Metagenomic Next Generation Sequencing in the Detection of Pathogens in Cerebrospinal Fluid of Patients After Alternative Donor Transplantation: A Feasibility Analysis

Abstract

Central nervous system (CNS) complications can occur in 9%–15% of patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The clinical manifestations of the CNS complications are non-specific, with most of them being disturbances of consciousness, convulsions, headaches, fever, and epilepsy, making it difficult to infer the cause of the complications based on clinical manifestations. We retrospectively analyzed the sensitivity and feasibility of metagenomic next generation sequencing (mNGS) in the diagnosis of CNS infections after allo-HSCT. Lumbar punctures were performed on 20 patients with CNS symptoms after receiving alternative donor HSCT(AD-HSCT) at the Affiliated Cancer Hospital of Zhengzhou University from February 2019 to December 2020, and their cerebrospinal fluid (CSF) was collected. The mNGS technique was used to detect pathogens in the CSF. Routine CSF testing, biochemical analyses, G experiments, GM experiments, ink staining, acid-fast staining, and bacterial cultures were carried out, and quantitative PCR (qPCR) tests were used to detect cytomegalovirus (CMV), Epstein-Barr virus (EBV), BK polyomavirus (BKPyV), and human alphaherpesvirus (HHV). A total of 29 tests were performed with 21 of them being positive. Of the five negative patients, three were diagnosed with a posterior reversible encephalopathy syndrome, one as having transplantation-associated thrombotic microangiopathy, and one with transient seizure caused by hypertension. Fifteen patients tested positive, of which four had single infections and eleven had mixed infections. Five cases of fungal infections, six cases of bacterial infections, and 13 cases of viral infections were detected. Among the 13 cases of viral infections, ten cases were CMV(HHV-5); three were BKPyV; two were Torque teno virus (TTV); Two were HHV-1,two were EBV(HHV4), and one each of HpyV5 and HHV-6B. Thirteen patients tested positive for virus while the qPCR detection method of 6 identical specimens were below the minimum detection limit(<1×103 U/ml). The mNGS technique is highly sensitive, and it can be used to diagnose CNS infections after allo-HSCT.

Metagenomic next-generation sequencing for the diagnosis of Pneumocystis jirovecii Pneumonia in critically pediatric patients

“…For DNA extraction from blood, we transferred 200 µL of plasma into a 2 mL sterile tube. Then the IDseq TM Micro DNA kit (Vision medicals, VM002-50D, China) was used to extract DNA based on standard procedures [ 26 ].…”

“…(3) Virus/ Brucella : SMRN ≥ 3, Pathogens detected in the negative control group were excluded but only if the detected SMRN was ≥ tenfold than that in the negative control group. (4) Mycobacterium tuberculosis : SMRN ≥ 1 [ 26 , 30 ].…”

Nanopore-Targeted Sequencing Improves the Diagnosis and Treatment of Patients with Serious Infections

“…Although HSCT centers provide a clean, hygienic, and protective environment, the risk of severe infection is still present ( 17 ) and may even lead to HSCT engraftment failure and death. It was reported that about 30% to 80% of patients developed a bacterial, fungal, or viral infection after receiving HSCT ( 18 – 20 ), and their cumulative incidences were 43.3%, 33.3%, and 23.3%, respectively ( 21 ). Bacterial infections always occur in the early graft period; both Gram-negative and Gram-positive bacteria may appear in this phase, while Haemophilus influenzae and Streptococcus pneumoniae are more frequent in the late period, as they are encapsulated pathogens ( 22 ).…”

Pathogen Detection Using Metagenomic Next-Generation Sequencing of Plasma Samples from Patients with Sepsis in Uganda

“…This was not surprising to us and can likely explained by several factors, including the read depth, limited sample volume, age of the samples (10+ years), different sample types (sequencing on plasma versus qPCR on whole blood), stringency of the background filtering thresholds used, and the potential presence of inhibitory substances to sequencing in human plasma, which have all been previously described ( 21 – 23 ). Several studies have shown similarly variable sensitivity and specificity of mNGS versus PCR ( 24 – 27 ). Additionally, our extraction and library preparation methods aimed to sequence both RNA and DNA viruses, bacteria, and fungi by using RNA sequencing, which may have preferentially favored some pathogens over others, as previously described ( 27 – 29 ).…”