Nucleic acid testing for tuberculosis at the point-of-care in high-burden countries

Abstract

Early diagnosis of tuberculosis (TB) facilitates appropriate treatment initiation and can limit the spread of this highly contagious disease. However, commonly used TB diagnostic methods are slow, often insensitive, cumbersome and inaccessible to most patients in TB endemic countries that lack necessary resources. This review discusses nucleic acid amplification technologies, which are being developed for rapid near patient TB diagnosis, that are in the market or undergoing clinical evaluation. They are based on PCR or isothermal methods and are implemented as manual assays or partially/fully integrated instrument systems, with associated tradeoffs between clinical performance, cost, robustness, quality assurance and usability in remote settings by minimally trained personnel. Unmet needs prevail for the identification of drug-resistant TB and for TB diagnosis in HIV-positive and pediatric patients.

Figure 1. Healthcare access pyramid for tuberculosis control in the developing world

Reproduced with permission from [103].

Figure 2. Levels of laboratory infrastructure in high tuberculosis-burden countries

(A) Tuberculosis testing in a district hospital with biosafety precautions entailing a simple dead air box. (B) Open sample processing at a crowded bench in a peripheral laboratory. (C) Community-level healthcare provider and (D) vehicle for ambulatory patient care. (E) Storage of laboratory supplies and (F) bio-hazardous waste disposal exemplify logistical challenges of clinical diagnostics in low-resource settings. Photographs provided courtesy of Gerard Cangelosi, Tanya Ferguson and PATH. Image (C) is reprinted with permission from [19].

Figure 3. Schematic representation of the Cepheid GeneXpert cartridge

Fluid transfer within the cartridge is controlled via a syringe plunger and a rotating valve. Sample preparation is executed in the bottom of the cartridge, whereas PCR amplification with real-time detection occurs in the rectangular PCR tube protruding from the back of the cartridge. Most of the cartridge body is used for reagent storage. Modified with permission from [49].

Figure 4. Process for tuberculosis diagnosis using the Eiken Loopamp Tuberculosis Complex Detection Reagent Kit, in conjunction with the Loopamp PURE DNA Extraction Kit

During sample preparation, a 40 μl volume of either raw or processed sputum sample is inserted into the heating tube, and mycobacteria are killed and heat lysed at 95°C for 5 min in the PureLAMP heater. The sample is transferred into an adsorbent tube, wherein contaminants are removed from the sample (precise technical specifications were not available for this review). DNA-containing filtrate (30 μl) is then transferred into a reaction tube containing dried LAMP TB assay mastermix. Sample preparation requires approximately 10 min, followed by isothermal amplification for 40 min, with either real-time detection using the Loopamp LF-160 or through visual end point detection using the PureLAMP Heater, which includes a UV lamp. LAMP: Loop-mediated isothermal amplification. Modified with permission from [53].

Figure 5. The XCP Nucleic Acid Detection Device (Ustar)

(A) Schematic cross-section, showing the amplicon cartridge containing a plastic bulb with lateral flow running buffer, and a 200 μl tube containing the reaction mix, inserted into the detection chamber holding the lateral flow test-strip. The detection chamber is then closed using a levered lid, which pushes the amplicon cartridge down into the detection chamber body, where the plastic bulb containing the nucleic acid lateral flow running buffer and the reaction tube are pierced as the lid snaps shut. The two fluids combine and then run up the lateral flow test strip. Results can be visually interpreted after 5–10 min. (B) The device on the left shows a negative reaction with the test line T remaining blank while the control line C indicates appropriate flow and test reagent reactivity for internal quality control of the lateral flow strip. In the device on the right, the test line is apparent in addition to the control line, indicating a positive cross-priming amplification test result. Images reprinted with permission from Ustar Biotechnologies and D Boyle, PATH.

Figure 6. The Molbio TrueLab Mycobacterium tuberculosis detection system

(A) The Trueprep^™-MAG and (B) the Truelab^™ Uno analyzer. The red-capped container is a sputum sample cup containing lyophilized reagents to liquefy the sputum. After DNA extraction 5 μl is transferred to the disposable reaction cartridge, which is then entered into the machine for processing and analysis. Images reprinted with permission from Bigtech Private Ltd (India).

Figure 7. Key components of Epistem’s Genedrive^® Mycobacterium tuberculosis iD^® system

(A) The Genedrive instrument, with tripartite port at the front for the insertion of the test cartridge. (B) The Genedrive test cartridge, with three protruding clear reaction tubes that are inserted into the instrument port prior to test execution. (C) The user interface displays test results for Mycobacterium tuberculosis identification and rifampin drug resistance testing in an easily interpretable and clinically actionable form. Images reprinted with permission from Epistem (UK).