March 15th, 2023
Molecular Diagnosis of Active Tuberculosis Using CRISPR Gene-Editing TechnologyBy Aldon Li, MD, FIDSA
Manipulation of clustered regularly interspaced short palindromic repeats (CRISPR) into a method to cut DNA at any specified sequence has earned a Nobel prize for the authors of this innovation. The authors of a study in The Lancet Microbe adapted the CRISPR cleavage activity to release a fluorescent probe to increase the detection of low concentration cell-free DNA in blood samples with the aim to use this adaptation to diagnose tuberculosis and evaluate tuberculosis treatment responses. Prior to this study, methods using polymerase chain reaction (PCR)-based technology to detect Mycobacterium tuberculosis (MTB)-cell-free DNA from serum samples had problems with sensitivity given low circulating MTB-cell-free DNA in the blood.
The authors identified two cohorts with cryopreserved serum samples and diagnosis through either MTB culture or PCR, or clinical tuberculosis diagnosis through National Health Institute pediatric tuberculosis case definitions or World Health Organization guidelines for adults.
In the cohort that included both adults and children, the authors analyzed the sera of 30 patients with a microbiological diagnosis, 4 patients with a clinical diagnosis, and 39 patients without a microbiological or clinical diagnosis of tuberculosis that served as controls, finding a 96% sensitivity and 94% specificity in the adult group and 83%/95% sensitivity/specificity respectively in the pediatric group. Extrapulmonary tuberculosis cases involving lymph node, bone, joint, and pleura were found in this cohort.
The other cohort included only children younger than 12 years who were hospitalized with HIV and antiretroviral therapy naïve. In this cohort, the authors analyzed the sera of 153 patients that excluded central nervous system involvement, but since true negative controls were unavailable in this cohort, sensitivity and specificity were not calculated. However, longitudinal cryopreserved sera were available for analysis in this cohort, providing data to quantify changes in cell-free DNA concentrations before and after tuberculosis treatment. CRISPR fluorescent assay detected all children with confirmed tuberculosis, and MTB-cell-free DNA concentrations decreased following tuberculosis treatment.
Because MTB infection is on the differential diagnosis of many clinical syndromes, and diagnosis is often difficult to prove, the CRISPR fluorescent assay may be able to provide an easier diagnostic method through a blood test. It may also have a role in tuberculosis treatment monitoring given the finding of decreasing MTB-cell-free DNA concentrations with therapy. While it is exciting to see development of a rapid diagnostic tool for MTB infections, further calibration of CRISPR techniques in larger sample sizes may be needed to verify testing accuracy.
