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Chapter 40 : Point-of-Care Technologies for the Diagnosis of Active Tuberculosis

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Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, Page 1 of 2

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Abstract:

Tuberculosis (TB) is a preventable and curable disease, yet it is responsible for over 1.5 million deaths every year (1). In 2012, 6 million new cases of TB were diagnosed, yet an estimated two-thirds cases were missed. Almost half of the TB cases in the world are in Brazil, Russia, India, China, and South Africa, with the highest incidence in sub-Saharan Africa. Although TB is readily curable, the failure to diagnose more cases rapidly means that patients have poorer outcomes and prolonged infectiousness (2). TB therefore remains a global threat to public health.

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 1
FIGURE 1

Pipeline of new and emerging commercial tests for tuberculosis. Only selected tests are shown. Tests marked with a blue line are endorsed by the WHO, whereas those marked with an orange line have been reviewed but not endorsed, and those marked with a red line have not been reviewed nor endorsed. Adapted from references and . Abbreviations: Ab, antibody; Ag, antigen; DST, drug susceptibility test; LAM, lipoarabinomannan; LED, light-emitting diode; LPA, line probe assay; RT-PCR, real-time PCR; MDR-TB, multidrug-resistant tuberculosis; TB, tuberculosis; VOC, volatile organic compound.

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 2
FIGURE 2

Examples of peripheral tuberculosis microscopy centers in Uganda (A), India (B, C), and Kenya (D). Point-of-care or near-care tests for TB in most of the 22 high-burden countries will need to be performed in such facilities. Republished with permission from reference .

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 3
FIGURE 3

Characteristics of peripheral microscopy centers in 22 high-tuberculosis-burden countries. Questions are related to environmental conditions (Is temperature or humidity not a concern?); infrastructure (Is stable power supply, clean water supply present?); presence of equipment (Are N95 respirator, micropipettes, refrigerator, incubator, centrifuge, hot water bath, or biosafety hood present?) and skills (Are staff able to operate a micropipette or computer or perform a PCR test?); and the presence of means of communication (Is landline, mobile network, or Internet present?). Additional questions were asked about whether quality assurance measures were established and which smear methods were currently used. Countries are sorted by increasing purchasing power parity. The BRICS countries are Brazil, Russia, India, China, and South Africa. Republished with permission from reference .

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 4
FIGURE 4

The Xpert MTB/RIF system for the detection of TB and resistance to rifampin, which is the first automated nucleic acid amplification platform endorsed by the WHO. (A) Detailed illustration of the Xpert MTB/RIF cartridge body, showing the reagent reservoirs and the PCR amplification chamber, the front-view of an Xpert MTB/RIF cartridge, which is single use, and a GeneXpert four-module machine. (B) Specimen preparation procedure. (C) Five molecular beacons span the 81-bp rifampin resistance-determining region within the gene of . (D) The stem-loop structure within each beacon hybridizes to its complementary region and, after each amplification cycle, the quencher separates from the fluorophore, which after excitation emits light. (E) Examples of two results from the GeneXpert system. The first result is positive for and, because all beacons successfully bound to their amplicons, is found not to contain any rifampin-resistance-causing mutations, and is hence called rifampin-susceptible. The second example shows a failure of probe B to hybridize and amplify, presumably due to the presence of a mutation. This specimen is therefore detected as positive but rifampin-resistant. The bacillary load in the specimen is judged by the software to be “low,” “medium,” “high,” or “very high” based on the cycle threshold values generated by the reaction. The cartridge diagram in (A) is republished with permission from reference . Other images in (A) and (B) are republished with permission from Cepheid.

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 5
FIGURE 5

The TB-LAMP system for the detection of TB. (A) TB-LAMP test overview. Bacilli are first lysed using temperature and an extraction buffer before the lysate is mixed with the buffer in the absorbent tube and injected into reaction tubes, which contain the PCR reagents. The mixture is then incubated and the amplified product visualized by fluorescence under UV light. (B) TB-LAMP amplifies DNA using a novel strand-displacement polymerase and specially designed primers that contain oligonucleotides that hybridize to both the sense and antisense strands of the regions flanking the target sequence. The forward and back inner primers (FIP and BIP) first amplify the target sequencing and add a 5′ region that is complementary to the sequence downstream of the primer hybridization site. Once these strands are displaced by the DNA polymerase, they form stem-loop structures, which the FIP and BIP can hybridize to and, after elongation, serve as a template for further amplification. (C) The amplified DNA, which has a high molecular weight due to its complex secondary structure, is visualized by the titration of manganese by pyrophosphate, which is produced during the reaction, which allows the calcein marker within the reaction tubes to fluoresce. In this example, the middle four tubes are positive. (A) is republished with permission from reference , (B) is republished with permission from references and , and (C) is republished with permission from reference .

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 6
FIGURE 6

Urine lipoarabinomannan (LAM) strip test and reference scale card. The reference scale card, provided with each 100-strip packet, illustrates six cut-off points (visual grades 0 to 5) categorized by different band intensities appearing in the patient window. To optimize the specificity of the test, it is recommended that the grade 2 cut-point is used ( ). Republished with permission from reference .

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 7
FIGURE 7

Selected NAAT platforms currently under evaluation or in development. (A) The Epistem Genedrive system for the detection of , into which the cartridge with three ports that serve as reaction tubes are inserted. The results screen is also shown. (B) A schematic of the XCP Nucleic Acid Device (Ustar Biotechnologies), which is used in conjunction with the EasyNAT TB test (Ustar Biotechnologies) cartridge. The cartridge contains a plastic bulb with both the reaction mix and a lateral flow running buffer. This is inserted into the detection chamber holding the lateral flow test strip. After 5 to 10 minutes the result is read. Examples of negative and positive test results with control bands are shown. (C) The equipment made by the Molbio Group (India) required to process specimens and extract DNA (Trueprep) and monitor amplification (Truelab UNO real-time PCR analyzer) and the chip used for the detection of DNA. (D) The Fluorocycler (Hain Lifesciences) which is used for the semi-automated detection of TB using the Fluorotype MTB test. (E) The Alere Q system and cartridge, which are currently being developed for the detection of TB. It performs on-board sample processing, lysis, DNA extraction, and TB detection. Sputum is collected in a special container that is attached to the test cartridge, which is then inserted into the machine. A and B are republished with permission from reference . C, D, and E are republished with permission from reference .

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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Image of FIGURE 8
FIGURE 8

The Twista diagnostic platform. The left panel shows the battery-powered portable fluorometer for monitoring the progress of the recombinase polymerase amplification reaction. The right panel shows the mechanisms of amplification, in which three core proteins (a recombinase, single-strand DNA binding protein [SSB], and strand-displacing polymerase) isothermally amplify DNA ( ). The right-hand panel was created by TwistDx Ltd (http://www.twistdx.co.uk/our_technology/) and is licensed under a Creative Commons Attribution 3.0 United States License.

Citation: Theron G. 2016. Point-of-Care Technologies for the Diagnosis of Active Tuberculosis, p 556-579. In Persing D, Tenover F, Hayden R, Ieven M, Miller M, Nolte F, Tang Y, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555819071.ch40
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