A 44-year-old woman presented with persistent neck and back pain for over one year. On initial admission one year ago, Magnetic resonance imaging (MRI) suggested destruction of the thoracic vertebrae (T11 and T12) (Figures 1A,B), and computed tomography (CT) scans revealed destruction of the right 12th rib (Figure 2). Radiology consult suggested the findings resembled spinal tuberculosis. The patient underwent surgical removal of focal necrosis in the thoracic spine and thoracolumbar vertebroplasty. Though biopsy of the necrotic tissue did not reveal Mycobacterium tuberculosis, the patient received empirical antituberculosis therapy in the setting of her disease manifestations and no history of Bacillus Calmette-Guerin (BCG) vaccine. During follow-up, an abscess developed adjacent to the back wound and the patient underwent an open debridement of abscess six months after the surgery.
The wound of the debridement healed, but repetitive neck and back pain struck the patients once again one month before the current admission, accompanied by numbness of bilateral upper limbs and neck immobility. Physical examination revealed a 20 cm healed surgical wound in her back. Tenderness and percussion pain were noted in the interspinous and spinous process of each cervical cone. Muscle tone was found in the bilateral trapezius, rhomboid and sternocleidomastoid muscles. MRI showed multiple cystic masses in the craniocervical junction region and effusion around the lumbar vertebrae (Figure 1C). Nothing special was noted in the abdominal contrast enhanced CT of the patient (Supplementary Figure S1). Blood tests suggested mildly elevated white blood cells with normal eosinophil count. Fecal examination suggested mildly positive occult blood. Multiple attempts were made to detect tuberculosis and other pathogens through both puncture of the effusion and cerebrospinal fluid, through culture and histopathological examination, but the results were all negative.
Though spinal tuberculosis was the initial leading diagnosis in the case of this patient, the negative results from multiple biopsies led us question our assumptions and conduct further testing. Therefore, we adopted mNGS to further screen for potential pathogens in this case, and the results revealed high reads count and coverage rate for Echinococcus multilocularis, which was further verified by PCR and Sanger sequencing.
To investigate the underlying reason of abscess formation, a DNBseq  mNGS detection of the punctured effusion was performed. The detailed protocol is established as follows. First, 7.2 mul lysozyme was added into 0.6 ml effusion sample for wall-breaking reaction. Then we added the above solution to a 1.5 ml microcentrifuge tube with 250 mul 0.5 mm glass beads and placed into the FastPrep-24  5G bead beating grinder and lysis system for vigorous agitation. Next, we separated 0.3 ml sample into a new 1.5 ml microcentrifuge tube and used the TIANamp Micro DNA Kit (DP316, TIANGEN BIOTECH) to extract DNA. DNA libraries were constructed through DNA-fragmentation, end-repair, adapter-ligation and PCR amplification. Agilent 2,100 was used for quality control of the DNA libraries. Quality qualified libraries were pooled, DNA Nanoball was made and sequenced by MGISEQ-2000 platform. High-quality sequencing data were generated by removing low-quality reads, followed by computational subtraction of human host sequences mapped to the human reference genome (hg19) using Burrows-Wheeler Alignment. The remaining data were classified by simultaneously aligning to the Pathogens metagenomics Database, consisting of bacteria, fungi, viruses and parasites. The classification reference databases were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/). The total number of reads detected was 176,712,306, of which 73,044 aligned to microorganisms. Among them, the detected reads from the specimen mapped well with the Echinococcus multilocularis genome (Figure 3A). As for the constitution, Echinococcus multilocularis was identified as the most predominant pathogen taking up to 99.74% (3,938 reads) of the total sequence reads (Figure 3B).
To verify the mNGS result, we performed Sanger sequencing on the extracted sample DNA. According to the mNGS result, we designed a pair of PCR primers (EfF: 5'-GATTCCTTCTTTAGTTTTGTTGTTGATTAG-3' and EfR: 5'-CGAACTAAAAACTCTAGAAACACCTGCT-3') to specifically amplify the COX1 gene of Echinococcus multilocularis (GenBankTM accession number: AB813188). The sequencing results are consistent with the sequence alignment of the tapeworm cox1 gene, which further confirmed the mNGS results (Figure 4).
After diagnosis of vertebral AE, the antituberculosis therapy was ceased and a continuous Benzimidazole treatment with Albendazole 400 mg twice per day was imitated immediately. The local symptom of the patient improved and was waiting for the next period of follow-up. No adverse events were noted one month after the Benzimidazole therapy. Our case was reported based on the CARE reporting standard (Supplementary Table S1). The whole timeline of the patients was displayed in Supplementary Figure S2.