Background Lentiviral vectors are commonly used in engineering CAR-T cells; however, they can sometimes lead to false positive results for Human Immunodeficiency Virus (HIV) following CAR-T infusion. Identifying such false positives can be challenging.
Methods We report the first case of a false positive HIV-1 detected by metagenomic next-generation sequencing (mNGS) after CAR-T therapy. We analyzed a case involving a five-year-old boy diagnosed with B-cell acute lymphoblastic leukemia who developed cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) three days post-CAR-T infusion. Given the difficulty in distinguishing CRS from sepsis, mNGS was performed on blood and cerebrospinal fluid samples. Both samples tested positive for HIV-1 DNA sequences via mNGS.
Results The detected DNA sequences only covered partial sites of the HIV genome (as shown in Figure 1). Meanwhile, no HIV sequences were detected through blood metatranscriptome sequencing (RNA-seq). High-sensitivity HIV-1 RNA tests were negative, and multiple HIV antibody immunoassays conducted over the patient’s history confirmed the results as false positives. The patient did not initiate any anti-HIV treatments. We also used mNGS to extract partial genomic sequences of HIV for further analysis.
Conclusions Several reports have described instances of false positive HIV detection following CAR-T therapy. These studies employed nucleic acid amplification techniques (NAAT), but due to proprietary reasons, specific details about the vectors used in CAR-T products are limited. Compared to NAAT, mNGS offers a visualization method to represent the coverage of a pathogen’s genome, providing a more intuitive and interpretable approach for clinical assessments. Especially when significant portions of the genome are not covered, mNGS can help differentiate nonspecific sequence detection and false positives. If needed, bioinformatics further analyzes and uses BLAST to compare identified nucleotide sequences. The comprehensive genomic view capability of mNGS is a unique advantage over NAAT, which could be further explored in future clinical practice.
Expert Commentary
Professor Jun Lu: This case study serves as a valuable reference for clinical physicians in departments such as ICU and infectious diseases. The incidence of CRS following CAR-T therapy exceeds 90%, making it difficult to distinguish the fever and infection caused by the severe inflammatory response.These can be identified through culture or mNGS to detect various pathogens. This study discovered the viral sequence of HIV, which typically raises questions among clinical doctors: How could a patient experience CRS infection 2 to 4 months after CAR-T infusion? In reality, it’s not an infection but rather the presence of HIV sequences accompanying the CAR-T infusion process. So in the future, when encountering such situations, a correct interpretation can be provided, which serves as excellent guidance for us as clinical physicians.
