Raeesa Gupte, PhD
Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (MTB), is a major global health problem. According to the World Health Organization (WHO), an estimated 10 million people contracted TB in 2018 and 1.5 million died of it.1 Approximately 30 countries contribute to 87 percent of the global TB burden. In these countries, TB is commonly diagnosed using sputum smear microscopy and bacterial culture.
Smear microscopy produces rapid results but is limited by low sensitivity and specificity. It can neither distinguish between Mycobacterium species nor provide information on drug resistance. More sensitive than microscopy, culture remains the gold standard in TB detection. However, it requires adequate laboratory infrastructure and takes four to eight weeks to produce conclusive results.
Rapid and accurate detection is crucial in initiating treatment and reducing disease transmission. Advances in microbiology and genetics have led to the development of novel molecular diagnostic tools.
Molecular diagnostic tests
Molecular diagnostics are more accurate than microscopy and significantly faster than culture methods. They are capable of detecting mutations associated with drug resistance and may also be used on non-respiratory specimens. Commercially available and upcoming molecular testing technologies are discussed below.
Nucleic acid amplification tests (NAATs)
Polymerase chain reaction (PCR) or reverse transcriptase PCR (RT-PCR) are among the most common molecular diagnostic tests for TB. Several commercial and laboratory-developed tests are available. These tests target sequences from genes encoding 16S rRNA, IS6110, hsp65, and dnaJ among others.2,3 The WHO recommends using a MTB/RIF test as the initial diagnostic test for simultaneous detection of TB and drug resistance. The assay amplifies a fragment of the ß subunit ofMTB RNA polymerase (rpoB) and probes it for rifampicin resistance-associated mutations. In a systematic review of 27 studies, the MTB/RIF test demonstrated pooled specificity of 99 percent and 89 percent sensitiv-ity in smear positive samples or 67 percent sensitivity in smear-negative samples. Pooled sensitivity of the assay for smear-positive and culture-positive samples was 98 percent.4 It had a pooled sensitivity of 94 percent and sensitivity of 98 percent in detecting rifampicin resistance. The test provides results in less than two hours.
Unlike PCR-based assays, loop-mediated isothermal amplification (LAMP) does not require a thermal cycler. LAMP is a highly sensitive method that amplifies target DNA at a constant temperature using a set of four specially designed primers and DNA polymerase with strand displacement activity. Due to the large output of amplification products, the result can be qualitatively observed by the naked eye or quantified using turbidity, colorimetry, or fluorescence detection methods.5 A meta-analysis of 13 studies showed that TB-LAMP had similar specificity but higher sensitivity than both sputum smear microscopy and the MTB/RIF assay.6 Therefore, the WHO recommends using this test as a replacement for microscopy during the diagnosis of pulmonary TB in symptomatic adults. Several LAMP-based assays have now been developed that target the gyrB, rrs, rim, IS6110, hspX, mpb64 and sdaA genes of MTB. These tests can provide results within one hour.
Whole genome sequencing (WGS)
WGS enables detection of single nucleotide polymorphisms, insertions, and deletions in the entire genome of an organism. Therefore, it can provide information on disease transmission, bacterial evolution, and drug resistance. For WGS analysis, MTB strains obtained from clinical samples (such as sputum) are grown in culture. DNA is extracted from the cultured isolates. Following enzymatic processing, the multiple DNA fragments obtained are sequenced in parallel. The individual fragments are then mapped to a reference genome in order to identify specific alterations in the genetic code of the test organism.7
A systematic review of 20 publications reported high specificity and sensitivity (pooled estimates over 95 percent) of WGS in detecting resistance to the first-line drugs rifampicin and isoniazid.8 However, these studies rely on bacterial culture that may delay results by several weeks. Direct WGS of sputum provided results on drug resistance within five days, compared to 11 days using cultured isolates. However, only 74 percent of sputum samples generated whole genomes of adequate quality compared to 100 percent of cultured samples.9 Therefore, the diagnostic workflow needs to be optimized before WGS is routinely used for clinical decisions.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have been extensively used for gene editing owing to their ability to function as molecular scissors. The discovery of the Cas12 and Cas13 family of proteins has spurred the development of CRISPR-based diagnostics. These diagnostics harness the “collateral cleavage” potential of Cas12 and Cas13. The endonuclease activity of Cas12 cleaves DNA and Cas13 cleaves RNA. The Cas proteins are coupled to guide RNA that targets a complementary sequence within a pathogen’s genome. The Cas proteins are activated once they cleave the targeted nucleotide sequence and continue to cleave nearby non-targeted DNA or RNA sequences. The reaction is visualized by introducing DNA and RNA reporters that fluoresce when cleaved.
A CRISPR-MTB assay was developed recently.10 DNA extracted from clinical samples was amplified at constant temperature using recombinase polymerase amplification. The amplified product was added to the CRISPR reaction mix containing Cas12a, guide RNA, and fluorescent reporter DNA. In order to enhance test sensitivity, guide RNA targeted the IS6110 gene since each MTB genome contains six to 10 copies of this gene. The CRISPR-MTB assay detected pulmonary TB with 90 percent sensitivity and had overall specificity of 98 percent. Results were obtained within 1.5 hours on average. Efforts are underway to develop a lateral flow assay that uses a simple paper test strip for point-of-care testing in low resource settings.11
Pros and cons of molecular testing technologies
Rapid turn-around time of NAAT systems facilitates testing and treatment initiation in the same visit, reducing cases of loss to follow up. LAMP tests are cost-effective and do not need specially trained personnel or elaborate infrastructure. This makes them ideal for use in resource-limited settings for point-of-care testing. Although several NAATs are available for testing MTB resistance to first-line drugs, drug susceptibility tests for second-line agents are limited. In countries with high TB burden, second-line drugs become crucial in treatment. With the ability to comprehensively detect multiple drug resistance genes at once, WGS may play an important role in such clinical settings.
The major limitation of PCR-based NAATs such as the MTB/RIF assay is their need for specialized instruments like thermal cyclers. The cost of test cartridges, advanced instrumentation, and availability of trained personnel are drawbacks for resource-limited countries with high TB burden. Similarly, WGS suffers from high costs associated with computing infrastructure and bioinformatics training. No commercial WGS kits are available and the diagnostic workflow needs to be optimized. In addition, the slow growth of bacterial culture may delay treatment initiation and lead to poor health outcomes. However, enrichment of DNA directly from clinical samples is challenging. Lastly, CRISPR-based diagnostic tests are still in their infancy. They need to undergo extensive optimization and standardization before their clinical validity is established.
1. Global tuberculosis report 2019. Geneva: World Health Organization (2019)
2. Singh, Anamika, and Vijendra Kumar Kashyap. "Specific andrapid detection of Mycobacterium tuberculosis complex inclinical samples by polymerase chain reaction." Interdisciplinary Perspectives on Infectious Diseases (2012).
3. Tevere, Vincent J., et al. "Detection of Mycobacterium tuberculosis by PCR amplification with pan-Mycobacterium primers and hybridization to an M. tuberculosis-specific probe." Journal of Clinical Microbiology 34.4 (1996): 918-923.
4. Steingart, Karen R., et al. "Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults." Cochrane Database of Systematic Reviews 1 (2013).
5. Notomi, Tsugunori, et al. "Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects." Journal of Microbiology 53.1 (2015): 1-5.
6. Shete, Priya B., et al. "Diagnostic accuracy of TB-LAMP forpulmonary tuberculosis: a systematic review and meta-analysis." BMC Infectious Diseases 19.1 (2019): 268.
7. World Health Organization.“The use of next-generation sequencing technologies for the detection of mutations associated with drug resistance in Mycobacterium tuberculosis complex: technical guide.” No. WHO/CDS/TB/2018.19.World Health Organization, 2018.
8. Papaventsis, D., et al. "Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review." Clinical Microbiology and Infection 23.2 (2017): 61-68.
9. Doyle, Ronan M., et al. "Direct whole-genome sequencing of sputum accurately identifies drug-resistant Mycobacterium tuberculosis faster than MGIT culture sequencing."Journal of Clinical Microbiology 56.8 (2018): e00666-18.
10. Ai, Jing-Wen, et al. "CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis." Emerging Microbes & Infections 8.1 (2019): 1361-1369.
11. Gronowski, Ann M. "Who or what is SHERLOCK?." EJIF-CC 29.3 (2018): 201.