blood and excised valve tissue prevent cross-reactions between biologically similar antigens. Of note, diagnoses of mixed pathogenic infections cannot be excluded in patients with C. burnetii or Bartonella endocarditis. Thus, the use of mNGS for broad pathogenic detection has diagnostic value compared with organism-specific PCR. However, mNGS exhibited lower specificity than that observed in conventional pathogenic identification examinations, including culture and serological IFAs, which may have resulted from contamination during the sample collection or molecular procedures or from the presence of chronic, persistent bacterial DNA in vivo [13]. Thus, mNGS results should be cautiously interpreted within the full clinical context and correlated with the findings of all other laboratory tests. The statistical scoring and filtering of microbiological and complex mNGS data sets can help inherently differentiate between microbiological contaminants and true infectious organisms. Sanger sequencing for pathogenic diagnostic verification was essential when the mNGS result was confusing. mNGS allows pathogenic identification within one working day, which facilitates timely diagnosis and the appropriate administration of antibiotics for CNE caused by C. burnetii and Bartonella species, which can improve the prognosis in a substantial number of patients.
Interestingly, we summarized the visual characteristics of C. burnetii and Bartonella endocarditis ( Figure 4). Of note, the endomembrane of the abscess was smooth and integrated, and less exudate was detected in the abscess than in other paravalvular abscesses caused by Staphylococcus aureus or Streptococcus. This phenomenon may be related to the diversity of infection and the invasiveness of different bacterial strains [14]. Here, we offer three short surgical videos showing the differences in vegetation and paravalvular abscesses in different etiological forms of endocarditis (Supplementary Video S1). However, our study was an observational study with a small sample and was quite subjective for use in attending decisions. Additionally, the study only included endocarditis patients who underwent cardiac surgery for valve tissue acquisition. This is the natural bias in patient selection. Further study involving more specimens and a more rigorous design is required for the verification of our results.
pathogenic DNA fragments obtained from both blood and excised valve tissue prev cross-reactions between biologically similar antigens. Of note, diagnoses of mixed path genic infections cannot be excluded in patients with C. burnetii or Bartonella endocardi Thus, the use of mNGS for broad pathogenic detection has diagnostic value compar with organism-specific PCR. However, mNGS exhibited lower specificity than that o served in conventional pathogenic identification examinations, including culture and rological IFAs, which may have resulted from contamination during the sample collecti or molecular procedures or from the presence of chronic, persistent bacterial DNA in v [13]. Thus, mNGS results should be cautiously interpreted within the full clinical cont and correlated with the findings of all other laboratory tests. The statistical scoring a filtering of microbiological and complex mNGS data sets can help inherently differenti between microbiological contaminants and true infectious organisms. Sanger sequenci for pathogenic diagnostic verification was essential when the mNGS result was confusin mNGS allows pathogenic identification within one working day, which facilitates tim diagnosis and the appropriate administration of antibiotics for CNE caused by C. burn and Bartonella species, which can improve the prognosis in a substantial number of p tients.
Interestingly, we summarized the visual characteristics of C. burnetii and Barton endocarditis (Figure 4). Of note, the endomembrane of the abscess was smooth and in grated, and less exudate was detected in the abscess than in other paravalvular absces caused by Staphylococcus aureus or Streptococcus. This phenomenon may be related to diversity of infection and the invasiveness of different bacterial strains [14]. Here, we of three short surgical videos showing the differences in vegetation and paravalvular a scesses in different etiological forms of endocarditis (Supplementary Video S1). Howev our study was an observational study with a small sample and was quite subjective use in attending decisions. Additionally, the study only included endocarditis patie who underwent cardiac surgery for valve tissue acquisition. This is the natural bias patient selection. Further study involving more specimens and a more rigorous design required for the verification of our results.

Conclusions
CNE caused by C. burnetii and Bartonella species is challenging to diagnose and has a poor outcome due to delayed treatment. In response, mNGS, characterized by high sensitivity and rapid results, is an effective alternative method for the etiological identification of C. burnetii and Bartonella endocarditis.