Clinical metagenomic sequencing for diagnosis of pulmonary tuberculosis.

OBJECTIVES: The aim of this study is to investigate the clinical usefulness of metagenomic Next-generation sequencing (mNGS) on bronchoalveolar lavage fluid (BALF) samples to discriminate pulmonary tuberculosis (PTB) from Non-TB community-acquired pneumonia (CAP) in PTB suspects. METHODS: We investigate the performance of mNGS on BALF samples from 110 PTB suspects, in comparison with conventional microbiological testing (solid media culture, acid-fast bacilli staining (AFS), Xpert) of BALF or sputum samples and final clinical diagnosis. RESULTS: We finally clinically diagnosed 48 cases of pulmonary tuberculosis patients and 62 cases of non-tuberculosis patients. Comparing to the final clinical diagnosis, mNGS produced a sensitivity of 47.92%, which was similar to that of Xpert (45.83%) and culture (46.81%), but much higher than that of AFS (29.17%) for TB diagnosis in BALF samples. Apart from detecting Mycobacterium tuberculosis, mNGS also identified mixed infections in PTB patients, including 3 fungal cases and 1 bacteria case. Meanwhile, mNGS efficiently identified 14 of 22 (63.63%) cases of non-tuberculous mycobacteria (NTM), 7 cases of fungi, 1 case of viral infection, and other common bacterial pathogens in Non-PTB group. Finally, mNGS identified 67.23% infection cases within 3 days, while the conventional methods identified 49.58% infection cases for over 90 days. CONCLUSION: Our data show that mNGS of BALF represents a potentially effective tool for the rapid diagnosis of PTB suspects.

Establishment of a human colorectal cancer cell line P6C with stem cell properties and resistance to chemotherapeutic drugs.

AIM: Cancer stem cells have the capacity to initiate and sustain tumor growth. In this study, we established a CD44(+) colorectal cancer stem cell line with particular emphasis on its self-renewal capacity, enhanced tumor initiation and drug resistance. METHODS: Fresh colon cancer and paired normal colon tissues were collected from 13 patients who had not received chemotherapy or radiotherapy prior to surgery. Among the 6 single-cell derived clones, only the P6C cell line was cultured for more than 20 passages in serial culture and formed holoclones with high efficiency, and then the stemness gene expression, colony formation, tumorigenicity and drug sensitivities of the P6C cell line were examined. RESULTS: Stemness proteins, including c-Myc, Oct3/4, Nanog, Lgr5, and SOX2, were highly expressed in the P6C cell line. Oct3/4-positive P6C cells mostly generated holoclones through symmetric division, while a small number of P6C cells generated meroclones through asymmetric division. P6C cells stably expressed CD44 and possessed a high capacity to form tumor spheres. A single cell-derived sphere was capable of generating xenograft tumors in nude mice. Compared to SW480 and HCT116 colorectal cancer cells, P6C cells were highly resistant to Camptothecin and 5-fluorouracil, the commonly used chemotherapeutic agents to treat colorectal cancers. CONCLUSION: We established a colorectal cancer stem cell line P6C with a high tumorigenic capacity and the characteristics of normal stem cells. It will benefit the mechanistic studies on cancer stem cells and the development of drugs that specifically target the cancer stem cells.