A 77-year-old Asian male presented with a 2-month duration of pain and swelling in the medial aspect of the left thigh and fever. Before referring, two aspirations were performed by a community hospital, but the pain recurred after temporary relief. The patient's medical history was noteworthy for degenerative lumbar disk disease and he had undergone inguinal herniorrhaphy 16 years prior. He was not on any long-term medication. At the time of admission, the patient had been suffering aggravating pain and lameness. Upon physical examination, the movement of the patient's left hip joint was restricted and the Patrick's test was positive. The patient had a temperature of 37.3 C without chills. The patient's bloodwork was not clinically significant except hemoglobin 118 g/L (reference: 120-160 g/L), erythrocyte sedimentation rate (ESR) 60 mm/hour (reference: 0-15 mm/hour), and C-reactive protein (CRP) > 150 mg/L (reference: 0-5 mg/L). Blood cultures tested for aerobic and anaerobic bacteria were negative. Magnetic resonance imaging (MRI) revealed a small amount of effusion in the left acetabular cavity. An increased T2 signal of the left iliopsoas was also revealed by MRI, indicating infection. No joint space narrowing was observed. Computed tomography (CT) showed spotted calcification in the upper and lower lobes of both lungs, which hinted at a history of pulmonary tuberculosis (TB). An interferon-gamma release assay (TSPOT. TB) was performed and the positive reaction indicated latent or active TB. The affected hip was assumed to be the result of prior pulmonary infection with Mycobacterium tuberculosis (MTB), which reached the joint via hematogenous spread.
Based on this evidence, the patient began to receive anti-TB therapy with a combination of oral isoniazid, rifampicin, pyrazinamide, and ethambutol. Although the patient's pain was partially relieved and the MRI revealed a smaller lesion, his temperature continued to rise and the pain aggravated after five days of treatment. The patient then underwent joint clearance surgery and vacuum sealing drainage (VSD) to alleviate the symptoms. During the surgery, granulomatous inflammation was found and caseous materials were removed from the femoral neck, femoral head, and joint capsule. The caseous materials and synovial fluid were collected for microbiological examination. Although a biopsy specimen showed histopathological features of mycobacterial arthritis, microscopy for acid-fast bacilli and culture were negative.
The patient's whole blood and synovial fluid were kept for bacteriological confirmation using mNGS with high sequencing depth to increase the sensitivity. Briefly, nucleic acids were extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) and libraries were constructed using the NEBNext Ultra DNA Library Prep Kit (NEB, Ipswich, MA, USA). Libraries were sequenced using an Illumina HiSeq2500 sequencer with paired-end reads of an average 150 bp in length. In-house Perl scripts were used to filter low-quality reads and Bowtie 2 to remove reads with similarity to the human genome. The workflow and criteria used in this study were described previously. BLASTn was used to align the remainder against the NCBI non-redundant nucleotide database with an E-value < 10-5, and hits were analyzed and extracted by MEGAN 5 software. The post-filtering data sets have been stored in the NCBI Sequence Read Archive and are available from BioSample accession number SAMN13246068 and SAMN13246069 under BioProject PRJNA588503.
Sequencing the libraries from synovial fluid and whole blood yield 32,629,075 and 36,759,980 clean paired-end reads representing 9.79 GB and 11.03 GB of nucleotide data, respectively. Following quality control and eliminating human sequences, for the synovial fluid and whole blood sample, the filtered sequence data contain 53,887 and 109,198 reads, among which 681 and 144 reads yield alignments to bacterial sequences, respectively. Among all hits, only two paired-end reads from synovial fluid samples were identified as MTB. Remaining reads belonging to genera of Corynebacterium, Brevundimonas, Propionibacterium, Micrococcus, Kocuria,and Stenotrophomonasa account for the majority of the bacterial hits (Figure 1). Most of the viral reads belong to Torque teno virus (TTV), a member of the family Anelloviridae. Detailed proportions of each bacterial and viral taxa were listed in Supplementary materials (Table S1). Due to the low possibility of contamination with mycobacteria, the criterion for MTB being considered positive by mNGS was at least one read mapped to its genome. Meanwhile, except MTB, other identified bacteria are regarded as contaminants commonly found in laboratory environments, and furthermore, none of them should be classified as pathogen, because they were present at levels equivalent to the background. For viruses, infections with TTV are ubiquitous in human population and there are no convincing pieces of evidence showing that TTV is associated with any specific diseases. Considering all the above, we conclude that the results of mNGS can provide evidence of MTB infection.
After surgery, the patient was treated for a surgical site infection with isoniazid and the oral anti-TB therapy continued. Four weeks after surgery, MRI revealed a reduced range of the lesion compared to before the surgery and the CRP gradually decreased. The patient's hip pain was relieved and temperature stabilized. At the patient's request, he was allowed to leave the hospital. A timeline showing the patient's entire clinical course and the results of important tests is provided in Supplementary materials (Figure S1).