Outlook for CRISPR-based tuberculosis assays now in their infancy

Abstract

Tuberculosis (TB) remains a major underdiagnosed public health threat worldwide, being responsible for more than 10 million cases and one million deaths annually. TB diagnosis has become more rapid with the development and adoption of molecular tests, but remains challenging with traditional TB diagnosis, but there has not been a critical review of this area. Here, we systematically review these approaches to assess their diagnostic potential and issues with the development and clinical evaluation of proposed CRISPR-based TB assays. Based on these observations, we propose constructive suggestions to improve sample pretreatment, method development, clinical validation, and accessibility of these assays to streamline future assay development and validation studies.

Keywords: CRISPR; challenge and outlook; diagnosis; point-of-care; tuberculosis.

Figure 1

Current CRISPR-TB assay parameters and performance. (A) Methodological details of 12 studies employing CRISPR-TB assays. (B) Summary of QUADAS assessment results for the risk of bias in assessing data quality. Nine studies performed clinical sample evaluations (answers to QUADAS questions are listed in Table S1 ). The bar length reflects the frequency of an answer for each question. DC, disease contacts; DR, drug-resistant; HC, healthy control; LAMP, loop-mediated isothermal amplification; LOD, the limit of detection; NAA, nucleic acid amplification; OD, other diseases; PCR, polymerase chain reaction; RPA, recombinase polymerase amplification; UR, unreported. (C) Forest plot for the sensitivity and specificity of the CRISPR-TB assay and GeneXpert MTB/RIF (Xpert) results when compared with a composite reference standard. Details of the ten clinical specimen or strain validation studies, including control group information and diagnostic performance, are summarized in Table S2 .

Figure 2

Characteristics desired for future CRISPR-TB assays. Future CRISPR-TB assays should ideally analyze noninvasive or minimally invasive diagnostic specimens (e.g., breath aerosol, saliva, sputum, swab, blood, urine and stool samples) and employ an integrated platform with lyophilized reagents to minimize cold chain concerns. These assays should integrate sample treatment and target detection reactions in a streamlined workflow to provide assay results within minutes. The assay readout should also provide quantitative results when read by a smart terminal, and this device should be able to report these results to a central system to facilitate TB control efforts or telemedicine interventions. Mtb, Mycobacterium tuberculosis.